Relation between Assertiveness, Academic Self-Efficacy, and Psychosocial Adjustment among International Graduate Students.

ERIC Educational Resources Information Center

Poyrazli, Senel; Arbona, Consuelo; Nora, Amaury; McPherson, Robert; Pisecco, Stewart

2002-01-01

Rathus Assertiveness Schedule, Academic Self-Efficacy Scale, The Inventory for Student Adjustment Strain, and UCLA Loneliness Scale were used to examine a total of 122 graduate international students. Findings indicate that English proficiency, assertiveness, and academic self-efficacy contributed uniquely to the variance in students' general…

The effect of high and low assertiveness on locus of control and health problems.

PubMed

Williams, J M; Stout, J K

1985-03-01

The effect of high and low assertiveness on locus of control and health problems was examined with 78 direct-service workers in mental health and mental retardation settings in northeastern Pennsylvania. The direct-service workers completed the Rathus (1973) Assertiveness Schedule, the Rotter (1966) Internal-External Locus of Control Scale, and a health-problems inventory. Highly assertive individuals were found to be more internally controlled and to experience fewer health problems than were individuals low in assertiveness.

Is modified brief assertiveness training for nurses effective? A single-group study with long-term follow-up.

PubMed

Yoshinaga, Naoki; Nakamura, Yohei; Tanoue, Hiroki; MacLiam, Fionnula; Aoishi, Keiko; Shiraishi, Yuko

2018-01-01

To evaluate the long-term effectiveness of modified brief assertiveness training (with cognitive techniques) for nurses. Most assertiveness training takes a long time to conduct; thus, briefer training is required for universal on-the-job training in the workplace. In this single-group study, nurses received two 90-min training sessions with a 1-month interval between sessions. The degree of assertiveness was assessed by using the Rathus Assertiveness Schedule as the primary outcome, at four time points: pre- and post-training, 3-month follow-up and 6-month follow-up. A total of 33 nurses received the training, and the mean Rathus Assertiveness Schedule score improved from -14.2 (SD = 16.5) pre-training to -10.5 (SD = 18.0) post-training (p < .05). These improvements were maintained until the 6-month follow-up. The pre-post effect size of 0.22 (indicating small effect) was larger than the effect sizes ranging from -0.56 to 0.17 (no effect) reported in previous studies that used brief training. Modified brief assertiveness training seems feasible and may achieve long-term favourable outcomes in improving assertiveness among nurses. The ease of implementation of assertiveness training is important because creating an open environment for communication leads to improved job satisfaction, improved nursing care and increased patient safety. © 2017 The Authors. Journal of Nursing Management Published by John Wiley & Sons Ltd.

Factors affecting assertiveness among student nurses.

PubMed

Ibrahim, Sanaa Abd El Azim

2011-05-01

This study aimed to investigate the factors affecting assertiveness among student nurses. The study was carried out at Faculty of Nursing, Port-Said University, on 207 student nurses from four different grades. Rathus Assertiveness Schedule, consisted of 30 items, was used to measure the students' assertiveness level and a 12-item scale developed by Spreitzer was used to measure students' psychological empowerment. The study results showed that 60.4% of the students were assertive, while about half of the students were empowered. A positive relation between student assertiveness and psychological empowerment was detected. Moreover, positive relations regarding family income and students' assertiveness and psychological empowerment were determined. The study recommended introduction of specific courses aiming at enhancing the acquisition of assertiveness skills, in addition, nurse educators must motivate their students to express their opinion and personal rights and also they must pay attention for students' empowerment and enhance students' autonomy. Copyright © 2010 Elsevier Ltd. All rights reserved.

Palonosetron as an anti-emetic and anti-nausea agent in oncology.

PubMed

Aapro, Matti S

2007-12-01

Palonosetron (Aloxi(®), Onicit(®), Paloxi(®)) is a second-generation 5-HT(3) receptor antagonist (RA) with an extended half-life of ~40 hours and high binding affinity for the 5-HT₃ receptor that is markedly different from other 5-HT(3) RAs. Phase III trials demonstrate that a single dose of palonosetron compared with traditional 5-HT₃ RAs is more effective in preventing chemotherapy-induced nausea and vomiting (CINV) during the first 24 hours following chemotherapy (acute CINV), and also exhibits prolonged efficacy to provide significantly better protection from CINV in the delayed and overall phases. This superior and extended protection from CINV conferred by palonosetron following a single intravenous dose before chemotherapy simplifies dosing schedules. Recent research has focused on optimization of palonosetron-based antiemetic regimens, particularly in combination with steroids and neurokinin-1 RAs. The available clinical data indicate high control rates for palonosetron, suggesting a synergistic potential for protection in patients scheduled to receive emetogenic drug regimens.

Risk and protective factors of dissocial behavior in a probability sample.

PubMed

Moral de la Rubia, José; Ortiz Morales, Humberto

2012-07-01

The aims of this study were to know risk and protective factors for dissocial behavior keeping in mind that the self-report of dissocial behavior is biased by the impression management. A probability sample of adolescents that lived in two neighborhoods with high indexes of gangs and offenses (112 male and 86 women) was collected. The 27-item Dissocial Behavior Scale (ECODI27; Pacheco & Moral, 2010), Balanced Inventory of Desirable Responding, version 6 (BIDR-6; Paulhus, 1991), Sensation Seeking Scale, form V (SSS-V; Zuckerman, Eysenck, & Eysenck, 1978), Parent-Adolescent Communication Scale (PACS; Barnes & Olson, 1982), 30-item Rathus Assertiveness Schedule (RAS; Rathus, 1973), Interpersonal Reactivity Index (IRI; Davis, 1983) and a social relationship questionnaire (SRQ) were applied. Binary logistic regression was used for the data analysis. A third of the participants showed dissocial behavior. Belonging to a gang in the school (schooled adolescents) or to a gang out of school and job (total sample) and desinhibition were risk factors; being woman, perspective taking and open communication with the father were protective factors. School-leaving was a differential aspect. We insisted on the need of intervention on these variables.

Influence of Scheduling Variations on the Effectiveness of a Group Assertion-Training Program for Women.

ERIC Educational Resources Information Center

Berah, Ellen F.

1981-01-01

Explored the applicability of laboratory findings on the effects of massed versus distributed practice to an applied situation. Four groups were compared: massed practice, distributed practice, combination massed and distributed practice assertion-training groups, and no-treatment control groups. No differences between the different types of…

Synchronization of Leisure Conflicts in the Family Schedule.

ERIC Educational Resources Information Center

Kelly, John R.

The model of the family as a set of contingent careers is brought into focus by Wilbert Moore's suggestions that in schedule synchronization the family takes priority in claiming time, the wife-mother asserts such claims and arranges the schedule, and common leisure activities symbolize the solidarity of the unit. The perception of parents of the…

The relationship between psychosocial maturity and assertiveness in males and females.

PubMed

Goldman, J A; Olczak, P V

1981-02-01

The relationship between psychosocial maturity (psychological health) and assertiveness was investigated in a sample of United States college males and females. Results revealed a moderately high positive relationship between psychosocial maturity (PSM) and self-reported assertiveness on the Rathus and Galassi scales for both sexes. This relationship was slightly stronger (in terms of variance accounted for) for males than females, significant differences being obtained for Intimacy on the Rathus scale and PSM and Intimacy on the Galassi scale. Multiple regression analyses revealed that the personality components most consistently accounting for major portions of the variance in predicting male assertiveness scores on both the Rathus Assertiveness Schedule and the College Self-Expression Scale were Intimacy and Initiative, while in predicting female assertiveness, only Initiative was involved. The findings were related to previous research, recent work on the androgyny construct (instrumental vs. expressive behaviors), and exhortations for increased cooperation between schools of psychotherapy to establish it as a more unified discipline.

75 FR 16337 - Standards for Business Practices for Interstate Natural Gas Pipelines

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-01

... transportation by power plant operators more difficult. In response to this need, in early 2004, NAESB... receipt and delivery points; and (3) changes to the intraday nomination schedule to increase the number of... conference on the issue of intraday pipeline nomination schedules. In this regard, NGSA asserts that NAESB...

Freshwater Recirculating Aquaculture System Operations Drive Biofilter Bacterial Community Shifts around a Stable Nitrifying Consortium of Ammonia-Oxidizing Archaea and Comammox Nitrospira

PubMed Central

Bartelme, Ryan P.; McLellan, Sandra L.; Newton, Ryan J.

2017-01-01

Recirculating aquaculture systems (RAS) are unique engineered ecosystems that minimize environmental perturbation by reducing nutrient pollution discharge. RAS typically employ a biofilter to control ammonia levels produced as a byproduct of fish protein catabolism. Nitrosomonas (ammonia-oxidizing), Nitrospira, and Nitrobacter (nitrite-oxidizing) species are thought to be the primary nitrifiers present in RAS biofilters. We explored this assertion by characterizing the biofilter bacterial and archaeal community of a commercial scale freshwater RAS that has been in operation for >15 years. We found the biofilter community harbored a diverse array of bacterial taxa (>1000 genus-level taxon assignments) dominated by Chitinophagaceae (~12%) and Acidobacteria (~9%). The bacterial community exhibited significant composition shifts with changes in biofilter depth and in conjunction with operational changes across a fish rearing cycle. Archaea also were abundant, and were comprised solely of a low diversity assemblage of Thaumarchaeota (>95%), thought to be ammonia-oxidizing archaea (AOA) from the presence of AOA ammonia monooxygenase genes. Nitrosomonas were present at all depths and time points. However, their abundance was >3 orders of magnitude less than AOA and exhibited significant depth-time variability not observed for AOA. Phylogenetic analysis of the nitrite oxidoreductase beta subunit (nxrB) gene indicated two distinct Nitrospira populations were present, while Nitrobacter were not detected. Subsequent identification of Nitrospira ammonia monooxygenase alpha subunit genes in conjunction with the phylogenetic placement and quantification of the nxrB genotypes suggests complete ammonia-oxidizing (comammox) and nitrite-oxidizing Nitrospira populations co-exist with relatively equivalent and stable abundances in this system. It appears RAS biofilters harbor complex microbial communities whose composition can be affected directly by typical system operations while supporting multiple ammonia oxidation lifestyles within the nitrifying consortium. PMID:28194147

The Depressive Experiences Questionnaire: validity and psychological correlates in a clinical sample.

PubMed

Riley, W T; McCranie, E W

1990-01-01

This study sought to compare the original and revised scoring systems of the Depressive Experiences Questionnaire (DEQ) and to assess the construct validity of the Dependent and Self-Critical subscales of the DEQ in a clinically depressed sample. Subjects were 103 depressed inpatients who completed the DEQ, the Beck Depression Inventory (BDI), the Hopelessness Scale, the Automatic Thoughts Questionnaire (ATQ), the Rathus Assertiveness Schedule (RAS), and the Minnesota Multiphasic Personality Inventory (MMPI). The original and revised scoring systems of the DEQ evidenced good concurrent validity for each factor scale, but the revised system did not sufficiently discriminate dependent and self-critical dimensions. Using the original scoring system, self-criticism was significantly and positively related to severity of depression, whereas dependency was not, particularly for males. Factor analysis of the DEQ scales and the other scales used in this study supported the dependent and self-critical dimensions. For men, the correlation of the DEQ with the MMPI scales indicated that self-criticism was associated with psychotic symptoms, hostility/conflict, and a distress/exaggerated response set, whereas dependency did not correlate significantly with any MMPI scales. Females, however, did not exhibit a differential pattern of correlations between either the Dependency or the Self-Criticism scales and the MMPI. These findings suggest possible gender differences in the clinical characteristics of male and female dependent and self-critical depressive subtypes.

Scheduling nursing personnel on a microcomputer.

PubMed

Liao, C J; Kao, C Y

1997-01-01

Suggests that with the shortage of nursing personnel, hospital administrators have to pay more attention to the needs of nurses to retain and recruit them. Also asserts that improving nurses' schedules is one of the most economic ways for the hospital administration to create a better working environment for nurses. Develops an algorithm for scheduling nursing personnel. Contrary to the current hospital approach, which schedules nurses on a person-by-person basis, the proposed algorithm constructs schedules on a day-by-day basis. The algorithm has inherent flexibility in handling a variety of possible constraints and goals, similar to other non-cyclical approaches. But, unlike most other non-cyclical approaches, it can also generate a quality schedule in a short time on a microcomputer. The algorithm was coded in C language and run on a microcomputer. The developed software is currently implemented at a leading hospital in Taiwan. The response to the initial implementation is quite promising.

Depression, automatic thoughts, alexithymia, and assertiveness in patients with tension-type headache.

PubMed

Yücel, Basak; Kora, Kaan; Ozyalçín, Süleyman; Alçalar, Nilüfer; Ozdemir, Ozay; Yücel, Aysen

2002-03-01

The role of psychological factors related to headache has long been a focus of investigation. The aim of this study was to evaluate depression, automatic thoughts, alexithymia, and assertiveness in persons with tension-type headache and to compare the results with those from healthy controls. One hundred five subjects with tension-type headache (according to the criteria of the International Headache Society classification) and 70 controls were studied. The Beck Depression Inventory, Automatic Thoughts Scale, Toronto Alexithymia Scale, and Rathus Assertiveness Schedule were administered to both groups. Sociodemographic variables and headache features were evaluated via a semistructured scale. Compared with healthy controls, the subjects with headache had significantly higher scores on measures of depression, automatic thoughts, and alexithymia and lower scores on assertiveness. Subjects with chronic tension-type headache had higher depression and automatic thoughts scores than those with episodic tension-type headache. These findings suggested that persons with tension-type headache have high depression scores and also may have difficulty with expression of their emotions. Headache frequency appears to influence the likelihood of coexisting depression.

Women's attitudes toward forcible rape.

PubMed

Tolor, A

1978-01-01

This study assessed the attitudes of women with diverse backgrounds toward possible responses to an attempted sexual assault and also ascertained their beliefs about how society should handle a convicted rapist. Additionally, the role which the personality dimensions of assertiveness and internal-external expectamcy play in shaping attitudes toward rape was investigated. Seventy-seven women and 25 men were administered the Rathus Assertiveness Schedule, the Rotter I-E Scale, and a Rape Inventory. Women relied on a narrow range of options to deal with a rapist's attack, depending mostly on less active modes. They advocated humane approaches be taken with convicted rapists. Personality played only a minor role.

Rationale for combination therapy in hypertension management: focus on angiotensin receptor blockers and thiazide diuretics.

PubMed

Nash, David T

2007-04-01

Despite recognition that hypertension is a major risk factor for cardiovascular events and mortality, blood pressure control rates remain low in the US population. Reflecting clinical trial results, hypertension management guidelines assert the clinical benefit of achieving current blood pressure goals and indicate that most patients will require 2 or more drugs to reach goal. Well-designed drug combinations counter hypertension via complementary mechanisms that increase antihypertensive efficacy, potentially with lower rates of adverse events than higher dose monotherapy regimens. Lower adverse event rates, in turn, may contribute to greater adherence with treatment. The combination of a low-dose diuretic with agents that block the effects of the renin-angiotensin system (RAS), such as angiotensin receptor blockers, has been found in numerous clinical trials to be highly effective for lowering blood pressure in patients with uncomplicated as well as high-risk hypertension, with a comparable favorable side effect profile compared with monotherapy. Moreover, agents that block the RAS are associated with a lower risk of new-onset diabetes mellitus than other antihypertensive classes. Complementary combinations of antihypertensive agents provide an efficient and effective approach to hypertension management.

IAA RAS Radio Telescope Monitoring System

NASA Astrophysics Data System (ADS)

Mikhailov, A.; Lavrov, A.

2007-07-01

Institute of Applied Astronomy of the Russian Academy of Sciences (IAA RAS) has three identical radio telescopes, the receiving complex of which consists of five two-channel receivers of different bands, six cryogen systems, and additional devices: four local oscillators, phase calibration generators and IF commutator. The design, hardware and data communication protocol are described. The most convenient way to join the devices of the receiving complex into the common monitoring system is to use the interface which allows to connect numerous devices to the data bus. For the purpose of data communication regulation and to exclude conflicts, a data communication protocol has been designed, which operates with complex formatted data sequences. Formation of such sequences requires considerable data processing capability. That is provided by a microcontroller chip in each slave device. The test version of the software for the central computer has been developed in IAA RAS. We are developing the Mark IV FS software extension modules, which will allow us to control the receiving complex of the radio telescope by special SNAP commands from both operator input and schedule files. We are also developing procedures of automatic measurements of SEFD, system noise temperature and other parameters, available both in VLBI and single-dish modes of operation. The system described has been installed on all IAA RAS radio telescopes at "Svetloe", "Zelenchukskaya" and "Badary" observatories. It has proved to be working quite reliably and to show the perfonmance expected.

Vaccine-Related Beliefs and Practices of Parents of Children with Autism Spectrum Disorders

ERIC Educational Resources Information Center

Bazzano, Alicia; Zeldin, Ari; Schuster, Erica; Barrett, Christopher; Lehrer, Danise

2012-01-01

Although the assertion of a link between vaccines and autism has been scientifically rejected, the theory continues to be popular and may influence the attitudes of parents of children with autism spectrum disorders. The authors sought to assess how often parents change or discontinue their child's vaccine schedule after autism spectrum disorder…

The schedule effect: can recurrent peak infections be reduced without vaccines, quarantines or school closings?

PubMed

Diedrichs, Danilo R; Isihara, Paul A; Buursma, Doeke D

2014-02-01

Using a basic, two transmission level seasonal SIR model, we introduce mathematical evidence for the schedule effect which asserts that major recurring peak infections can be significantly reduced by modification of the traditional school calendar. The schedule effect is observed first in simulated time histories of the infectious population. Schedules with higher average transmission rate may exhibit reduced peak infections. Splitting vacations changes the period of the oscillating transmission function and can confine limit cycles in the proportion susceptible/proportion infected phase plane. Numerical analysis of the phase plane shows the relationship between the transmission period and the maximum recurring infection peaks and period of the response. For certain transmission periods, this response may exhibit period-doubling and chaos, leading to increased peaks. Non-monotonic infectious response is also observed in conjunction with changing birth rate. We discuss how to take these effects into consideration to design an optimum school schedule with particular reference to a hypothetical developing world context. Copyright © 2013 Elsevier Inc. All rights reserved.

Identification of Psychological Dysfunctions and Eating Disorders in Obese Women Seeking Weight Loss: Cross-Sectional Study

PubMed Central

Panchaud Cornut, Maude; Szymanski, Jennifer; Marques-Vidal, Pedro; Giusti, Vittorio

2014-01-01

Objective. The aim of this study is to analyse associations between eating behaviour and psychological dysfunctions in treatment-seeking obese patients and identify parameters for the development of diagnostic tools with regard to eating and psychological disorders. Design and Methods. Cross-sectional data were analysed from 138 obese women. Bulimic Investigatory Test of Edinburgh and Eating Disorder Inventory-2 assessed eating behaviours. Beck Depression Inventory II, Spielberger State-Trait Anxiety Inventory, form Y, Rathus Assertiveness Schedule, and Marks and Mathews Fear Questionnaire assessed psychological profile. Results. 61% of patients showed moderate or major depressive symptoms and 77% showed symptoms of anxiety. Half of the participants presented with a low degree of assertiveness. No correlation was found between psychological profile and age or anthropometric measurements. The prevalence and severity of depression, anxiety, and assertiveness increased with the degree of eating disorders. The feeling of ineffectiveness explained a large degree of score variance. It explained 30 to 50% of the variability of assertiveness, phobias, anxiety, and depression. Conclusion. Psychological dysfunctions had a high prevalence and their severity is correlated with degree of eating disorders. The feeling of ineffectiveness constitutes the major predictor of the psychological profile and could open new ways to develop screening tools. PMID:24737999

A Preliminary Investigation of the Reinforcement Function of Signal Detections in Simulated Baggage Screening: Further Support for the Vigilance Reinforcement Hypothesis

ERIC Educational Resources Information Center

Hogan, Lindsey C.; Bell, Matthew; Olson, Ryan

2009-01-01

The vigilance reinforcement hypothesis (VRH) asserts that errors in signal detection tasks are partially explained by operant reinforcement and extinction processes. VRH predictions were tested with a computerized baggage screening task. Our experiment evaluated the effects of signal schedule (extinction vs. variable interval 6 min) and visual…

Pricing the Computing Resources: Reading Between the Lines and Beyond

NASA Technical Reports Server (NTRS)

Nakai, Junko; Veronico, Nick (Editor); Thigpen, William W. (Technical Monitor)

2001-01-01

Distributed computing systems have the potential to increase the usefulness of existing facilities for computation without adding anything physical, but that is realized only when necessary administrative features are in place. In a distributed environment, the best match is sought between a computing job to be run and a computer to run the job (global scheduling), which is a function that has not been required by conventional systems. Viewing the computers as 'suppliers' and the users as 'consumers' of computing services, markets for computing services/resources have been examined as one of the most promising mechanisms for global scheduling. We first establish why economics can contribute to scheduling. We further define the criterion for a scheme to qualify as an application of economics. Many studies to date have claimed to have applied economics to scheduling. If their scheduling mechanisms do not utilize economics, contrary to their claims, their favorable results do not contribute to the assertion that markets provide the best framework for global scheduling. We examine the well-known scheduling schemes, which concern pricing and markets, using our criterion of what application of economics is. Our conclusion is that none of the schemes examined makes full use of economics.

DOD Financial Management: Improved Documentation Needed to Support the Air Force’s Military Payroll and Meet Audit Readiness Goals

DTIC Science & Technology

2015-12-01

balances to match Treasury balances. 19The financial reporting system collects and consolidates information for financial statement presentation...2The financial reporting system collects and consolidates information for financial statement presentation. 3Subsistence...efforts to achieve auditability of its financial statements , the Air Force in July 2014 asserted audit readiness for its Schedule of Budgetary

Base Closure and Realignment Act (BRAC) Cleanup Plan, Sacramento Army Depot, Sacramento, California

DTIC Science & Technology

1995-10-01

and remedial actions ( RAs ) presented in this document are believed to be accurate and have been reviewed by the regulatory agencies. However, this...of items to be competed, a time-phased schedule, and source selection criteria. The competition will begin as soon as possible. The Communications...allowed to be emitted into the region’s air from all permitted sources . If new sources are sited within these non-attainment areas, they cannot emit more

RESISTANCE TO EXTINCTION AND RELAPSE IN COMBINED STIMULUS CONTEXTS

PubMed Central

Podlesnik, Christopher A; Bai, John Y. H; Elliffe, Douglas

2012-01-01

Reinforcing an alternative response in the same context as a target response reduces the rate of occurrence but increases the persistence of that target response. Applied researchers who use such techniques to decrease the rate of a target problem behavior risk inadvertently increasing the persistence of the same problem behavior. Behavioral momentum theory asserts that the increased persistence is a function of the alternative reinforcement enhancing the Pavlovian relation between the target stimulus context and reinforcement. A method showing promise for reducing the persistence-enhancing effects of alternative reinforcement is to train the alternative response in a separate stimulus context before combining with the target stimulus in extinction. The present study replicated previous findings using pigeons by showing that combining an “alternative” richer VI schedule (96 reinforcers/hr) with a “target” leaner VI schedule (24 reinforcers/hr) reduced resistance to extinction of target responding compared with concurrent training of the alternative and target responses (totaling 120 reinforcers/hr). We also found less relapse with a reinstatement procedure following extinction with separate-context training, supporting previous findings that training conditions similarly influence both resistance to extinction and relapse. Finally, combining the alternative stimulus context was less disruptive to target responding previously trained in the concurrent schedule, relative to combining with the target response trained alone. Overall, the present findings suggest the technique of combining stimulus contexts associated with alternative responses with those associated with target responses disrupts target responding. Furthermore, the effectiveness of this disruption is a function of training context of reinforcement for target responding, consistent with assertions of behavioral momentum theory. PMID:23008521

Schools That Do Too Much: Wasting Time and Money in Schools and What We Can All Do about It.

ERIC Educational Resources Information Center

Kralovec, Etta

This book asserts that time and money are systematically misspent in U.S. schools, suggesting that this is a result of a variety of factors. From class schedules that fragment students' time, to budgets that invest money in dozens of activities that are essentially distractions from learning, schools try to do too much and end up delivering too…

Autonomous power expert system

NASA Technical Reports Server (NTRS)

Walters, Jerry L.; Petrik, Edward J.; Roth, Mary Ellen; Truong, Long Van; Quinn, Todd; Krawczonek, Walter M.

1990-01-01

The Autonomous Power Expert (APEX) system was designed to monitor and diagnose fault conditions that occur within the Space Station Freedom Electrical Power System (SSF/EPS) Testbed. APEX is designed to interface with SSF/EPS testbed power management controllers to provide enhanced autonomous operation and control capability. The APEX architecture consists of three components: (1) a rule-based expert system, (2) a testbed data acquisition interface, and (3) a power scheduler interface. Fault detection, fault isolation, justification of probable causes, recommended actions, and incipient fault analysis are the main functions of the expert system component. The data acquisition component requests and receives pertinent parametric values from the EPS testbed and asserts the values into a knowledge base. Power load profile information is obtained from a remote scheduler through the power scheduler interface component. The current APEX design and development work is discussed. Operation and use of APEX by way of the user interface screens is also covered.

Non-traditional Sensor Tasking for SSA: A Case Study

NASA Astrophysics Data System (ADS)

Herz, A.; Herz, E.; Center, K.; Martinez, I.; Favero, N.; Clark, C.; Therien, W.; Jeffries, M.

Industry has recognized that maintaining SSA of the orbital environment going forward is too challenging for the government alone. Consequently there are a significant number of commercial activities in various stages of development standing-up novel sensors and sensor networks to assist in SSA gathering and dissemination. Use of these systems will allow government and military operators to focus on the most sensitive space control issues while allocating routine or lower priority data gathering responsibility to the commercial side. The fact that there will be multiple (perhaps many) commercial sensor capabilities available in this new operational model begets a common access solution. Absent a central access point to assert data needs, optimized use of all commercial sensor resources is not possible and the opportunity for coordinated collections satisfying overarching SSA-elevating objectives is lost. Orbit Logic is maturing its Heimdall Web system - an architecture facilitating “data requestor” perspectives (allowing government operations centers to assert SSA data gathering objectives) and “sensor operator” perspectives (through which multiple sensors of varying phenomenology and capability are integrated via machine -machine interfaces). When requestors submit their needs, Heimdall’s planning engine determines tasking schedules across all sensors, optimizing their use via an SSA-specific figure-of-merit. ExoAnalytic was a key partner in refining the sensor operator interfaces, working with Orbit Logic through specific details of sensor tasking schedule delivery and the return of observation data. Scant preparation on both sides preceded several integration exercises (walk-then-run style), which culminated in successful demonstration of the ability to supply optimized schedules for routine public catalog data collection – then adapt sensor tasking schedules in real-time upon receipt of urgent data collection requests. This paper will provide a narrative of the joint integration process - detailing decision points, compromises, and results obtained on the road toward a set of interoperability standards for commercial sensor accommodation.

Autism Screening With Online Decision Support by Primary Care Pediatricians Aided by M-CHAT/F.

PubMed

Sturner, Raymond; Howard, Barbara; Bergmann, Paul; Morrel, Tanya; Andon, Lindsay; Marks, Danielle; Rao, Patricia; Landa, Rebecca

2016-09-01

Autism spectrum disorders (ASDs) often go undetected in toddlers. The Modified Checklist for Autism in Toddlers (M-CHAT) With Follow-up Interview (M-CHAT/F) has been shown to improve detection and reduce over-referral. However, there is little evidence supporting the administration of the interview by a primary care pediatrician (PCP) during typical checkups. The goal of this study was to evaluate the feasibility, validity, and reliability of the M-CHAT/F by PCPs with online prompts at the time of a positive M-CHAT screen. Forty-seven PCPs from 22 clinics completed 197 M-CHAT/Fs triggered by positive M-CHAT screens via the same secure Web-based platform that parents used to complete M-CHATs before an 18- or 24-month well-child visit. A second M-CHAT/F was administered live or by telephone by trained research assistants (RAs) at the Kennedy Krieger Institute Center for Autism and Related Disorders. The Autism Diagnostic Observation Schedule, Second Edition, and the Mullen Scales of Early Learning were administered as criterion measures. Measures of agreement between PCPs and RAs were calculated, and measures of test performance compared. There was 86.6% agreement between PCPs and RAs, with a Cohen's κ of 0.72. Comparison of sensitivity, specificity, positive predictive value (PPV), and overall accuracy for M-CHAT/F between PCPs and RAs showed significant equivalence for all measures. Use of the M-CHAT/F by PCPs resulted in significant improvement in PPV compared with the M-CHAT alone. Minimally trained PCPs can administer the M-CHAT/F reliably and efficiently during regular well-child visits, increasing PPV without compromising detection. Copyright © 2016 by the American Academy of Pediatrics.

The Human Response to the Gander Military Air Disaster: A Summary Report

DTIC Science & Technology

1987-12-01

should be asserted by leaders in that it refocuses thinking toward the future. Training Strategies oo Formal classes on grief leadership should be...2. Family support group members should be given the option to volunteer their assistance to the formal death notification teams. This includes...chaplains avoided burnout by trusting others in helping roles to take over for them, scheduling time for rest, and sharing feelings and experiences with

Ras proteins have multiple functions in vegetative cells of Dictyostelium.

PubMed

Bolourani, Parvin; Spiegelman, George; Weeks, Gerald

2010-11-01

During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG⁻ cells are only partially deficient in chemotaxis, whereas rasC⁻/rasG⁻ cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG⁻, rasC⁻, and rasC⁻/rasG⁻ cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative rasG⁻ and rasC⁻/rasG⁻ cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of rasG⁻ and rasC⁻/rasG⁻ cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells.

The Function of Embryonic Stem Cell-expressed RAS (E-RAS), a Unique RAS Family Member, Correlates with Its Additional Motifs and Its Structural Properties.

PubMed

Nakhaei-Rad, Saeideh; Nakhaeizadeh, Hossein; Kordes, Claus; Cirstea, Ion C; Schmick, Malte; Dvorsky, Radovan; Bastiaens, Philippe I H; Häussinger, Dieter; Ahmadian, Mohammad Reza

2015-06-19

E-RAS is a member of the RAS family specifically expressed in embryonic stem cells, gastric tumors, and hepatic stellate cells. Unlike classical RAS isoforms (H-, N-, and K-RAS4B), E-RAS has, in addition to striking and remarkable sequence deviations, an extended 38-amino acid-long unique N-terminal region with still unknown functions. We investigated the molecular mechanism of E-RAS regulation and function with respect to its sequence and structural features. We found that N-terminal extension of E-RAS is important for E-RAS signaling activity. E-RAS protein most remarkably revealed a different mode of effector interaction as compared with H-RAS, which correlates with deviations in the effector-binding site of E-RAS. Of all these residues, tryptophan 79 (arginine 41 in H-RAS), in the interswitch region, modulates the effector selectivity of RAS proteins from H-RAS to E-RAS features. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of Ras Exchange Factors and Cellular Localization of Ras Activation by Lipid Messengers in T Cells

PubMed Central

Jun, Jesse E.; Rubio, Ignacio; Roose, Jeroen P.

2013-01-01

The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs) catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and Son of Sevenless (SOS)-family GEFs. Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood. One large group of biomolecules critically involved in the control of RasGEFs functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR) stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells. PMID:24027568

Farnesyl transferase inhibitors: a major breakthrough in anticancer therapy? Naples, 12 April 2002.

PubMed

Caponigro, Francesco

2002-09-01

An international meeting focused on farnesyl transferase inhibitors (FTIs) was held in Naples on 12 April 2002 and represented an excellent occasion to gather most of the clinicians who are involved in clinical trials with this class of new compounds. Oncogene mutations of the gene occur in approximately 30% of all human cancers and may have prognostic significance. Ras protein is normally synthesized as pro-Ras, which undergoes a number of post-translational modifications, among which farnesylation. Processed Ras proteins localize to the inner surface of the plasma membrane, and function as a molecular switch that cycles between an inactive and an active form. When in its active form, either because of the binding of an external ligand or because of its constitutive activation, Ras activates several downstream effectors, such as Raf-1, Rac, Rho and phospahtidylinositol-3 kinase, which mediate important cellular functions, such as proliferation, cytoskeletal organization and others. Interruption of the Ras signaling pathway can be basically achieved in three ways, i.e. inhibition of Ras protein expression through antisense oligonucleotides, prevention of Ras membrane localization and inhibition of Ras downstream effectors. SCH 66336 (lonafarnib; Sarasar), a tricyclic orally active FTI, has been the first of these compounds to undergo clinical development. The toxicity profile observed in all completed phase I/II trials has been fairly similar, since gastrointestinal tract toxicity (nausea, vomiting and diarrhea) and fatigue have generally qualified as dose-limiting toxicity (DLT). One objective response in a patient with pretreated non-small cell lung cancer (NSCLC) was observed. Based on preclinical evidence of synergism between lonafarnib and other anticancer agents, combination studies have been started. In particular, lonafarnib has been combined both with gemcitabine and with paclitaxel in phase I studies. Nausea, vomiting, diarrhea and myelosuppression represented DLTs in these studies, in which an encouraging clinical activity was observed, in particular in pancreatic carcinoma (lonafarnib plus gemcitabine) and in NSCLC (lonafarnib plus paclitaxel). R115777 (Zarnestra) is another novel orally active FT competitive inhibitor in clinical development. Single-agent phase I/II studies have shown that myelotoxicity and neurotoxicity are DLTs, intermittent schedule is probably better tolerated and antitumor activity is observed particularly in breast cancer. A number of combination studies with R115777 have been carried out; taken as a whole, they show that the drug can be easily combined with several anticancer agents and phase III trials exploring the potential benefit from incorporation of R115777 into active chemotherapy regimens are indicated. Two other FTIs are in an earlier stage of clinical development. BMS-214662 has the main advantage of being cytotoxic in nature, rather than cytostatic; in particular, potent antitumor activity in human tumor xenografts of different histologies has been reported. A major drawback for BMS-214662 is its severe gastrointestinal and liver toxicities, which prevent the achievement of adequate systemic exposures following the oral route. L-778,123 has been stopped in its clinical development due to its severe and unexpected toxicity, i.e. grade 4 thrombocytopenia and significant Q-T prolongation.

H-Ras and K-Ras Oncoproteins Induce Different Tumor Spectra When Driven by the Same Regulatory Sequences.

PubMed

Drosten, Matthias; Simón-Carrasco, Lucía; Hernández-Porras, Isabel; Lechuga, Carmen G; Blasco, María T; Jacob, Harrys K C; Fabbiano, Salvatore; Potenza, Nicoletta; Bustelo, Xosé R; Guerra, Carmen; Barbacid, Mariano

2017-02-01

Genetic studies in mice have provided evidence that H-Ras and K-Ras proteins are bioequivalent. However, human tumors display marked differences in the association of RAS oncogenes with tumor type. Thus, to further assess the bioequivalence of oncogenic H-Ras and K-Ras, we replaced the coding region of the murine K-Ras locus with H-Ras G12V oncogene sequences. Germline expression of H-Ras G12V or K-Ras G12V from the K-Ras locus resulted in embryonic lethality. However, expression of these genes in adult mice led to different tumor phenotypes. Whereas H-Ras G12V elicited papillomas and hematopoietic tumors, K-Ras G12V induced lung tumors and gastric lesions. Pulmonary expression of H-Ras G12V created a senescence-like state caused by excessive MAPK signaling. Likewise, H-Ras G12V but not K-Ras G12V induced senescence in mouse embryonic fibroblasts. Label-free quantitative analysis revealed that minor differences in H-Ras G12V expression levels led to drastically different biological outputs, suggesting that subtle differences in MAPK signaling confer nonequivalent functions that influence tumor spectra induced by RAS oncoproteins. Cancer Res; 77(3); 707-18. ©2016 AACR. ©2016 American Association for Cancer Research.

The Tumor Suppressor DiRas3 Forms a Complex with H-Ras and C-RAF Proteins and Regulates Localization, Dimerization, and Kinase Activity of C-RAF*

PubMed Central

Baljuls, Angela; Beck, Matthias; Oenel, Ayla; Robubi, Armin; Kroschewski, Ruth; Hekman, Mirko; Rudel, Thomas; Rapp, Ulf R.

2012-01-01

The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity. PMID:22605333

A distinct class of dominant negative Ras mutants: cytosolic GTP-bound Ras effector domain mutants that inhibit Ras signaling and transformation and enhance cell adhesion.

PubMed

Fiordalisi, James J; Holly, Stephen P; Johnson, Ronald L; Parise, Leslie V; Cox, Adrienne D

2002-03-29

Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a beta(1)-subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Ras-induced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF- or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first extensive characterization of this functionally distinct class of DN Ras inhibitor proteins.

Fendiline Inhibits K-Ras Plasma Membrane Localization and Blocks K-Ras Signal Transmission

PubMed Central

van der Hoeven, Dharini; Cho, Kwang-jin; Ma, Xiaoping; Chigurupati, Sravanthi; Parton, Robert G.

2013-01-01

Ras proteins regulate signaling pathways important for cell growth, differentiation, and survival. Oncogenic mutant Ras proteins are commonly expressed in human tumors, with mutations of the K-Ras isoform being most prevalent. To be active, K-Ras must undergo posttranslational processing and associate with the plasma membrane. We therefore devised a high-content screening assay to search for inhibitors of K-Ras plasma membrane association. Using this assay, we identified fendiline, an L-type calcium channel blocker, as a specific inhibitor of K-Ras plasma membrane targeting with no detectable effect on the localization of H- and N-Ras. Other classes of L-type calcium channel blockers did not mislocalize K-Ras, suggesting a mechanism that is unrelated to calcium channel blockade. Fendiline did not inhibit K-Ras posttranslational processing but significantly reduced nanoclustering of K-Ras and redistributed K-Ras from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. Fendiline significantly inhibited signaling downstream of constitutively active K-Ras and endogenous K-Ras signaling in cells transformed by oncogenic H-Ras. Consistent with these effects, fendiline blocked the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-Ras. Taken together, these results suggest that inhibitors of K-Ras plasma membrane localization may have utility as novel K-Ras-specific anticancer therapeutics. PMID:23129805

Dominant negative Ras attenuates pathological ventricular remodeling in pressure overload cardiac hypertrophy

PubMed Central

Ramos-Kuri, Manuel; Rapti, Kleopatra; Mehel, Hind; Zhang, Shihong; Dhandapany, Perundurai S.; Liang, Lifan; García-Carrancá, Alejandro; Bobe, Regis; Fischmeister, Rodolphe; Adnot, Serge; Lebeche, Djamel; Hajjar, Roger J.; Lipskaia, Larissa; Chemaly, Elie R.

2015-01-01

The importance of the oncogene Ras in cardiac hypertrophy is well appreciated. The hypertrophic effects of the constitutively active mutant Ras-Val12 are revealed by clinical syndromes due to the Ras mutations and experimental studies. We examined the possible anti-hypertrophic effect of Ras inhibition in vitro using rat neonatal cardiomyocytes (NRCM) and in vivo in the setting of pressure-overload left ventricular (LV) hypertrophy (POH) in rats. Ras functions were modulated via adenovirus directed gene transfer of active mutant Ras-Val12 or dominant negative mutant N17-DN-Ras (DN-Ras). Ras-Val12 expression in vitro activates NFAT resulting in pro-hypertrophic and cardio-toxic effects on NRCM beating and Z-line organization. In contrast, the DN-Ras was antihypertrophic on NRCM, inhibited NFAT and exerted cardio-protective effects attested by preserved NRCM beating and Z line structure. Additional experiments with silencing H-Ras gene strategy corroborated the antihypertrophic effects of siRNA-H-Ras on NRCM. In vivo, with the POH model, both Ras mutants were associated with similar hypertrophy two weeks after simultaneous induction of POH and Ras-mutant gene transfer. However, LV diameters were higher and LV fractional shortening lower in the Ras-Val12 group compared to control and DN-Ras. Moreover, DN-Ras reduced the cross-sectional area of cardiomyocytes in vivo, and decreased the expression of markers of pathologic cardiac hypertrophy. In isolated adult cardiomyocytes after 2 weeks of POH and Ras-mutant gene transfer, DN-Ras improved sarcomere shortening and calcium transients compared to Ras-Val12. Overall, DN-Ras promotes a more physiological form of hypertrophy, suggesting an interesting therapeutic target for pathological cardiac hypertrophy. PMID:26260012

Involvement of H- and N-Ras isoforms in transforming growth factor-{beta}1-induced proliferation and in collagen and fibronectin synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez-Salgado, Carlos; Fuentes-Calvo, Isabel; Instituto 'Reina Sofia' de Investigacion Nefrologica, Universidad de Salamanca, 37007 Salamanca

2006-07-01

Transforming growth factor {beta}1 (TGF-{beta}1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-{beta} and Ras signaling pathways are closely related: TGF-{beta}1 overcomes Ras mitogenic effects and Ras counteracts TGF-{beta} signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-{beta}1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras {sup -/-}/N-ras {sup -/-}) isoforms andmore » from heterozygote mice (H-ras {sup +/-}/N-ras {sup +/-}). ECM synthesis is increased in basal conditions in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts, this increase being higher after stimulation with TGF-{beta}1. TGF-{beta}1-induced fibroblast proliferation is smaller in H-ras {sup -/-}/N-ras {sup -/-} than in H-ras {sup +/-}/N-ras {sup +/-} fibroblasts. Erk activation is decreased in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.« less

The PANDA study: a randomized phase II study of first-line FOLFOX plus panitumumab versus 5FU plus panitumumab in RAS and BRAF wild-type elderly metastatic colorectal cancer patients.

PubMed

Battaglin, Francesca; Schirripa, Marta; Buggin, Federica; Pietrantonio, Filippo; Morano, Federica; Boscolo, Giorgia; Tonini, Giuseppe; Lutrino, Eufemia Stefania; Lucchetti, Jessica; Salvatore, Lisa; Passardi, Alessandro; Cremolini, Chiara; Arnoldi, Ermenegildo; Scartozzi, Mario; Pella, Nicoletta; Boni, Luca; Bergamo, Francesca; Zagonel, Vittorina; Loupakis, Fotios; Lonardi, Sara

2018-01-25

Few data are available regarding the treatment of metastatic colorectal cancer elderly patients with anti-EGFR agents in combination with chemotherapy. FOLFOX plus panitumumab is a standard first-line option for RAS wild-type metastatic colorectal cancer. Slight adjustments in chemo-dosage are commonly applied in clinical practice to elderly patients, but those modified schedules have never been prospectively tested. Clinical definition of elderly (≥70 years old) patients that may deserve a more or less intensive combination therapy is still debated. Several geriatric screening tools have been developed to predict survival and risk of toxicity from treatment. Among those, the G8 screening tool has been tested in cancer patients showing the strongest prognostic value for overall survival, while the CRASH score can stratify patients according to an estimated risk of treatment-related toxicities. The PANDA study is a prospective, open-label, multicenter, randomized phase II trial of first-line therapy with panitumumab in combination with dose-adjusted FOLFOX or with 5-fluorouracil monotherapy, in previously untreated elderly patients (≥70 years) with RAS and BRAF wild-type unresectable metastatic colorectal cancer. RAS and BRAF analyses are centralized. Geriatric assessment by means of G8 and CRASH score is planned at baseline and G8 will be re-evaluated at disease progression. The primary endpoint is duration of progression-free survival in both arms. Secondary endpoints include prospective evaluation of the prognostic role of G8 score and the correlation of CRASH risk categories with toxicity. The PANDA study aims at exploring safety and efficacy of panitumumab in combination with FOLFOX or with 5FU/LV in elderly patients affected by RAS and BRAF wild-type metastatic colorectal cancer, to identify the most promising treatment strategy in this setting. Additionally, this is the first trial in which the prognostic role of the G8 score will be prospectively evaluated. Results of this study will drive further experimental developments for one or both combinations. PANDA is registered at Clinicaltrials.gov : NCT02904031 , July 11, 2016. PANDA is registered at EudraCT-No.: 2015-003888-10, September 3, 2015.

What makes Ras an efficient molecular switch: a computational, biophysical, and structural study of Ras-GDP interactions with mutants of Raf.

PubMed

Filchtinski, Daniel; Sharabi, Oz; Rüppel, Alma; Vetter, Ingrid R; Herrmann, Christian; Shifman, Julia M

2010-06-11

Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (Ras(GTP)) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras(GDP)) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with Ras(GDP). Most of our designed mutations narrow the gap between the affinity of Raf for Ras(GTP) and Ras(GDP), producing the desired shift in binding specificity towards Ras(GDP). A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras(GDP). The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras(GDP) bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras(GDP).Raf mutant complex is found in a conformation similar to that of Ras(GTP) and not Ras(GDP). Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras(GTP) is likely to explain the natural low affinity of Raf and other Ras effectors to Ras(GDP). Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch. Copyright 2010 Elsevier Ltd. All rights reserved.

RasGRP3 regulates the migration of glioma cells via interaction with Arp3

PubMed Central

Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Poisson, Laila M.; Blumberg, Peter M.; Brodie, Chaya

2015-01-01

Glioblastoma (GBM), the most aggressive primary brain tumors, are highly infiltrative. Although GBM express high Ras activity and Ras proteins have been implicated in gliomagenesis, Ras-activating mutations are not frequent in these tumors. RasGRP3, an important signaling protein responsive to diacylglycerol (DAG), increases Ras activation. Here, we examined the expression and functions of RasGRP3 in GBM and glioma cells. RasGRP3 expression was upregulated in GBM specimens and glioma stem cells compared with normal brains and neural stem cells, respectively. RasGRP3 activated Ras and Rap1 in glioma cells and increased cell migration and invasion partially via Ras activation. Using pull-down assay and mass spectroscopy we identified the actin-related protein, Arp3, as a novel interacting protein of RasGRP3. The interaction of RasGRP3 and Arp3 was validated by immunofluorescence staining and co-immunoprecipitation, and PMA, which activates RasGRP3 and induces its translocation to the peri-nuclear region, increased the association of Arp3 and RasGRP3. Arp3 was upregulated in GBM, regulated cell spreading and migration and its silencing partially decreased these effects of RasGRP3 in glioma cells. In summary, RasGRP3 acts as an important integrating signaling protein of the DAG and Ras signaling pathways and actin polymerization and represents an important therapeutic target in GBM. PMID:25682201

Capns1, a new binding partner of RasGAP-SH3 domain in K-Ras(V12) oncogenic cells: modulation of cell survival and migration.

PubMed

Pamonsinlapatham, Perayot; Gril, Brunilde; Dufour, Sylvie; Hadj-Slimane, Réda; Gigoux, Véronique; Pethe, Stéphanie; L'hoste, Sébastien; Camonis, Jacques; Garbay, Christiane; Raynaud, Françoise; Vidal, Michel

2008-11-01

Ras GTPase-activating protein (RasGAP) is hypothesized to be an effector of oncogenic Ras stimulating numerous downstream cellular signaling cascades involved in survival, proliferation and motility. In this study, we identified calpain small subunit-1 (Capns1) as a new RasGAP-SH3 domain binding partner, using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation assay and was found specific to cells expressing oncogenic K-Ras. We used confocal microscopy to analyze our stably transfected cell model producing mutant Ras (PC3Ras(V12)). Staining for RasGAP-SH3/Capns1 co-localization was two-fold stronger in the protrusions of Ras(V12) cells than in PC3 cells. RasGAP or Capns1 knockdown in PC3Ras(V12) cells induced a two- to three-fold increase in apoptosis. Capns1 gene silencing reduced the speed and increased the persistence of movement in PC3Ras(V12) cells. In contrast, RasGAP knockdown in PC3Ras(V12) cells increased cell migration. Knockdown of both proteins altered the speed and directionality of cell motility. Our findings suggest that RasGAP and Capns1 interaction in oncogenic Ras cells is involved in regulating migration and cell survival.

Mutation-Specific RAS Oncogenicity Explains N-RAS Codon 61 Selection in Melanoma

PubMed Central

Burd, Christin E.; Liu, Wenjin; Huynh, Minh V.; Waqas, Meriam A.; Gillahan, James E.; Clark, Kelly S.; Fu, Kailing; Martin, Brit L.; Jeck, William R.; Souroullas, George P.; Darr, David B.; Zedek, Daniel C.; Miley, Michael J.; Baguley, Bruce C.; Campbell, Sharon L.

2014-01-01

N-RAS mutation at codon 12, 13 or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an N-RasQ61R knock-in allele to similarly designed K-RasG12D and N-RasG12D alleles. With concomitant p16INK4a inactivation, K-RasG12D or N-RasQ61R expression efficiently promoted melanoma in vivo, whereas N-RasG12D did not. Additionally, N-RasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of N-RasQ61R and N-RasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, N-RasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity and increased stability when compared to N-RasG12D. This work identifies a faithful model of human N-RAS mutant melanoma, and suggests that the increased melanomagenecity of N-RasQ61R over N-RasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways. PMID:25252692

K-Ras Populates Conformational States Differently from Its Isoform H-Ras and Oncogenic Mutant K-RasG12D.

PubMed

Parker, Jillian A; Volmar, Alicia Y; Pavlopoulos, Spiro; Mattos, Carla

2018-06-05

Structures of wild-type K-Ras from crystals obtained in the presence of guanosine triphosphate (GTP) or its analogs have remained elusive. Of the K-Ras mutants, only K-RasG12D and K-RasQ61H are available in the PDB representing the activated form of the GTPase not in complex with other proteins. We present the crystal structure of wild-type K-Ras bound to the GTP analog GppCH 2 p, with K-Ras in the state 1 conformation. Signatures of conformational states obtained by one-dimensional proton NMR confirm that K-Ras has a more substantial population of state 1 in solution than H-Ras, which predominantly favors state 2. The oncogenic mutant K-RasG12D favors state 2, changing the balance of conformational states in favor of interactions with effector proteins. Differences in the population of conformational states between K-Ras and H-Ras, as well as between K-Ras and its mutants, can provide a structural basis for focused targeting of the K-Ras isoform in cancer-specific strategies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Overexpression of K-p21Ras play a prominent role in lung cancer

NASA Astrophysics Data System (ADS)

Zhang, Peng-bo; Zhou, Xin-liang; Yang, Ju-lun

2018-06-01

The proto-oncogene ras product, p21Ras, has been found overexpression in many human tumors. However, the subtypes of overexpressed p21Ras still remain unclear. The purpose of this study was to investigate overexpressed isoforms of p21Ras and their roles in the progress of lung cancer. Method: The expression of total p21Ras in normal lung tissues and lung cancers was determined by immunohistochemically staining with monoclonal antibody (Mab) KGHR-1 which could recognize and broad spectrum reaction with the (K/H/N) ras protein. Then, the isoforms of p21Ras was examined by specific Mab for each p21Ras subtypes. Results: Low expression of total p21Ras was found in 26.67% (8/30) of normal lung tissues, and 81.31% (87/107) of adenocarcinoma harbored overexpressed total p21Ras. Besides, 70.00% (35/50) of squamous cell carcinoma were detected overexpressed total p21Ras. In addition, 122 lung cancer tissues from overexpression of total p21Ras protein were selected to detect the expression of each subtype. And all the 122 lung cancer tissues were K-p21Ras overexpression. Moreover, there was a statistical significance difference between the expression level of total p21Ras and differentiation, and the same results were observed between the expression level of total p21Ras and lymph node metastasis (P<0.05). However, there was no correlation between the expression level of total p21Ras and gender, age, tumor size (P>0.05). Conclusions: Overexpression of K-p21Ras plays a prominent role in the progress of lung cancer and it is suggested that the p21Ras could serve as a promising treatment target in lung cancer.

Resistance of R-Ras knockout mice to skin tumour induction

PubMed Central

May, Ulrike; Prince, Stuart; Vähätupa, Maria; Laitinen, Anni M.; Nieminen, Katriina; Uusitalo-Järvinen, Hannele; Järvinen, Tero A. H.

2015-01-01

The R-ras gene encodes a small GTPase that is a member of the Ras family. Despite close sequence similarities, R-Ras is functionally distinct from the prototypic Ras proteins; no transformative activity and no activating mutations of R-Ras in human malignancies have been reported for it. R-Ras activity appears inhibitory towards tumour proliferation and invasion, and to promote cellular quiescence. Contrary to this, using mice with a deletion of the R-ras gene, we found that R-Ras facilitates DMBA/TPA-induced skin tumour induction. The tumours appeared in wild-type (WT) mice on average 6 weeks earlier than in R-Ras knockout (R-Ras KO) mice. WT mice developed almost 6 times more tumours than R-Ras KO mice. Despite strong R-Ras protein expression in the dermal blood vessels, no R-Ras could be detected in the epidermis from where the tumours arose. The DMBA/TPA skin tumourigenesis-model is highly dependent upon inflammation, and we found a greatly attenuated skin inflammatory response to DMBA/TPA-treatment in the R-Ras KO mice in the context of leukocyte infiltration and proinflammatory cytokine expression. Thus, these data suggest that despite its characterised role in promoting cellular quiescence, R-Ras is pro-tumourigenic in the DMBA/TPA tumour model and important for the inflammatory response to DMBA/TPA treatment. PMID:26133397

Focal adhesions and Ras are functionally and spatially integrated to mediate IL-1 activation of ERK

PubMed Central

Wang, Qin; Downey, Gregory P.; McCulloch, Christopher A.

2011-01-01

In connective tissue cells, IL-1-induced ERK activation leading to matrix metalloproteinase (MMP)-3 expression is dependent on cooperative interactions between focal adhesions and the endoplasmic reticulum (ER). As Ras can be activated on the ER, we investigated the role of Ras in IL-1 signaling and focal adhesion formation. We found that constitutively active H-Ras, K-Ras or N-Ras enhanced focal adhesion maturation and β1-integrin activation. IL-1 promoted the accumulation of Ras isoforms in ER and focal adhesion fractions, as shown in cells cotransfected with GFP-tagged Ras isoforms and YFP-ER protein and by analysis of subcellular fractions enriched for ER or focal adhesion proteins. Dominant-negative H-Ras or K-Ras reduced accumulation of H-Ras and K-Ras in focal adhesions induced by IL-1 and also blocked ERK activation and focal adhesion maturation. Ras-GRF was enriched constitutively in focal adhesion fractions and was required for Ras recruitment to focal adhesions. We conclude that Ras activation and IL-1 signaling are interactive processes that regulate the maturation of focal adhesions, which, in turn, is required for ERK activation.—Wang, Q., Downey, G. P., McCulloch, C. A. Focal adhesions and Ras are functionally and spatially integrated to mediate IL-1 activation of ERK. PMID:21719512

Targeting the RAS oncogene

PubMed Central

Takashima, Asami

2013-01-01

Introduction The Ras proteins (K-Ras, N-Ras, H-Ras) are GTPases that function as molecular switches for a variety of critical cellular activities and their function is tightly and temporally regulated in normal cells. Oncogenic mutations in the RAS genes, which create constitutively-active Ras proteins, can result in uncontrolled proliferation or survival in tumor cells. Areas covered The paper discusses three therapeutic approaches targeting the Ras pathway in cancer: 1) Ras itself, 2) Ras downstream pathways, and 3) synthetic lethality. The most adopted approach is targeting Ras downstream signaling, and specifically the PI3K-AKT-mTOR and Raf-MEK pathways, as they are frequently major oncogenic drivers in cancers with high Ras signaling. Although direct targeting of Ras has not been successful clinically, newer approaches being investigated in preclinical studies, such as RNA interference-based and synthetic lethal approaches, promise great potential for clinical application. Expert opinion The challenges of current and emerging therapeutics include the lack of “tumor specificity” and their limitation to those cancers which are “dependent” upon aberrant Ras signaling for survival. While the newer approaches have the potential to overcome these limitations, they also highlight the importance of robust preclinical studies and bidirectional translational research for successful clinical development of Ras-related targeted therapies. PMID:23360111

A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors.

PubMed

Loboda, Andrey; Nebozhyn, Michael; Klinghoffer, Rich; Frazier, Jason; Chastain, Michael; Arthur, William; Roberts, Brian; Zhang, Theresa; Chenard, Melissa; Haines, Brian; Andersen, Jannik; Nagashima, Kumiko; Paweletz, Cloud; Lynch, Bethany; Feldman, Igor; Dai, Hongyue; Huang, Pearl; Watters, James

2010-06-30

Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors.

A Histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1.

PubMed

Vercoulen, Yvonne; Kondo, Yasushi; Iwig, Jeffrey S; Janssen, Axel B; White, Katharine A; Amini, Mojtaba; Barber, Diane L; Kuriyan, John; Roose, Jeroen P

2017-09-27

RasGRPs are guanine nucleotide exchange factors that are specific for Ras or Rap, and are important regulators of cellular signaling. Aberrant expression or mutation of RasGRPs results in disease. An analysis of RasGRP1 SNP variants led to the conclusion that the charge of His 212 in RasGRP1 alters signaling activity and plasma membrane recruitment, indicating that His 212 is a pH sensor that alters the balance between the inactive and active forms of RasGRP1. To understand the structural basis for this effect we compared the structure of autoinhibited RasGRP1, determined previously, to those of active RasGRP4:H-Ras and RasGRP2:Rap1b complexes. The transition from the autoinhibited to the active form of RasGRP1 involves the rearrangement of an inter-domain linker that displaces inhibitory inter-domain interactions. His 212 is located at the fulcrum of these conformational changes, and structural features in its vicinity are consistent with its function as a pH-dependent switch.

Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1

PubMed Central

Iwig, Jeffrey S; Vercoulen, Yvonne; Das, Rahul; Barros, Tiago; Limnander, Andre; Che, Yan; Pelton, Jeffrey G; Wemmer, David E; Roose, Jeroen P; Kuriyan, John

2013-01-01

RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals. DOI: http://dx.doi.org/10.7554/eLife.00813.001 PMID:23908768

Antigastric parietal cell and antithyroid autoantibodies in patients with recurrent aphthous stomatitis.

PubMed

Wu, Yang-Che; Wu, Yu-Hsueh; Wang, Yi-Ping; Chang, Julia Yu-Fong; Chen, Hsin-Ming; Sun, Andy

2017-01-01

Anti-gastric parietal cell antibody (GPCA), anti-thyroglobulin antibody (TGA), and anti-thyroid microsomal antibody (TMA) have not yet been reported in patients with recurrent aphthous stomatitis (RAS). This study mainly assessed the frequencies of the presence of serum GPCA, TGA, and TMA in different types of RAS patients. Serum GPCA, TGA, and TMA levels were measured in 355 RAS patients of different subtypes and in 355 age- and sex-matched healthy control individuals. We found that 13.0%, 19.4%, and 19.7% of 355 RAS patients, 16.7%, 23.3%, and 21.7% of 60 major-typed RAS patients, 12.2%, 18.6%, and 19.3% of 295 minor-typed RAS patients, 18.1%, 20.0%, and 21.9% of 160 atrophic glossitis-positive RAS (AG+/RAS) patients, and 8.7%, 19.0%, and 17.9% of 195 AG-negative RAS (AG-/RAS) patients had the presence of GPCA, TGA, and TMA in their sera, respectively. RAS, major-typed RAS, minor-typed RAS, AG+/RAS, and AG-/RAS patients all had a significantly higher frequency of GPCA, TGA, or TMA positivity than healthy control individuals (all p < 0.001). Of 65 TGA/TMA-positive RAS patients whose serum thyroid-stimulating hormone (TSH) levels were measured, 76.9%, 12.3%, and 10.8% of these TGA/TMA-positive RAS patients had normal, lower, and higher serum TSH levels, respectively. We conclude that approximately one-third RAS patients may have GPCA/TGA/TMA positivity in their sera. Because some GPCA-positive patients may develop pernicious anemia, autoimmune atrophic gastritis, and gastric carcinoma, and some TGA/TMA-positive patients may have thyroid dysfunction such as hyperthyroidism and hypothyroidism, these patients should be referred to doctors for further management. Copyright © 2016. Published by Elsevier B.V.

Ras and relatives--job sharing and networking keep an old family together.

PubMed

Ehrhardt, Annette; Ehrhardt, Götz R A; Guo, Xuecui; Schrader, John W

2002-10-01

Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.

Importance of the REM (Ras exchange) domain for membrane interactions by RasGRP3.

PubMed

Czikora, Agnes; Kedei, Noemi; Kalish, Heather; Blumberg, Peter M

2017-12-01

RasGRP comprises a family of guanine nucleotide exchange factors, regulating the dissociation of GDP from Ras GTPases to enhance the formation of the active GTP-bound form. RasGRP1 possesses REM (Ras exchange), GEF (catalytic), EF-hand, C1, SuPT (suppressor of PT), and PT (plasma membrane-targeting) domains, among which the C1 domain drives membrane localization in response to diacylglycerol or phorbol ester and the PT domain recognizes phosphoinositides. The homologous family member RasGRP3 shows less plasma membrane localization. The objective of this study was to explore the role of the different domains of RasGRP3 in membrane translocation in response to phorbol esters. The full-length RasGRP3 shows limited translocation to the plasma membrane in response to PMA, even when the basic hydrophobic cluster in the PT domain, reported to be critical for RasGRP1 translocation to endogenous activators, is mutated to resemble that of RasGRP1. Moreover, exchange of the C-termini (SuPT-PT domain) of the two proteins had little effect on their plasma membrane translocation. On the other hand, while the C1 domain of RasGRP3 alone showed partial plasma membrane translocation, truncated RasGRP3 constructs, which contain the PT domain and are missing the REM, showed stronger translocation, indicating that the REM of RasGRP3 was a suppressor of its membrane interaction. The REM of RasGRP1 failed to show comparable suppression of RasGRP3 translocation. The marked differences between RasGRP3 and RasGRP1 in membrane interaction necessarily will contribute to their different behavior in cells and are relevant to the design of selective ligands as potential therapeutic agents. Published by Elsevier B.V.

A cross-sectional study examining the expression of splice variants K-RAS4A and K-RAS4B in advanced non-small-cell lung cancer patients.

PubMed

Aran, Veronica; Masson Domingues, Pedro; Carvalho de Macedo, Fabiane; Moreira de Sousa, Carlos Augusto; Caldas Montella, Tatiane; de Souza Accioly, Maria Theresa; Ferreira, Carlos Gil

2018-02-01

Mammalian cells differently express 4 RAS isoforms: H-RAS, N-RAS, K-RAS4A and K-RAS4B, which are important in promoting oncogenic processes when mutated. In lung cancer, the K-RAS isoform is the most frequently altered RAS protein, being also a difficult therapeutic target. Interestingly, there are two K-RAS splice variants (K-RAS4A and K-RAS4B) and little is known about the role of K-RAS4A. Most studies targeting K-RAS, or analysing it as a prognostic factor, have not taken into account the two isoforms. Consequently, the in-depth investigation of them is needed. The present study analysed 98 specimens from advanced non-small cell lung cancer (NSCLC) adenocarcinoma patients originated from Brazil. The alterations present in K-RAS at the DNA level (Sanger sequencing) as well as the expression of the splicing isoforms at the RNA (qRT-PCR) and protein levels (immunohistochemistry analysis), were evaluated. Possible associations between clinicopathological features and the molecular findings were also investigated. Our results showed that in the non-smoking population, the cancer incidence was higher among women. In contrast, in smokers and former smokers, the incidence was higher among men. Regarding sequencing results, 10.5% of valid samples presented mutations in exon 2, being all wild-type for exon 3, and the most frequently occurring base change was the transversion G → T. Our qRT-PCR and immunohistochemical analysis showed that both, K-RAS4A and K-RAS4B, were differently expressed in NSCLC tumour samples. For example, tumour specimens showed higher K-RAS4A mRNA expression in relation to commercial normal lung control than did K-RAS4B. In addition, K-RAS4B protein expression was frequently stronger than K-RAS4A in the patients analysed. Our results highlight the differential expression of K-RAS4A and K-RAS4B in advanced adenocarcinoma NSCLC patients and underline the need to further clarify the enigma behind their biological significance in various cancer types, including NSCLC. Copyright © 2017 Elsevier B.V. All rights reserved.

Downregulation of Ras C-terminal processing by JNK inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mouri, Wataru; Department of Neurosurgery, Yamagata University School of Medicine, Yamagata 990-9585; Biology Division, National Cancer Center Research Institute, Tokyo 104-0045

2008-06-27

After translation, Ras proteins undergo a series of modifications at their C-termini. This post-translational C-terminal processing is essential for Ras to become functional, but it remains unknown whether and how Ras C-terminal processing is regulated. Here we show that the C-terminal processing and subsequent plasma membrane localization of H-Ras as well as the activation of the downstream signaling pathways by H-Ras are prevented by JNK inhibition. Conversely, JNK activation by ultraviolet irradiation resulted in promotion of C-terminal processing of H-Ras. Furthermore, increased cell density promoted C-terminal processing of H-Ras most likely through an autocrine/paracrine mechanism, which was also blocked undermore » JNK-inhibited condition. Ras C-terminal processing was sensitive to JNK inhibition in the case of H- and N-Ras but not K-Ras, and in a variety of cell types. Thus, our results suggest for the first time that Ras C-terminal processing is a regulated mechanism in which JNK is involved.« less

Review: Intracardiac intracellular angiotensin system in diabetes

PubMed Central

Kumar, Rajesh; Yong, Qian Chen; Thomas, Candice M.

2012-01-01

The renin-angiotensin system (RAS) has mainly been categorized as a circulating and a local tissue RAS. A new component of the local system, known as the intracellular RAS, has recently been described. The intracellular RAS is defined as synthesis and action of ANG II intracellularly. This RAS appears to differ from the circulating and the local RAS, in terms of components and the mechanism of action. These differences may alter treatment strategies that target the RAS in several pathological conditions. Recent work from our laboratory has demonstrated significant upregulation of the cardiac, intracellular RAS in diabetes, which is associated with cardiac dysfunction. Here, we have reviewed evidence supporting an intracellular RAS in different cell types, ANG II's actions in cardiac cells, and its mechanism of action, focusing on the intracellular cardiac RAS in diabetes. We have discussed the significance of an intracellular RAS in cardiac pathophysiology and implications for potential therapies. PMID:22170614

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation

PubMed Central

Lim, Chinten James; Spiegelman, George B.; Weeks, Gerald

2001-01-01

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC– cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC– cells stimulated by 2′-deoxy-cAMP, but is produced in response to GTPγS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC– cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC– cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC– cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC– cells, suggesting that AleA may activate RasC. PMID:11500376

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

PubMed

Lim, C J; Spiegelman, G B; Weeks, G

2001-08-15

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

Deconstructing Ras Signaling in the Thymus

PubMed Central

Kortum, Robert L.; Sommers, Connie L.; Pinski, John M.; Alexander, Clayton P.; Merrill, Robert K.; Li, Wenmei; Love, Paul E.

2012-01-01

Thymocytes must transit at least two distinct developmental checkpoints, governed by signals that emanate from either the pre-T cell receptor (pre-TCR) or the TCR to the small G protein Ras before emerging as functional T lymphocytes. Recent studies have shown a role for the Ras guanine exchange factor (RasGEF) Sos1 at the pre-TCR checkpoint. At the second checkpoint, the quality of signaling through the TCR is interrogated to ensure the production of an appropriate T cell repertoire. Although RasGRP1 is the only confirmed RasGEF required at the TCR checkpoint, current models suggest that the intensity and character of Ras activation, facilitated by both Sos and RasGRP1, will govern the boundary between survival (positive selection) and death (negative selection) at this stage. Using mouse models, we have assessed the independent and combined roles for the RasGEFs Sos1, Sos2, and RasGRP1 during thymocyte development. Although Sos1 was the dominant RasGEF at the pre-TCR checkpoint, combined Sos1/RasGRP1 deletion was required to effectively block development at this stage. Conversely, while RasGRP1 deletion efficiently blocked positive selection, combined RasGRP1/Sos1 deletion was required to block negative selection. This functional redundancy in RasGEFs during negative selection may act as a failsafe mechanism ensuring appropriate central tolerance. PMID:22586275

H-Ras Exerts Opposing Effects on Type I Interferon Responses Depending on Its Activation Status.

PubMed

Chen, Guann-An; Lin, Yun-Ru; Chung, Hai-Ting; Hwang, Lih-Hwa

2017-01-01

Using shRNA high-throughput screening, we identified H-Ras as a regulator of antiviral activity, whose depletion could enhance Sindbis virus replication. Further analyses indicated that depletion of H-Ras results in a robust increase in vesicular stomatitis virus infection and a decrease in Sendai virus (SeV)-induced retinoic acid-inducible gene-I-like receptor (RLR) signaling. Interestingly, however, ectopic expression of wild-type H-Ras results in a biphasic mode of RLR signaling regulation: while low-level expression of H-Ras enhances SeV-induced RLR signaling, high-level expression of H-Ras significantly inhibits this signaling. The inhibitory effects correlate with the activation status of H-Ras. As a result, oncogenic H-Ras, H-RasV12, strongly inhibits SeV-induced IFN-β promoter activity and type I interferon signaling. Conversely, the positive effects exerted by H-Ras on RLR signaling are independent of its signaling activity, as a constitutively inactive form of H-Ras, H-RasN17, also positively regulates RLR signaling. Mechanistically, we demonstrate that depletion of H-Ras reduces the formation of MAVS-TNF receptor-associated factor 3 signaling complexes. These results reveal that the H-Ras protein plays a role in promoting MAVS signalosome assembly in the mitochondria, whereas oncogenic H-Ras exerts a negative effect on type I IFN responses.

Plasma ctDNA RAS mutation analysis for the diagnosis and treatment monitoring of metastatic colorectal cancer patients

PubMed Central

Vidal, J; Muinelo, L; Dalmases, A; Jones, F; Edelstein, D; Iglesias, M; Orrillo, M; Abalo, A; Rodríguez, C; Brozos, E; Vidal, Y; Candamio, S; Vázquez, F; Ruiz, J; Guix, M; Visa, L; Sikri, V; Albanell, J; Bellosillo, B; López, R; Montagut, C

2017-01-01

Abstract Background RAS assessment is mandatory for therapy decision in metastatic colorectal cancer (mCRC) patients. This determination is based on tumor tissue, however, genotyping of circulating tumor (ct)DNA offers clear advantages as a minimally invasive method that represents tumor heterogeneity. Our study aims to evaluate the use of ctDNA as an alternative for determining baseline RAS status and subsequent monitoring of RAS mutations during therapy as a component of routine clinical practice. Patients and methods RAS mutational status in plasma was evaluated in mCRC patients by OncoBEAM™ RAS CRC assay. Concordance of results in plasma and tissue was retrospectively evaluated. RAS mutations were also prospectively monitored in longitudinal plasma samples from selected patients. Results Analysis of RAS in tissue and plasma samples from 115 mCRC patients showed a 93% overall agreement. Plasma/tissue RAS discrepancies were mainly explained by spatial and temporal tumor heterogeneity. Analysis of clinico-pathological features showed that the site of metastasis (i.e. peritoneal, lung), the histology of the tumor (i.e. mucinous) and administration of treatment previous to blood collection negatively impacted the detection of RAS in ctDNA. In patients with baseline mutant RAS tumors treated with chemotherapy/antiangiogenic, longitudinal analysis of RAS ctDNA mirrored response to treatment, being an early predictor of response. In patients RAS wt, longitudinal monitoring of RAS ctDNA revealed that OncoBEAM was useful to detect emergence of RAS mutations during anti-EGFR treatment. Conclusion The high overall agreement in RAS mutational assessment between plasma and tissue supports blood-based testing with OncoBEAM™ as a viable alternative for genotyping RAS of mCRC patients in routine clinical practice. Our study describes practical clinico-pathological specifications to optimize RAS ctDNA determination. Moreover, OncoBEAM™ is useful to monitor RAS in patients undergoing systemic therapy to detect resistance and evaluate the efficacy of particular treatments. PMID:28419195

Aurora kinase A interacts with H-Ras and potentiates Ras-MAPK signaling | Office of Cancer Genomics

Cancer.gov

In cancer, upregulated Ras promotes cellular transformation and proliferation in part through activation of oncogenic Ras-MAPK signaling. While directly inhibiting Ras has proven challenging, new insights into Ras regulation through protein-protein interactions may offer unique opportunities for therapeutic intervention. Here we report the identification and validation of Aurora kinase A (Aurora A) as a novel Ras binding protein. We demonstrate that the kinase domain of Aurora A mediates the interaction with the N-terminal domain of H-Ras.

A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors

PubMed Central

2010-01-01

Background Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. Methods We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. Results The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. Conclusions These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors. PMID:20591134

Targeting RAS Membrane Association: Back to the Future for Anti-RAS Drug Discovery?

PubMed Central

Cox, Adrienne D.; Der, Channing J.; Philips, Mark R.

2015-01-01

RAS proteins require membrane association for their biological activity, making this association a logical target for anti-RAS therapeutics. Lipid modification of RAS proteins by a farnesyl isoprenoid is an obligate step in that association, and is an enzymatic process. Accordingly, farnesyltransferase inhibitors (FTIs) were developed as potential anti-RAS drugs. The lack of efficacy of FTIs as anti-cancer drugs was widely seen as indicating that blocking RAS membrane association was a flawed approach to cancer treatment. However, a deeper understanding of RAS modification and trafficking has revealed that this was an erroneous conclusion. In the presence of FTIs, KRAS and NRAS, which are the RAS isoforms most frequently mutated in cancer, become substrates for alternative modification, can still associate with membranes, and can still function. Thus, FTIs failed not because blocking RAS membrane association is an ineffective approach, but because FTIs failed to accomplish that task. Recent findings regarding RAS isoform trafficking and the regulation of RAS subcellular localization have rekindled interest in efforts to target these processes. In particular, improved understanding of the palmitoylation/depalmitoylation cycle that regulates RAS interaction with the plasma membrane, endomembranes and cytosol, and of the potential importance of RAS chaperones, have led to new approaches. Efforts to validate and target other enzymatically regulated post-translational modifications are also ongoing. In this review, we revisit lessons learned, describe the current state of the art, and highlight challenging but promising directions to achieve the goal of disrupting RAS membrane association and subcellular localization for anti-RAS drug development. PMID:25878363

AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2

PubMed Central

Cho, Kwang-jin; Casteel, Darren E.; Prakash, Priyanka; Tan, Lingxiao; van der Hoeven, Dharini; Salim, Angela A.; Kim, Choel; Capon, Robert J.; Lacey, Ernest; Cunha, Shane R.; Gorfe, Alemayehu A.

2016-01-01

K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function. PMID:27697864

In TCR-Stimulated T-cells, N-ras Regulates Specific Genes and Signal Transduction Pathways

PubMed Central

Lynch, Stephen J.; Zavadil, Jiri; Pellicer, Angel

2013-01-01

It has been recently shown that N-ras plays a preferential role in immune cell development and function; specifically: N-ras, but not H-ras or K-ras, could be activated at and signal from the Golgi membrane of immune cells following a low level T-cell receptor stimulus. The goal of our studies was to test the hypothesis that N-ras and H-ras played distinct roles in immune cells at the level of the transcriptome. First, we showed via mRNA expression profiling that there were over four hundred genes that were uniquely differentially regulated either by N-ras or H-ras, which provided strong evidence in favor of the hypothesis that N-ras and H-ras have distinct functions in immune cells. We next characterized the genes that were differentially regulated by N-ras in T cells following a low-level T-cell receptor stimulus. Of the large pool of candidate genes that were differentially regulated by N-ras downstream of TCR ligation, four genes were verified in qRT-PCR-based validation experiments (Dntt, Slc9a6, Chst1, and Lars2). Finally, although there was little overlap between individual genes that were regulated by N-ras in unstimulated thymocytes and stimulated CD4+ T-cells, there was a nearly complete correspondence between the signaling pathways that were regulated by N-ras in these two immune cell types. PMID:23755101

Ras GTPases Modulate Morphogenesis, Sporulation and Cellulase Gene Expression in the Cellulolytic Fungus Trichoderma reesei

PubMed Central

Zhang, Jiwei; Zhang, Yanmei; Zhong, Yaohua; Qu, Yinbo; Wang, Tianhong

2012-01-01

Background The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown. Methodology/Principal Findings Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. Conclusions/Significance Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the powerful competitive ability of plant cell wall degrading fungi in nature. PMID:23152805

Non-prescription proton-pump inhibitors for self-treating frequent heartburn:the role of the Canadian pharmacist

PubMed Central

Armstrong, David; Nakhla, Nardine

2016-01-01

Heartburn and acid regurgitation are the cardinal symptoms of gastroesophageal reflux and occur commonly in the Canadian population. Multiple non-prescription treatment options are available for managing these symptoms, including antacids, alginates, histamine-H2 receptor antagonists (H2RAs), and proton-pump inhibitors (PPIs). As a result, pharmacists are ideally positioned to recommend appropriate treatment options based upon an individual’s needs and presenting symptoms, prior treatment response, comorbid medical conditions, and other relevant factors. Individuals who experience mild heartburn and/or have symptoms that occur predictably in response to known precipitating factors can manage their symptoms by avoiding known triggers and using on-demand antacids and/or alginates or lower-dose non-prescription H2RAs (e.g. ranitidine 150 mg). For those with moderate symptoms, lifestyle changes, in conjunction with higher-dose non-prescription H2RAs, may be effective. However, for individuals with moderate-to-severe symptoms that occur frequently (i.e. ≥2 days/week), the non-prescription (Schedule II) PPI omeprazole 20 mg should be considered. The pharmacist can provide important support by inquiring about the frequency and severity of symptoms, identifying an appropriate treatment option, and recognizing other potential causes of symptoms, as well as alarm features and atypical symptoms that would necessitate referral to a physician. After recommending an appropriate treatment, the pharmacist can provide instructions for its correct use. Additionally, the pharmacist should inquire about recurrences, respond to questions about adverse events, provide monitoring parameters, and counsel on when referral to a physician is warranted. Pharmacists are an essential resource for individuals experiencing heartburn; they play a crucial role in helping individuals make informed self-care decisions and educating them to ensure that therapy is used in an optimal, safe, and effective manner. PMID:28042359

Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering

PubMed Central

Blaževitš, Olga; Mideksa, Yonatan G.; Šolman, Maja; Ligabue, Alessio; Ariotti, Nicholas; Nakhaeizadeh, Hossein; Fansa, Eyad K.; Papageorgiou, Anastassios C.; Wittinghofer, Alfred; Ahmadian, Mohammad R.; Abankwa, Daniel

2016-01-01

Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The latter activity may present an alternative mechanism for how overexpressed Gal-1 stimulates tumourigenesis. Here we revise the current model for the interaction of Gal-1 with H-ras. We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors. A computationally generated model of the Gal-1/C-Raf-RBD complex is validated by mutational analysis. Both cellular FRET as well as proximity ligation assay experiments confirm interaction of Gal-1 with Raf proteins in mammalian cells. Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering. In addition, an intact dimer interface of Gal-1 is required for it to positively regulate H-rasG12V nanoclustering, but negatively K-rasG12V nanoclustering. Our findings suggest stacked dimers of H-ras, Raf and Gal-1 as building blocks of GTP-H-ras-nanocluster at high Gal-1 levels. Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling. PMID:27087647

Metabolic Rate Regulation by the Renin-Angiotensin System: Brain vs. Body

PubMed Central

Grobe, Justin L.; Rahmouni, Kamal; Liu, Xuebo; Sigmund, Curt D.

2013-01-01

Substantial evidence supports a role for the renin-angiotensin system (RAS) in the regulation of metabolic function, but an apparent paradox exists where genetic or pharmacological inhibition of the RAS occasionally have similar physiological effects as chronic angiotensin infusion. Similarly, while RAS targeting in animal models has robust metabolic consequences, effects in humans are more subtle. Here we review the data supporting a role for the RAS in metabolic rate regulation and propose a model where the local brain RAS works in opposition to the peripheral RAS, thus helping to explain the paradoxically similar effects of RAS supplementation and inhibition. Selectively modulating the peripheral RAS or brain RAS may thus provide a more effective treatment paradigm for obesity and obesity-related disorders. PMID:22491893

The Affordable Medicines Facility-malaria (AMFm): are remote areas benefiting from the intervention?

PubMed

Ye, Yazoume; Arnold, Fred; Noor, Abdisalan; Wamukoya, Marilyn; Amuasi, John; Blay, Samuel; Mberu, Blessing; Ren, Ruilin; Kyobutungi, Catherine; Wekesah, Frederick; Gatakaa, Hellen; Toda, Mitsuru; Njogu, Julius; Evance, Illah; O'Connell, Kathryn; Shewchuk, Tanya; Thougher, Sarah; Mann, Andrea; Willey, Barbara; Goodman, Catherine; Hanson, Kara

2015-10-09

To assess the availability, price and market share of quality-assured artemisinin-based combination therapy (QAACT) in remote areas (RAs) compared with non-remote areas (nRAs) in Kenya and Ghana at end-line of the Affordable Medicines Facility-malaria (AMFm) intervention. Areas were classified by remoteness using a composite index computed from estimated travel times to three levels of service centres. The index was used to five categories of remoteness, which were then grouped into two categories of remote and non-remote areas. The number of public or private outlets with the potential to sell or distribute anti-malarial medicines, screened in nRAs and RAs, respectively, was 501 and 194 in Ghana and 9980 and 2353 in Kenya. The analysis compares RAs with nRAs in terms of availability, price and market share of QAACT in each country. QAACT were similarly available in RAs as nRAs in Ghana and Kenya. In both countries, there was no statistical difference in availability of QAACT with AMFm logo between RAs and nRAs in public health facilities (PHFs), while private-for-profit (PFP) outlets had lower availability in RA than in nRAs (Ghana: 66.0 vs 82.2 %, p < 0.0001; Kenya: 44.9 vs 63.5 %, p = <0.0001. The median price of QAACT with AMFm logo for PFP outlets in RAs (USD1.25 in Ghana and USD0.69 in Kenya) was above the recommended retail price in Ghana (US$0.95) and Kenya (US$0.46), and much higher than in nRAs for both countries. QAACT with AMFm logo represented the majority of QAACT in RAs and nRAs in Kenya and Ghana. In the PFP sector in Ghana, the market share for QAACT with AMFm logo was significantly higher in RAs than in nRAs (75.6 vs 51.4 %, p < 0.0001). In contrast, in similar outlets in Kenya, the market share of QAACT with AMFm logo was significantly lower in RAs than in nRAs (39.4 vs 65.1 %, p < 0.0001). The findings indicate the AMFm programme contributed to making QAACT more available in RAs in these two countries. Therefore, the AMFm approach can inform other health interventions aiming at reaching hard-to-reach populations, particularly in the context of universal access to health interventions. However, further examination of the factors accounting for the deep penetration of the AMFm programme into RAs is needed to inform actions to improve the healthcare delivery system, particularly in RAs.

Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

PubMed

Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

2015-12-15

Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.

Platelet-derived growth factor-dependent association of the GTPase-activating protein of Ras and Src.

PubMed Central

Schlesinger, T K; Demali, K A; Johnson, G L; Kazlauskas, A

1999-01-01

Here we report that the platelet-derived growth factor beta receptor (betaPDGFR) is not the only tyrosine kinase able to associate with the GTPase-activating protein of Ras (RasGAP). The interaction of non-betaPDGFR kinase(s) with RasGAP was dependent on stimulation with platelet-derived growth factor (PDGF) and seemed to require tyrosine phosphorylation of RasGAP. Because the tyrosine phosphorylation site of RasGAP is in a sequence context that is favoured by the Src homology 2 ('SH2') domain of Src family members, we tested the possibility that Src was the kinase that associated with RasGAP. Indeed, Src interacted with phosphorylated RasGAP fusion proteins; immunodepletion of Src markedly decreased the recovery of the RasGAP-associated kinase activity. Thus PDGF-dependent tyrosine phosphorylation of RasGAP results in the formation of a complex between RasGAP and Src. To begin to address the relevance of these observations, we focused on the consequences of the interaction of Src and RasGAP. We found that a receptor mutant that did not activate Src was unable to efficiently mediate the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Taken together, these observations support the following hypothesis. When RasGAP is recruited to the betaPDGFR, it is phosphorylated and associates with Src. Once bound to RasGAP, Src is no longer able to promote the phosphorylation of PLCgamma. This hypothesis offers a mechanistic explanation for our previously published findings that the recruitment of RasGAP to the betaPDGFR attenuates the tyrosine phosphorylation of PLCgamma. Finally, these findings suggest a novel way in which RasGAP negatively regulates signal relay by the betaPDGFR. PMID:10567236

Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.

PubMed

Lechuga, Carmen G; Simón-Carrasco, Lucía; Jacob, Harrys K C; Drosten, Matthias

2017-01-01

Signaling transmitted by the Ras family of small GTPases (H-, N-, and K-Ras) is essential for proliferation of mouse embryonic fibroblasts (MEFs). However, constitutive activation of the downstream Raf/Mek/Erk pathway can bypass the requirement for Ras proteins and allow cells to proliferate in the absence of the three Ras isoforms. Here we describe a protocol for a colony formation assay that permits evaluating the role of candidate proteins that are positive or negative regulators of cell proliferation mediated via Ras-independent Raf/Mek/Erk pathway activation. K-Ras lox (H-Ras -/- , N-Ras -/- , K-Ras lox/lox , RERT ert/ert ) MEFs are infected with retro- or lentiviral vectors expressing wild-type or constitutively activated candidate cDNAs, shRNAs, or sgRNAs in combination with Cas9 to ascertain the possibility of candidate proteins to function either as an activator or inhibitor of Ras-independent Raf/Mek/Erk activation. These cells are then seeded in the absence or presence of 4-Hydroxytamoxifen (4-OHT), which activates the resident CreERT2 alleles resulting in elimination of the conditional K-Ras alleles and ultimately generating Rasless cells. Colony formation in the presence of 4-OHT indicates cell proliferation via Ras-independent Raf/Mek/Erk activation.

Regulation of Son of sevenless by the membrane-actin linker protein ezrin

PubMed Central

Geißler, Katja J.; Jung, M. Juliane; Riecken, Lars Björn; Sperka, Tobias; Cui, Yan; Schacke, Stephan; Merkel, Ulrike; Markwart, Robby; Rubio, Ignacio; Than, Manuel E.; Breithaupt, Constanze; Peuker, Sebastian; Seifert, Reinhard; Kaupp, Ulrich Benjamin; Herrlich, Peter; Morrison, Helen

2013-01-01

Receptor tyrosine kinases participate in several signaling pathways through small G proteins such as Ras (rat sarcoma). An important component in the activation of these G proteins is Son of sevenless (SOS), which catalyzes the nucleotide exchange on Ras. For optimal activity, a second Ras molecule acts as an allosteric activator by binding to a second Ras-binding site within SOS. This allosteric Ras-binding site is blocked by autoinhibitory domains of SOS. We have reported recently that Ras activation also requires the actin-binding proteins ezrin, radixin, and moesin. Here we report the mechanism by which ezrin modulates SOS activity and thereby Ras activation. Active ezrin enhances Ras/MAPK signaling and interacts with both SOS and Ras in vivo and in vitro. Moreover, in vitro kinetic assays with recombinant proteins show that ezrin also is important for the activity of SOS itself. Ezrin interacts with GDP-Ras and with the Dbl homology (DH)/pleckstrin homology (PH) domains of SOS, bringing GDP-Ras to the proximity of the allosteric site of SOS. These actions of ezrin are antagonized by the neurofibromatosis type 2 tumor-suppressor protein merlin. We propose an additional essential step in SOS/Ras control that is relevant for human cancer as well as all physiological processes involving Ras. PMID:24297905

Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway.

PubMed

Han, Chang Woo; Jeong, Mi Suk; Jang, Se Bok

2017-09-16

Ras proteins are small GTPases that serve as master moderators of a large number of signaling pathways involved in various cellular processes. Activating mutations in Ras are found in about one-third of cancers. H-REV107, a K-Ras binding protein, plays an important role in determining K-Ras function. H-REV107 is a member of the HREV107 family of class II tumor suppressor genes and a growth inhibitory Ras target gene that suppresses cellular growth, differentiation, and apoptosis. Expression of H-REV107 was strongly reduced in about 50% of human carcinoma cell lines. However, the specific molecular mechanism by which H-REV107 inhibits Ras is still unknown. In the present study, we suggest that H-REV107 forms a strong complex with activating oncogenic mutation Q61H K-Ras from various biochemical binding assays and modeled structures. In addition, the interaction sites between K-Ras and H-REV107 were predicted based on homology modeling. Here, we found that some structure-based mutants of the K-Ras disrupted the complex formation with H-REV107. Finally, a novel molecular mechanism describing K-Ras and H-REV107 binding is suggested and insights into new K-Ras effector target drugs are provided. Copyright © 2017 Elsevier Inc. All rights reserved.

Renal microvascular disease determines the responses to revascularization in experimental renovascular disease.

PubMed

Chade, Alejandro R; Kelsen, Silvia

2010-08-01

Percutaneous transluminal renal angioplasty (PTRA) is the most frequent therapeutic approach to resolving renal artery stenosis (RAS). However, renal function recovers in only 30% of the cases. The causes of these poor outcomes are still unknown. We hypothesized that preserving the renal microcirculation distal to RAS will improve the responses to PTRA. RAS was induced in 28 pigs. In 14, vascular endothelial growth factor (VEGF)-165 0.05 microg/kg was infused intrarenally (RAS+VEGF). Single-kidney function was assessed in all pigs in vivo using ultrafast CT after 6 weeks. Observation of half of the RAS and RAS+VEGF pigs was completed. The other half underwent PTRA and repeated VEGF, and CT studies were repeated 4 weeks later. Pigs were then euthanized, the stenotic kidney removed, renal microvascular (MV) architecture reconstructed ex vivo using 3D micro-CT, and renal fibrosis quantified. The degree of RAS and hypertension were similar in RAS and RAS+VEGF. Renal function and MV density were decreased in RAS but improved in RAS+VEGF. PTRA largely resolved RAS, but the improvements of hypertension and renal function were greater in RAS+VEGF+PTRA than in RAS+PTRA, accompanied by a 34% increase in MV density and decreased fibrosis. Preservation of the MV architecture and function in the stenotic kidney improved the responses to PTRA, indicating that renal MV integrity plays a role in determining the responses to PTRA. This study indicates that damage and early loss of renal MV is an important determinant of the progression of renal injury in RAS and instigates often irreversible damage.

RENAL MICROVASCULAR DISEASE DETERMINES THE RESPONSES TO REVASCULARIZATION IN EXPERIMENTAL RENOVASCULAR DISEASE

PubMed Central

Chade, Alejandro R.; Kelsen, Silvia

2011-01-01

Background Percutaneous trasluminal renal angioplasty (PTRA) is the most frequent therapeutic approach to resolve renal artery stenosis (RAS). However, renal function recovers in only 30% of the cases. The causes of these poor outcomes are still unknown. We hypothesize that preserving the renal microcirculation distal to RAS will improve the responses to PTRA. Methods and Results RAS was induced in 28 pigs. In 14, vascular endothelial growth factor (VEGF)-165 was infused intra-renally (RAS+VEGF, 0.05 µg/kg). Single-kidney function was assessed in all pigs in vivo using ultra-fast CT after 6 weeks. Half of the RAS/RAS+VEGF completed their observation, and the other half underwent PTRA, VEGF was repeated, and CT studies repeated 4 weeks later. Pigs were then euthanized, the stenotic kidney removed, renal microvascular (MV) architecture reconstructed ex-vivo using 3D micro-CT, and renal fibrosis quantified. Degree of RAS and hypertension were similar in RAS and RAS+VEGF. Renal function and MV density were decreased in RAS but improved in RAS+VEGF. PTRA largely resolved RAS, but the improvements of hypertension and renal function were greater in RAS+VEGF+PTRA than in RAS+PTRA, accompanied by a 34% increase in MV density and decreased fibrosis. Conclusion Preservation of the MV architecture and function in the stenotic kidney improved the responses to PTRA, indicating that renal MV integrity plays a role in determining the responses to PTRA. This study indicates that damage and early loss of renal MV is an important determinant of the progression of renal injury in RAS and instigates often irreversible damage. PMID:20587789

Palmitoylation regulates vesicular trafficking of R-Ras to membrane ruffles and effects on ruffling and cell spreading

PubMed Central

Wurtzel, Jeremy G.T.; Kumar, Puneet; Goldfinger, Lawrence E.

2012-01-01

In this study we investigated the dynamics of R-Ras intracellular trafficking and its contributions to the unique roles of R-Ras in membrane ruffling and cell spreading. Wild type and constitutively active R-Ras localized to membranes of both Rab11- and transferrin-positive and -negative vesicles, which trafficked anterograde to the leading edge in migrating cells. H-Ras also co-localized with R-Ras in many of these vesicles in the vicinity of the Golgi, but R-Ras and H-Ras vesicles segregated proximal to the leading edge, in a manner dictated by the C-terminal membrane-targeting sequences. These segregated vesicle trafficking patterns corresponded to distinct modes of targeting to membrane ruffles at the leading edge. Geranylgeranylation was required for membrane anchorage of R-Ras, whereas palmitoylation was required for exit from the Golgi in post-Golgi vesicle membranes and trafficking to the plasma membrane. R-Ras vesicle membranes did not contain phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), whereas R-Ras co-localized with PtdIns(3,4,5)P3 in membrane ruffles. Finally, palmitoylation-deficient R-Ras blocked membrane ruffling, R-Ras/PI3-kinase interaction, enrichment of PtdIns(3,4,5)P3 at the plasma membrane, and R-Ras-dependent cell spreading. Thus, lipid modification of R-Ras dictates its vesicle trafficking, targeting to membrane ruffles, and its unique roles in localizing PtdIns(3,4,5)P3 to ruffles and promoting cell spreading. PMID:22751447

Lewis M. Rutherfurd and the First Photograph of Solar Granulation

NASA Astrophysics Data System (ADS)

Harvey, J. W.; Briggs, John W.; Prosser, Sian

2017-08-01

A major astronomical controversy of the mid-19th century was discordant descriptions of the small scale structure of the solar surface. Visual observers contradicted each other by describing the surface as consisting of “corrugations”, “willow leaves”, “rice grains”, “cumuli”, “thatch”, “granules”, etc. Early photographs of the solar surface were not good enough to settle the controversy. The French astronomer Jules Janssen is credited with the first 1876 photographs that clearly showed what we now call solar granulation (1876, CRAS 82, 1363). Upon seeing these images, New Yorker Lewis M. Rutherfurd (1878, MNRAS 38, 410) praised the high quality of Janssen’s images but asserted that he had also photographed granulation as early as 1871 using collodion wet plates. He sent copies of his best photograph to the Royal Astronomical Society to support his assertion. Curious about his claim, Briggs and Harvey set up Rutherfurd’s 13-inch achromatic refractor on Kitt Peak and found that it easily showed well-resolved solar granulation, so his claim might well have been justified. But without his plates we could not confirm the claim. For 140 years the copies of Rutherfurd’s best solar photograph remained in the archives of the Royal Astronomical Society and were recently discovered by Prosser (RAS Photographs A3/001B and A3/002). By coincidence a few days later, Briggs found the original August 11, 1871 plate. Despite poor condition these photographs show solar granulation. There are at least two other possible early claimants (Reade; Vogel) but their plates are almost certainly lost. Rutherfurd was a master of astronomical instrumentation and photography. He was reticent about his work, letting results speak for themselves, so it is satisfying to find that he was justified in making his claim of priority.

Dragging ras back in the ring.

PubMed

Stephen, Andrew G; Esposito, Dominic; Bagni, Rachel K; McCormick, Frank

2014-03-17

Ras proteins play a major role in human cancers but have not yielded to therapeutic attack. Ras-driven cancers are among the most difficult to treat and often excluded from therapies. The Ras proteins have been termed "undruggable," based on failures from an era in which understanding of signaling transduction, feedback loops, redundancy, tumor heterogeneity, and Ras' oncogenic role was poor. Structures of Ras oncoproteins bound to their effectors or regulators are unsolved, and it is unknown precisely how Ras proteins activate their downstream targets. These knowledge gaps have impaired development of therapeutic strategies. A better understanding of Ras biology and biochemistry, coupled with new ways of targeting undruggable proteins, is likely to lead to new ways of defeating Ras-driven cancers. Copyright © 2014 Elsevier Inc. All rights reserved.

The RAS mutation status predicts survival in patients undergoing hepatic resection for colorectal liver metastases: The results from a genetic analysis of all-RAS.

PubMed

Amikura, Katsumi; Akagi, Kiwamu; Ogura, Toshiro; Takahashi, Amane; Sakamoto, Hirohiko

2018-03-01

We investigated the impact of mutations in KRAS exons 3-4 and NRAS exons 2-3 in addition to KRAS exon 2, so-called all-RAS mutations, in patients with colorectal liver metastasis (CLM) undergoing hepatic resection. We analyzed 421 samples from CLM patients for their all-RAS mutation status to compare the overall survival rate (OS), recurrence-free survival rate (RFS), and the pattern of recurrence between the patients with and without RAS mutations. RAS mutations were detected in 191 (43.8%). Thirty-two rare mutations (12.2%) were detected in 262 patients with KRAS exon 2 wild-type. After excluding 79 patients who received anti-EGFR antibody therapy, 168 were classified as all-RAS wild-type, and 174 as RAS mutant-type. A multivariate analysis of factors associated with OS and RFS identified the RAS status as an independent factor (OS; hazard ratio [HR] = 1.672, P = 0.0031, RFS; HR = 1.703, P = 0.0024). Recurrence with lung metastasis was observed significantly more frequent in patients with RAS mutations than in patients with RAS wild-type (P = 0.0005). Approximately half of CLM patients may have a RAS mutation. CLM patients with RAS mutations had a significantly worse survival rate in comparison to patients with RAS wild-type, regardless of the administration of anti-EGFR antibody therapy. © 2017 Wiley Periodicals, Inc.

Ras, an Actor on Many Stages

PubMed Central

Arozarena, Imanol; Calvo, Fernando; Crespo, Piero

2011-01-01

Among the wealth of information that we have gathered about Ras in the past decade, the introduction of the concept of space in the field has constituted a major revolution that has enabled many pieces of the Ras puzzle to fall into place. In the early days, it was believed that Ras functioned exclusively at the plasma membrane. Today, we know that within the plasma membrane, the 3 Ras isoforms—H-Ras, K-Ras, and N-Ras—occupy different microdomains and that these isoforms are also present and active in endomembranes. We have also discovered that Ras proteins are not statically associated with these localizations; instead, they traffic dynamically between compartments. And we have learned that at these localizations, Ras is under site-specific regulatory mechanisms, distinctively engaging effector pathways and switching on diverse genetic programs to generate different biological responses. All of these processes are possible in great part due to the posttranslational modifications whereby Ras proteins bind to membranes and to regulatory events such as phosphorylation and ubiquitination that Ras is subject to. As such, space and these control mechanisms act in conjunction to endow Ras signals with an enormous signal variability that makes possible its multiple biological roles. These data have established the concept that the Ras signal, instead of being one single, homogeneous entity, results from the integration of multiple, site-specified subsignals, and Ras has become a paradigm of how space can differentially shape signaling. PMID:21779492

Rat embryo cells immortalized with transfected oncogenes are transformed by gamma irradiation.

PubMed

Endlich, B; Salavati, R; Sullivan, T; Ling, C C

1992-12-01

Cesium-137 gamma rays were used to transform rat embryo cells (REC) which were first transfected with activated c-myc or c-Ha-ras oncogenes to produce immortal cell lines (REC:myc and REC:ras). When exposed to 6 Gy of 137Cs gamma rays, some cells became morphologically transformed with focus formation frequencies of approximately 3 x 10(-4) for REC:myc and approximately 1 x 10(-4) for REC:ras, respectively. Cells isolated from foci of gamma-ray-transformed REC:myc (REC:myc:gamma) formed anchorage-independent colonies and were tumorigenic in nude mice, but foci from gamma-ray-transformed REC:ras (REC:ras:gamma) did not exhibit either of these criteria of transformation. Similar to the results with gamma irradiation, we observed a sequence-dependent phenomenon when myc and ras were transfected into REC, one at a time. REC immortalized by ras transfection were not converted to a tumorigenic phenotype by secondary transfection with myc, but REC transfected with myc were very susceptible to transformation by subsequent ras transfection. This suggests that myc-immortalized cells are more permissive to transformation via secondary treatments. In sequentially transfected REC, myc expression was high whether it was transfected first or second, whereas ras expression was highest when the ras gene was transfected secondarily into myc-containing REC. Molecular analysis of REC:ras:gamma transformants showed no alterations in structure of the transfected ras or of the endogenous ras, myc, p53, or fos genes. The expression of ras and p53 was increased in some isolates of REC:ras:gamma, but myc and fos expression were not affected. Similarly, REC:myc:gamma transformants did not demonstrate rearrangement or amplification of the transfected or the endogenous myc genes, or of the potentially cooperating Ha-, Ki-, or N-ras genes. Northern hybridization analysis revealed increased expression of N-ras in two isolates, REC:myc:gamma 33 and gamma 41, but no alterations in the expression of myc, raf, Ha-ras, or Ki-ras genes in any REC:myc transformant. DNA from several transformed REC:myc:gamma cell lines induced focus formation in recipient C3H 10T1/2 and NIH 3T3 cells. The NIH 3T3 foci tested positive when hybridized to a probe for rat repetitive DNA. A detailed analysis of the NIH 3T3 transformants generated from REC:myc:gamma 33 and gamma 41 DNA failed to detect Ha-ras, Ki-ras, raf, neu, trk, abl, fms, or src oncogenes of rat origin.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural Dynamics in Ras and Related Proteins upon Nucleotide Switching.

PubMed

Harrison, Rane A; Lu, Jia; Carrasco, Martin; Hunter, John; Manandhar, Anuj; Gondi, Sudershan; Westover, Kenneth D; Engen, John R

2016-11-20

Structural dynamics of Ras proteins contributes to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on the movement of a conserved structural motif, switch II. To understand Ras signaling and advance Ras-targeting strategies, experimental methods to measure Ras dynamics are required. Here, we demonstrate the utility of hydrogen-deuterium exchange (HDX) mass spectrometry (MS) to measure Ras dynamics by studying representatives from two branches of the Ras superfamily, Ras and Rho. A comparison of differential deuterium exchange between active (GMPPNP-bound) and inactive (GDP-bound) proteins revealed differences between the families, with the most notable differences occurring in the phosphate-binding loop and switch II. The P-loop exchange signature correlated with switch II dynamics observed in molecular dynamics simulations focused on measuring main-chain movement. HDX provides a means of evaluating Ras protein dynamics, which may be useful for understanding the mechanisms of Ras signaling, including activated signaling of pathologic mutants, and for targeting strategies that rely on protein dynamics. Copyright © 2016 Elsevier Ltd. All rights reserved.

RasGRP1 opposes proliferative EGFR–SOS1–Ras signals and restricts intestinal epithelial cell growth

PubMed Central

Depeille, Philippe; Henricks, Linda M.; van de Ven, Robert A. H.; Lemmens, Ed; Wang, Chih-Yang; Matli, Mary; Werb, Zena; Haigis, Kevin M.; Donner, David; Warren, Robert; Roose, Jeroen P.

2015-01-01

The character of EGFR signals can influence cell fate but mechanistic insights into intestinal EGFR-Ras signalling are limited. Here we show that two distinct Ras nucleotide exchange factors, RasGRP1 and SOS1, lie downstream of EGFR but act in functional opposition. RasGRP1 is expressed in intestinal crypts where it restricts epithelial growth. High RasGRP1 expression in colorectal cancer (CRC) patient samples correlates with a better clinical outcome. Biochemically, we find that RasGRP1 creates a negative feedback loop that limits proliferative EGFR–SOS1–Ras signals in CRC cells. Genetic Rasgrp1 depletion from mice with either an activating mutation in KRas or with aberrant Wnt signalling due to a mutation in Apc resulted in both cases in exacerbated Ras–ERK signalling and cell proliferation. The unexpected opposing cell biological effects of EGFR–RasGRP1 and EGFR–SOS1 signals in the same cell shed light on the intricacy of EGFR-Ras signalling in normal epithelium and carcinoma. PMID:26005835

Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Travers, Timothy; Lopez Bautista, Cesar Augusto; Van, Que

Activation of RAF kinase involves the association of its RAS-binding domain (RBD) and cysteine-rich domain (CRD) with membrane-anchored RAS. However, the overall architecture of the RAS/RBD/CRD ternary complex and the orientations of its constituent domains at the membrane remain unclear. Here in this paper, we have combined all-atom and coarse-grained molecular dynamics (MD) simulations with experimental data to construct and validate a model of membrane-anchored CRD, and used this as a basis to explore models of membrane-anchored RAS/RBD/CRD complex. First, simulations of the CRD revealed that it anchors to the membrane via insertion of its two hydrophobic loops, which ismore » consistent with our NMR measurements of CRD bound to nanodiscs. Simulations of the CRD in the context of membrane-anchored RAS/RBD then show how CRD association with either RAS or RBD could play an unexpected role in guiding the membrane orientations of RAS/RBD. This finding has implications for the formation of RAS-RAS dimers, as different membrane orientations of RAS expose distinct putative dimerization interfaces.« less

Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain

DOE PAGES

Travers, Timothy; Lopez Bautista, Cesar Augusto; Van, Que; ...

2018-05-31

Activation of RAF kinase involves the association of its RAS-binding domain (RBD) and cysteine-rich domain (CRD) with membrane-anchored RAS. However, the overall architecture of the RAS/RBD/CRD ternary complex and the orientations of its constituent domains at the membrane remain unclear. Here in this paper, we have combined all-atom and coarse-grained molecular dynamics (MD) simulations with experimental data to construct and validate a model of membrane-anchored CRD, and used this as a basis to explore models of membrane-anchored RAS/RBD/CRD complex. First, simulations of the CRD revealed that it anchors to the membrane via insertion of its two hydrophobic loops, which ismore » consistent with our NMR measurements of CRD bound to nanodiscs. Simulations of the CRD in the context of membrane-anchored RAS/RBD then show how CRD association with either RAS or RBD could play an unexpected role in guiding the membrane orientations of RAS/RBD. This finding has implications for the formation of RAS-RAS dimers, as different membrane orientations of RAS expose distinct putative dimerization interfaces.« less

p21ras independent down-regulation of ras-induced increases in natural antibody binding during tumor progression.

PubMed

Tough, D F; Feng, X; Chow, D A

1995-01-01

Selective outgrowth of v-H-ras-infected 10T1/2 cells based on the cointroduction of a gene for resistance to geneticin (G418), yielded cells which exhibited an increased capacity to bind polyclonal serum natural antibody (NAb). This demonstrated an NAb-susceptible phase of tumor development which would be a basic requirement for NAb-mediated surveillance of tumors. The ras-oncogene dependence of the high-NAb-binding phenotype provided a model for assessing NAb resistance against ras transformants in vivo and for a comparative analysis of phenotypic and genetic alterations contributing to the progression of ras transformants. Variants were developed through in vitro and in vivo models of tumor progression. T24-H-ras and v-H-ras transformants were isolated in vitro through more rigorous growth conditions, focus formation in the presence of untransformed cells with no selecting drug. These clones expressed p21ras but exhibited little or no increase in NAb binding. Variants recovered following growth from intravenous or threshold subcutaneous (s.c.) inocula of high-NAb-binding ras transformants in syngeneic C3H/HeN mice exhibited decreases in NAb binding but no uniform change in p21ras. Concurring inverse correlations between NAb binding and s.c. tumorigenicity were exhibited by the T24-H-ras transformant clones, the ras transformants grown in vivo, and the v-H-ras-transformed clones isolated in the presence versus the absence of untransformed cells. This consistent inverse correlation, together with the reduced NAb binding of the ras transformants grown in vivo, provides strong evidence that NAb participates in the defense against ras-transformed cells in vivo. The lack of any direct correlation between p21ras expression and the reduction in NAb binding or the increase in tumorigenicity of cells generated through progression in vivo suggested the regulatory action of additional genes. Hybridization studies between high- and low-NAb-binding clones implicated the activation of an additional oncogene and inactivation of an antioncogene in the down-regulation of the ras-induced increases in NAb binding associated with tumor progression.

EphA2 Drives the Segregation of Ras-Transformed Epithelial Cells from Normal Neighbors.

PubMed

Porazinski, Sean; de Navascués, Joaquín; Yako, Yuta; Hill, William; Jones, Matthew Robert; Maddison, Robert; Fujita, Yasuyuki; Hogan, Catherine

2016-12-05

In epithelial tissues, cells expressing oncogenic Ras (hereafter RasV12 cells) are detected by normal neighbors and as a result are often extruded from the tissue [1-6]. RasV12 cells are eliminated apically, suggesting that extrusion may be a tumor-suppressive process. Extrusion depends on E-cadherin-based cell-cell adhesions and signaling to the actin-myosin cytoskeleton [2, 6]. However, the signals underlying detection of the RasV12 cell and triggering extrusion are poorly understood. Here we identify differential EphA2 signaling as the mechanism by which RasV12 cells are detected in epithelial cell sheets. Cell-cell interactions between normal cells and RasV12 cells trigger ephrin-A-EphA2 signaling, which induces a cell repulsion response in RasV12 cells. Concomitantly, RasV12 cell contractility increases in an EphA2-dependent manner. Together, these responses drive the separation of RasV12 cells from normal cells. In the absence of ephrin-A-EphA2 signals, RasV12 cells integrate with normal cells and adopt a pro-invasive morphology. We also show that Drosophila Eph (DEph) is detected in segregating clones of RasV12 cells and is functionally required to drive segregation of RasV12 cells in vivo, suggesting that our in vitro findings are conserved in evolution. We propose that expression of RasV12 in single or small clusters of cells within a healthy epithelium creates ectopic EphA2 boundaries, which drive the segregation and elimination of the transformed cell from the tissue. Thus, deregulation of Eph/ephrin would allow RasV12 cells to go undetected and expand within an epithelium. Copyright © 2016 Elsevier Ltd. All rights reserved.

Impact of Emergent Circulating Tumor DNA RAS Mutation in Panitumumab-Treated Chemoresistant Metastatic Colorectal Cancer.

PubMed

Kim, Tae Won; Peeters, Marc; Thomas, Anne L; Gibbs, Peter; Hool, Kristina; Zhang, Jianqi; Ang, Agnes; Bach, Bruce Allen; Price, Timothy

2018-06-13

The accumulation of emergent RAS mutations during anti-epidermal growth factor receptor (EGFR) therapy is of interest as a mechanism for acquired resistance to anti-EGFR treatment. Plasma analysis of circulating tumor (ct) DNA is a minimally invasive and highly sensitive method to determine RAS mutational status. This biomarker analysis of the global phase III ASPECCT study used next-generation sequencing to detect expanded RAS ctDNA mutations in panitumumab-treated patients. Plasma samples collected at baseline and posttreatment were analyzed categorically for the presence of RAS mutations by the Plasma Select -R™ 64-gene panel at 0.1% sensitivity. Among panitumumab-treated patients with evaluable plasma samples at baseline (n = 238), 188 (79%) were wild-type (WT) RAS, and 50 (21%) were mutant RAS Of the 188 patients with baseline ctDNA WT RAS status, 164 had evaluable posttreatment results with a 32% rate of emergent RAS mutations. The median overall survival (OS) for WT and RAS mutant status by ctDNA at baseline was 13.7 (95% confidence interval: 11.5-15.4) and 7.9 months (6.4-9.6), respectively ( P < 0.0001). Clinical outcomes were not significantly different between patients with and without emergent ctDNA RAS mutations. Although patients with baseline ctDNA RAS mutations had worse outcomes than patients who were WT RAS before initiating treatment, emergent ctDNA RAS mutations were not associated with less favorable patient outcomes in panitumumab-treated patients. Further research is needed to determine a clinically relevant threshold for baseline and emergent ctDNA RAS mutations. Copyright ©2018, American Association for Cancer Research.

A New Strategy to Control and Eradicate "Undruggable" Oncogenic K-RAS-Driven Pancreatic Cancer: Molecular Insights and Core Principles Learned from Developmental and Evolutionary Biology.

PubMed

Van Sciver, Robert E; Lee, Michael P; Lee, Caroline Dasom; Lafever, Alex C; Svyatova, Elizaveta; Kanda, Kevin; Colliver, Amber L; Siewertsz van Reesema, Lauren L; Tang-Tan, Angela M; Zheleva, Vasilena; Bwayi, Monicah N; Bian, Minglei; Schmidt, Rebecca L; Matrisian, Lynn M; Petersen, Gloria M; Tang, Amy H

2018-05-14

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely "undruggable". Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future.

Does Harvey-Ras gene expression lead to oral squamous cell carcinoma? A clinicopathological aspect

PubMed Central

Krishna, Akhilesh; Singh, Shraddha; Singh, Vineeta; Kumar, Vijay; Singh, Uma Shankar; Sankhwar, Satya Narayan

2018-01-01

Background: Harvey-Ras (H-Ras) is an important guanosine triphosphatase protein for the regulation of cellular growth and survival. Altered Ras signaling has been observed in different types of cancer either by gene amplification and/or mutation. The H-Ras oncogene mutations are well reported, but expression of the H-Ras gene is still unknown. Objective: This study aimed to examine both protein and messenger-RNA (mRNA) expressions of H-Ras in oral squamous cell carcinoma (OSCC) and analyzed the association with risk habits and the clinicopathological profile of cases. Methodology: A total of 65 tissue specimens of OSCC (case group) and equal number of normal tissues (control group) were included in this study. H-Ras protein and mRNA expressions were analyzed using immunohistochemical and quantitative real time-polymerase chain reaction techniques, respectively. Results: The H-Ras protein was significantly overexpressed in the oral carcinoma group compared to the normal group (P = 0.03). Most of the OSCC cases showed positive staining with moderate expression, while negative and moderate staining was high in the control group. The majority of H-Ras positive cases were found in individuals with multiple risk habits including tobacco chewing. The risk of H-Ras positivity was 1.46 times higher in smokers than non-smokers. H-Ras positivity increased in cases affected with buccal mucosa site and higher grade of carcinoma. Relative mRNA level of H-Ras was significantly elevated in oral carcinoma as compared with the control group (P ≤ 0.001). Protein and mRNA levels of H-Ras in case group was poorly correlated. Conclusion: H-Ras oncogene expression was markedly higher in oral carcinoma, and it can be a prognostic marker and target for an effective molecular therapy. PMID:29731559

Ras Dimer Formation as a New Signaling Mechanism and Potential Cancer Therapeutic Target

PubMed Central

Chen, Mo; Peters, Alec; Huang, Tao; Nan, Xiaolin

2016-01-01

The K-, N-, and HRas small GTPases are key regulators of cell physiology and are frequently mutated in human cancers. Despite intensive research, previous efforts to target hyperactive Ras based on known mechanisms of Ras signaling have been met with little success. Several studies have provided compelling evidence for the existence and biological relevance of Ras dimers, establishing a new mechanism for regulating Ras activity in cells additionally to GTP-loading and membrane localization. Existing data also start to reveal how Ras proteins dimerize on the membrane. We propose a dimer model to describe Ras-mediated effector activation, which contrasts existing models of Ras signaling as a monomer or as a 5-8 membered multimer. We also discuss potential implications of this model in both basic and translational Ras biology. PMID:26423697

Endomembrane H-Ras Controls Vascular Endothelial Growth Factor-induced Nitric-oxide Synthase-mediated Endothelial Cell Migration*

PubMed Central

Haeussler, Dagmar J.; Pimentel, David R.; Hou, Xiuyun; Burgoyne, Joseph R.; Cohen, Richard A.; Bachschmid, Markus M.

2013-01-01

We demonstrate for the first time that endomembrane-delimited H-Ras mediates VEGF-induced activation of endothelial nitric-oxide synthase (eNOS) and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total H-Ras is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane-localized H-Ras unexplained. Knockdown of H-Ras blocked VEGF-induced PI3K-dependent Akt (Ser-473) and eNOS (Ser-1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of H-Ras. Activation of endogenous H-Ras led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous H-Ras was fully restored by modest overexpression of an endomembrane-delimited H-Ras palmitoylation mutant. These studies define a newly recognized role for endomembrane-localized H-Ras in mediating nitric oxide-dependent proangiogenic signaling. PMID:23548900

Differences in the Regulation of K-Ras and H-Ras Isoforms by Monoubiquitination*

PubMed Central

Baker, Rachael; Wilkerson, Emily M.; Sumita, Kazutaka; Isom, Daniel G.; Sasaki, Atsuo T.; Dohlman, Henrik G.; Campbell, Sharon L.

2013-01-01

Ras GTPases are signaling switches that control critical cellular processes including gene expression, differentiation, and apoptosis. The major Ras isoforms (K, H, and N) contain a conserved core GTPase domain, but have distinct biological functions. Among the three Ras isoforms there are clear differences in post-translational regulation, which contribute to differences in localization and signaling output. Modification by ubiquitination was recently reported to activate Ras signaling in cells, but the mechanisms of activation are not well understood. Here, we show that H-Ras is activated by monoubiquitination and that ubiquitination at Lys-117 accelerates intrinsic nucleotide exchange, thereby promoting GTP loading. This mechanism of Ras activation is distinct from K-Ras monoubiquitination at Lys-147, which leads to impaired regulator-mediated GTP hydrolysis. These findings reveal that different Ras isoforms are monoubiquitinated at distinct sites, with distinct mechanisms of action, but with a common ability to chronically activate the protein in the absence of a receptor signal or oncogenic mutation. PMID:24247240

Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

PubMed

Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2014-03-04

Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

Enhanced MET Translation and Signaling Sustains K-Ras-Driven Proliferation under Anchorage-Independent Growth Conditions.

PubMed

Fujita-Sato, Saori; Galeas, Jacqueline; Truitt, Morgan; Pitt, Cameron; Urisman, Anatoly; Bandyopadhyay, Sourav; Ruggero, Davide; McCormick, Frank

2015-07-15

Oncogenic K-Ras mutation occurs frequently in several types of cancers, including pancreatic and lung cancers. Tumors with K-Ras mutation are resistant to chemotherapeutic drugs as well as molecular targeting agents. Although numerous approaches are ongoing to find effective ways to treat these tumors, there are still no effective therapies for K-Ras mutant cancer patients. Here we report that K-Ras mutant cancers are more dependent on K-Ras in anchorage-independent culture conditions than in monolayer culture conditions. In seeking to determine mechanisms that contribute to the K-Ras dependency in anchorage-independent culture conditions, we discovered the involvement of Met in K-Ras-dependent, anchorage-independent cell growth. The Met signaling pathway is enhanced and plays an indispensable role in anchorage-independent growth even in cells in which Met is not amplified. Indeed, Met expression is elevated under anchorage-independent growth conditions and is regulated by K-Ras in a MAPK/ERK kinase (MEK)-dependent manner. Remarkably, in spite of a global downregulation of mRNA translation during anchorage-independent growth, we find that Met mRNA translation is specifically enhanced under these conditions. Importantly, ectopic expression of an active Met mutant rescues K-Ras ablation-derived growth suppression, indicating that K-Ras-mediated Met expression drives "K-Ras addiction" in anchorage-independent conditions. Our results indicate that enhanced Met expression and signaling is essential for anchorage-independent growth of K-Ras mutant cancer cells and suggests that pharmacological inhibitors of Met could be effective for K-Ras mutant tumor patients. ©2015 American Association for Cancer Research.

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

PubMed Central

Jeong, Ji Hun; Park, Soon Ho; Park, Mi Jung; Kim, Moon Jin; Kim, Kyung Hee; Park, Pil Whan; Seo, Yiel Hea; Lee, Jae Hoon; Park, Jinny; Hong, Junshik

2013-01-01

Background N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. Methods We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. Results The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. Conclusions There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses. PMID:23667841

Enhanced MET translation and signaling sustains K-Ras driven proliferation under anchorage-independent growth conditions

PubMed Central

Fujita-Sato, Saori; Galeas, Jacqueline; Truitt, Morgan; Pitt, Cameron; Urisman, Anatoly; Bandyopadhyay, Sourav; Ruggero, Davide; McCormick, Frank

2015-01-01

Oncogenic K-Ras mutation occurs frequently in several types of cancers including pancreatic and lung cancers. Tumors with K-Ras mutation are resistant to chemotherapeutic drugs as well as molecular targeting agents. Although numerous approaches are ongoing to find effective ways to treat these tumors, there are still no effective therapies for K-Ras mutant cancer patients. Here we report that K-Ras mutant cancers are more dependent on K-Ras in anchorage independent culture conditions than in monolayer culture conditions. In seeking to determine mechanisms that contribute to the K-Ras dependency in anchorage independent culture conditions, we discovered the involvement of Met in K-Ras-dependent, anchorage independent cell growth. The Met signaling pathway is enhanced and plays an indispensable role in anchorage independent growth even in cells in which Met is not amplified. Indeed, Met expression is elevated under anchorage-independent growth conditions and is regulated by K-Ras in a MAPK/ERK kinase (MEK)-dependent manner. Remarkably, in spite of a global down-regulation of mRNA translation during anchorage independent growth, we find that Met mRNA translation is specifically enhanced under these conditions. Importantly, ectopic expression of an active Met mutant rescues K-Ras ablation-derived growth suppression, indicating that K-Ras mediated Met expression drives “K-Ras addiction” in anchorage independent conditions. Our results indicate that enhanced Met expression and signaling is essential for anchorage independent growth of K-Ras mutant cancer cells and suggests that pharmacological inhibitors of Met could be effective for K-Ras mutant tumor patients. PMID:25977330

[Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene].

PubMed

Gui, Li-ming; Wei, Li-hui; Zhang, Ying-mei; Wang, Jian-liu; Wang, Ying; Chen, Ying; Ma, Da-long

2002-01-01

Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K-ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type [12Asp] K-ras4B gene. (1) Full-length [12Asp]K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3) To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and [3H]thymidine incorporation method. (1) The authors have successfully constructed eukaryotic expression plasmid pcDI-[12Asp] K-ras4B; (2) To confirm that [12Asp] K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3) After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-[12Asp] K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-[12Asp]K-ras4B cell (P < 0.05). However, RafCAAX mutant can enhance pcDI-[12Asp]K-ras4B cell growth (P < 0.05). (1) [12Asp]K-ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) [12Asp]K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.

Investigating RAS Signaling in Cancer | Office of Cancer Clinical Proteomics Research

Cancer.gov

CPTAC expertise has been charged to develop RAS specific targeted proteomic assays to study the important pathways of human cancer. The oncogene RAS is linked to 30 percent of human cancers, but the search for a targeted therapy for RAS has remained elusive. To advance our understanding of this oncogene and to develop improved targeted therapies against RAS pathway, the National Cancer Institute (NCI) has launched a RAS Initiative.

Inhibition of Ras for cancer treatment: the search continues

PubMed Central

Baines, Antonio T.; Xu, Dapeng; Der, Channing J.

2012-01-01

Background The RAS oncogenes (HRAS, NRAS and KRAS) comprise the most frequently mutated class of oncogenes in human cancers (33%), stimulating intensive effort in developing anti-Ras inhibitors for cancer treatment. Discussion Despite intensive effort, to date no effective anti-Ras strategies have successfully made it to the clinic. We present an overview of past and ongoing strategies to inhibit oncogenic Ras in cancer. Conclusions Since approaches to directly target mutant Ras have not been successful, most efforts have focused on indirect approaches to block Ras membrane association or downstream effector signaling. While inhibitors of effector signaling are currently under clinical evaluation, genome-wide unbiased genetic screens have identified novel directions for future anti-Ras drug discovery. PMID:22004085

Wild-type H- and N-Ras promote mutant K-Ras driven tumorigenesis by modulating the DNA damage response

PubMed Central

Grabocka, Elda; Pylayeva-Gupta, Yuliya; Jones, Mathew JK; Lubkov, Veronica; Yemanaberhan, Eyoel; Taylor, Laura; Jeng, Hao Hsuan; Bar-Sagi, Dafna

2014-01-01

SUMMARY Mutations in KRAS are prevalent in human cancers and universally predictive of resistance to anti-cancer therapeutics. Although it is widely accepted that acquisition of an activating mutation endows RAS genes with functional autonomy, recent studies suggest that the wild-type forms of Ras may contribute to mutant Ras-driven tumorigenesis. Here we show that downregulation of wild-type H-Ras or N-Ras in mutant K-Ras cancer cells leads to hyperactivation of the Erk/p90RSK and PI3K/Akt pathways, and consequently, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint and confers specific sensitization of mutant K-Ras cancer cells to DNA damage chemotherapeutic agents in vitro and in vivo. PMID:24525237

Electrostatic Interactions Positively Regulate K-Ras Nanocluster Formation and Function▿

PubMed Central

Plowman, Sarah J.; Ariotti, Nicholas; Goodall, Andrew; Parton, Robert G.; Hancock, John F.

2008-01-01

The organization of Ras proteins into plasma membrane nanoclusters is essential for high-fidelity signal transmission, but whether the nanoscale enviroments of different Ras nanoclusters regulate effector interactions is unknown. We show using high-resolution spatial mapping that Raf-1 is recruited to and retained in K-Ras-GTP nanoclusters. In contrast, Raf-1 recruited to the plasma membrane by H-Ras is not retained in H-Ras-GTP nanoclusters. Similarly, upon epidermal growth factor receptor activation, Raf-1 is preferentially recruited to K-Ras-GTP and not H-Ras-GTP nanoclusters. The formation of K-Ras-GTP nanoclusters is inhibited by phosphorylation of S181 in the C-terminal polybasic domain or enhanced by blocking S181 phosphorylation, with a concomitant reduction or increase in Raf-1 plasma membrane recruitment, respectively. Phosphorylation of S181 does not, however, regulate in vivo interactions with the nanocluster scaffold galectin-3 (Gal3), indicating separate roles for the polybasic domain and Gal3 in driving K-Ras nanocluster formation. Together, these data illustrate that Ras nanocluster composition regulates effector recruitment and highlight the importance of lipid/protein nanoscale environments to the activation of signaling cascades. PMID:18458061

Cognitive performance, symptoms and counter-regulation during hypoglycaemia in patients with type 1 diabetes and high or low renin-angiotensin system activity.

PubMed

Høi-Hansen, Thomas; Pedersen-Bjergaard, Ulrik; Andersen, Rikke Due; Kristensen, Peter Lommer; Thomsen, Carsten; Kjaer, Troels; Høgenhaven, Hans; Smed, Annelise; Holst, Jens Juul; Dela, Flemming; Boomsma, Frans; Thorsteinsson, Birger

2009-12-01

High basal renin-angiotensin system (RAS) activity is associated with increased risk of severe hypoglycaemia in type 1 diabetes. We tested whether this might be explained by more pronounced cognitive dysfunction during hypoglycaemia in patients with high RAS activity than in patients with low RAS activity. Nine patients with type 1 diabetes and high and nine with low RAS activity were subjected to hypoglycaemia and euglycaemia in a cross-over study using an intravenous insulin infusion protocol. Cognitive function, electroencephalography, auditory evoked potentials and hypoglycaemic symptoms were recorded. At a hypoglycaemic nadir of 2.2 (SD 0.3) mmol/L the high RAS group displayed significant deterioration in cognitive performance during hypoglycaemia in the three most complex reaction time tasks. In the low RAS group, hypoglycaemia led to cognitive dysfunction in only one reaction time task. The high RAS group reported lower symptom scores during hypoglycaemia than the low RAS group, suggesting poorer hypoglycaemia awareness. High RAS activity is associated with increased cognitive dysfunction and blunted symptoms during mild hypoglycaemia compared to low RAS activity. This may explain why high RAS activity is a risk factor for severe hypoglycaemia in type 1 diabetes.

CHMP6 and VPS4A mediate recycling of Ras to the plasma membrane to promote growth factor signaling

PubMed Central

Zheng, Ze-Yi; Cheng, Chiang-Min; Fu, Xin-Rong; Chen, Liuh-Yow; Xu, Lizhong; Terrillon, Sonia; Wong, Stephen T.; Bar-Sagi, Dafna; Songyang, Zhou; Chang, Eric C.

2011-01-01

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III, were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding-loop and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound N-Ras, but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked EGFR recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling. PMID:22231449

Inhibitors of Ras-SOS Interactions.

PubMed

Lu, Shaoyong; Jang, Hyunbum; Zhang, Jian; Nussinov, Ruth

2016-04-19

Activating Ras mutations are found in about 30 % of human cancers. Ras activation is regulated by guanine nucleotide exchange factors, such as the son of sevenless (SOS), which form protein-protein interactions (PPIs) with Ras and catalyze the exchange of GDP by GTP. This is the rate-limiting step in Ras activation. However, Ras surfaces lack any evident suitable pockets where a molecule might bind tightly, rendering Ras proteins still 'undruggable' for over 30 years. Among the alternative approaches is the design of inhibitors that target the Ras-SOS PPI interface, a strategy that is gaining increasing recognition for treating Ras mutant cancers. Herein we focus on data that has accumulated over the past few years pertaining to the design of small-molecule modulators or peptide mimetics aimed at the interface of the Ras-SOS PPI. We emphasize, however, that even if such Ras-SOS therapeutics are potent, drug resistance may emerge. To counteract this development, we propose "pathway drug cocktails", that is, drug combinations aimed at parallel (or compensatory) pathways. A repertoire of classified cancer, cell/tissue, and pathway/protein combinations would be beneficial toward this goal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Graded inhibition of oncogenic Ras-signaling by multivalent Ras-binding domains

PubMed Central

2014-01-01

Background Ras is a membrane-associated small G-protein that funnels growth and differentiation signals into downstream signal transduction pathways by cycling between an inactive, GDP-bound and an active, GTP-bound state. Aberrant Ras activity as a result of oncogenic mutations causes de novo cell transformation and promotes tumor growth and progression. Results Here, we describe a novel strategy to block deregulated Ras activity by means of oligomerized cognate protein modules derived from the Ras-binding domain of c-Raf (RBD), which we named MSOR for multivalent scavengers of oncogenic Ras. The introduction of well-characterized mutations into RBD was used to adjust the affinity and hence the blocking potency of MSOR towards activated Ras. MSOR inhibited several oncogenic Ras-stimulated processes including downstream activation of Erk1/2, induction of matrix-degrading enzymes, cell motility and invasiveness in a graded fashion depending on the oligomerization grade and the nature of the individual RBD-modules. The amenability to accurate experimental regulation was further improved by engineering an inducible MSOR-expression system to render the reversal of oncogenic Ras effects controllable. Conclusion MSOR represent a new tool for the experimental and possibly therapeutic selective blockade of oncogenic Ras signals. PMID:24383791

Resistance and resilience of small-scale recirculating aquaculture systems (RAS) with or without algae to pH perturbation

PubMed Central

Giatsis, Christos; Md Yusoff, Fatimah; Verreth, Johan; Verdegem, Marc

2018-01-01

The experimental set-up of this study mimicked recirculating aquaculture systems (RAS) where water quality parameters such as dissolved oxygen, pH, temperature, and turbidity were controlled and wastes produced by fish and feeding were converted to inorganic forms. A key process in the RAS was the conversion of ammonia to nitrite and nitrite to nitrate through nitrification. It was hypothesized that algae inclusion in RAS would improve the ammonia removal from the water; thereby improving RAS water quality and stability. To test this hypothesis, the stability of the microbiota community composition in a freshwater RAS with (RAS+A) or without algae (RAS-A) was challenged by introducing an acute pH drop (from pH 7 to 4 during three hours) to the system. Stigeoclonium nanum, a periphytic freshwater microalga was used in this study. No significant effect of the algae presence was found on the resistance to the acute pH drop on ammonia conversion to nitrite and nitrite conversion to nitrate. Also the resilience of the ammonia conversion to the pH drop disruption was not affected by the addition of algae. This could be due to the low biomass of algae achieved in the RAS. However, with regard to the conversion step of nitrite to nitrate, RAS+A was significantly more resilient than RAS-A. In terms of overall bacterial communities, the composition and predictive function of the bacterial communities was significantly different between RAS+A and RAS-A. PMID:29659617

RasGRP1 confers the phorbol ester-sensitive phenotype to EL4 lymphoma cells.

PubMed

Han, Shujie; Knoepp, Stewart M; Hallman, Mark A; Meier, Kathryn E

2007-01-01

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk mitogen-activated protein kinase activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells but not in resistant cells lacking Ras-GRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of small interfering RNA (siRNA) for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA-sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function

PubMed Central

Hocker, Harrison J.; Cho, Kwang-Jin; Chen, Chung-Ying K.; Rambahal, Nandini; Sagineedu, Sreenivasa Rao; Shaari, Khozirah; Stanslas, Johnson; Hancock, John F.; Gorfe, Alemayehu A.

2013-01-01

Aberrant signaling by oncogenic mutant rat sarcoma (Ras) proteins occurs in ∼15% of all human tumors, yet direct inhibition of Ras by small molecules has remained elusive. Recently, several small-molecule ligands have been discovered that directly bind Ras and inhibit its function by interfering with exchange factor binding. However, it is unclear whether, or how, these ligands could lead to drugs that act against constitutively active oncogenic mutant Ras. Using a dynamics-based pocket identification scheme, ensemble docking, and innovative cell-based assays, here we show that andrographolide (AGP)—a bicyclic diterpenoid lactone isolated from Andrographis paniculata—and its benzylidene derivatives bind to transient pockets on Kirsten-Ras (K-Ras) and inhibit GDP–GTP exchange. As expected for inhibitors of exchange factor binding, AGP derivatives reduced GTP loading of wild-type K-Ras in response to acute EGF stimulation with a concomitant reduction in MAPK activation. Remarkably, however, prolonged treatment with AGP derivatives also reduced GTP loading of, and signal transmission by, oncogenic mutant K-RasG12V. In sum, the combined analysis of our computational and cell biology results show that AGP derivatives directly bind Ras, block GDP–GTP exchange, and inhibit both wild-type and oncogenic K-Ras signaling. Importantly, our findings not only show that nucleotide exchange factors are required for oncogenic Ras signaling but also demonstrate that inhibiting nucleotide exchange is a valid approach to abrogating the function of oncogenic mutant Ras. PMID:23737504

Blood-based detection of RAS mutations to guide anti-EGFR therapy in colorectal cancer patients: concordance of results from circulating tumor DNA and tissue-based RAS testing.

PubMed

Schmiegel, Wolff; Scott, Rodney J; Dooley, Susan; Lewis, Wendy; Meldrum, Cliff J; Pockney, Peter; Draganic, Brian; Smith, Steve; Hewitt, Chelsee; Philimore, Hazel; Lucas, Amanda; Shi, Elva; Namdarian, Kateh; Chan, Timmy; Acosta, Danilo; Ping-Chang, Su; Tannapfel, Andrea; Reinacher-Schick, Anke; Uhl, Waldemar; Teschendorf, Christian; Wolters, Heiner; Stern, Josef; Viebahn, Richard; Friess, Helmut; Janssen, Klaus-Peter; Nitsche, Ulrich; Slotta-Huspenina, Julia; Pohl, Michael; Vangala, Deepak; Baraniskin, Alexander; Dockhorn-Dworniczak, Barbara; Hegewisch-Becker, Susanne; Ronga, Philippe; Edelstein, Daniel L; Jones, Frederick S; Hahn, Stephan; Fox, Stephen B

2017-02-01

An accurate blood-based RAS mutation assay to determine eligibility of metastatic colorectal cancer (mCRC) patients for anti-EGFR therapy would benefit clinical practice by better informing decisions to administer treatment independent of tissue availability. The objective of this study was to determine the level of concordance between plasma and tissue RAS mutation status in patients with mCRC to gauge whether blood-based RAS mutation testing is a viable alternative to standard-of-care RAS tumor testing. RAS testing was performed on plasma samples from newly diagnosed metastatic patients, or from recurrent mCRC patients using the highly sensitive digital PCR technology, BEAMing (beads, emulsions, amplification, and magnetics), and compared with DNA sequencing data of respective FFPE (formalin-fixed paraffin-embedded) tumor samples. Discordant tissue RAS results were re-examined by BEAMing, if possible. The prevalence of RAS mutations detected in plasma (51%) vs. tumor (53%) was similar, in accord with the known prevalence of RAS mutations observed in mCRC patient populations. The positive agreement between plasma and tumor RAS results was 90.4% (47/52), the negative agreement was 93.5% (43/46), and the overall agreement (concordance) was 91.8% (90/98). The high concordance of plasma and tissue results demonstrates that blood-based RAS mutation testing is a viable alternative to tissue-based RAS testing. © 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Hemodynamic responses to acute and gradual renal artery stenosis in pigs.

PubMed

Rognant, Nicolas; Rouvière, Olivier; Janier, Marc; Lê, Quoc Hung; Barthez, Paul; Laville, Maurice; Juillard, Laurent

2010-11-01

Reduction of renal blood flow (RBF) due to a renal artery stenosis (RAS) can lead to renal ischemia and atrophy. However in pigs, there are no data describing the relationship between the degree of RAS, the reduction of RBF, and the increase of systemic plasma renin activity (PRA). Therefore, we conducted a study in order to measure the effect of acute and gradual RAS on RBF, mean arterial pressure (MAP), and systemic PRA in pigs. RAS was induced experimentally in six pigs using an occluder placed around the renal artery downstream of an ultrasound flow probe. The vascular occluder was inflated gradually to reduce RBF. At each inflation step, percentage of RAS was measured by digital subtraction angiography (DSA) with simultaneous measurements of RBF, MAP, and PRA. Data were normalized to baseline values obtained before RAS induction. Piecewise regression analysis was performed between percentage of RAS and relative RBF, MAP, and PRA, respectively. In all pigs, the relationship between the degree of RAS and RBF was similar. RBF decreased over a threshold of 42% of RAS, with a rapid drop in RBF when RAS reached 70%. PRA increased dramatically over a threshold of 58% of RAS (+1,300% before occlusion). MAP increased slightly (+15% before occlusion) without identifiable threshold. This study emphasizes that the relation between the degree of RAS and RBF and systemic PRA is not linear and that a high degree of RAS must be reached before the occurrence of significant hemodynamic and humoral effects.

Outcomes of a Postexposure Prophylaxis Program at the Korle-Bu Teaching Hospital in Ghana: A Retrospective Cohort Study.

PubMed

Tetteh, Raymond A; Nartey, Edmund T; Lartey, Margaret; Mantel-Teeuwisse, Aukje K; Leufkens, Hubert G M; Nortey, Priscilla A; Dodoo, Alexander N O

2015-01-01

The risk for occupational exposure to HIV is a serious public health problem that is well characterized in the developed world, but less so in the developing countries such as Ghana. This study was undertaken to examine the characteristics of occupational exposure to HIV and the utilization of a risk assessment system (RAS)-based postexposure prophylaxis (PEP) among health care workers (HCWs) and health care students (HCSs) in the Korle-Bu Teaching Hospital (KBTH). During the study period (January 2005-December 2010), a total of 260 and 35 exposures were reported by HCWs and HCSs, respectively. Ward attendants reported the highest incidence rate of 6.46 of 100 person-years (P-Y). The incidence of high-risk exposures was 0.33 of 100 P-Y (n = 65); 60.0% occurred during a procedure of disposing of a needle and 24.6% during a cannula insertion. A total of 289 of the 295 individuals were administered PEP, of which 181 (62.6%) completed the 6-month follow-up testing schedule and none sero-converted. This shows that with a good RAS in place, it is possible to deploy an effective PEP program in a typical African teaching hospital like the KBTH in Accra, Ghana. © The Author(s) 2013.

The RAS Initiative

Cancer.gov

NCI established the RAS Initiative to explore innovative approaches for attacking the proteins encoded by mutant forms of RAS genes and to ultimately create effective, new therapies for RAS-related cancers.

K-ras p21 expression and activity in lung and lung tumors.

PubMed

Ramakrishna, G; Sithanandam, G; Cheng, R Y; Fornwald, L W; Smith, G T; Diwan, B A; Anderson, L M

2000-12-01

Although K-ras is mutated in many human and mouse lung adenocarcinomas, the function of K-ras p21 in lung is not known. We sought evidence for the prevalent hypothesis that K-ras p21 activates raf, which in turn passes the signal through the extracellular signal regulated kinases (Erks) to stimulate cell division, and that this pathway is upregulated when K-ras is mutated. Results from both mouse lung tumors and immortalized cultured E10 and C10 lung type II cells failed to substantiate this hypothesis. Lung tumors did not have more total K-ras p21 or K-ras p21 GTP than normal lung tissue, nor were high levels of these proteins found in tumors with mutant K-ras. Activated K-ras p21-GTP levels did not correlate with proliferating cell nuclear antigen. Special features of tumors with mutant K-ras included small size of carcinomas compared with carcinomas lacking this mutation, and correlation of proliferating cell nuclear antigen with raf-1. In nontransformed type II cells in culture, both total and activated K-ras p21 increased markedly at confluence but not after serum stimulation, whereas both Erk1/2 and the protein kinase Akt were rapidly activated by the serum treatment. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays of K-ras mRNA indicated an increase in confluent and especially in postconfluent cells. Together the findings indicate that normal K-ras p21 activity is associated with growth arrest of lung type II cells, and that the exact contribution of mutated K-ras p21 to tumor development remains to be discovered.

Effect of stressful life events on the onset and duration of recurrent aphthous stomatitis.

PubMed

Huling, Laura B; Baccaglini, Lorena; Choquette, Linda; Feinn, Richard S; Lalla, Rajesh V

2012-02-01

Recurrent aphthous stomatitis (RAS) is a common and painful oral mucosal disease. Possible etiologies include genetics, vitamin deficiencies, trauma, immune dysfunction, and stress. The goal of this study was to examine the relationship between the occurrence, type, and magnitude of stressful events and the onset and duration of RAS episodes. One hundred and sixty subjects with a history of RAS completed a weekly phone survey for up to 1 year, providing data on the occurrence of RAS episodes and details of any stressful events they experienced during the previous week. During RAS episodes, subjects also completed daily paper diaries that recorded incidence and duration of the RAS episode. Stressful events were quantified using the validated Recent Life Changes Questionnaire (RLCQ) and were classified as mental or physical stressors. Stressful life events were significantly associated with the onset of RAS episodes (P < 0.001), however, not with the duration of the RAS episodes. Experiencing a stressful life event increased the odds of an RAS episode by almost three times (OR = 2.72; 95% CI = 2.04-3.62). When controlled for each other, mental stressors had a larger effect (OR = 3.46, 95% CI = 2.54-4.72) than physical stressors (OR = 1.44; 95% CI = 1.04-1.99) on the occurrence of RAS episodes. RAS episodes did not occur more frequently or last longer with increasing stress severity. In patients with a history of RAS, stressful events may mediate changes involved in the initiation of new RAS episodes. Mental stressors are more strongly associated with RAS episodes than physical stressors. © 2011 John Wiley & Sons A/S.

Involvement of H-ras in erythroid differentiation of TF1 and human umbilical cord blood CD34 cells.

PubMed

Ge, Y; Li, Z H; Marshall, M S; Broxmeyer, H E; Lu, L

1998-06-01

To investigate the role of the ras gene in erythroid differentiation, a human erythroleukemic cell line, TF1, was transduced with a selectable retroviral vector carrying a mammalian wild type H-ras gene or a cytoplasmic dominant negative RAS1 gene. Transduction of TF1 cells with the wild type H-ras gene resulted in changes of cell types and up-regulation of erythroid-specific gene expression similar to that seen in differentiating erythroid cells. The number of red blood cell containing colonies derived from TF1 cells transduced with wild type H-ras cDNA was significantly increased and the cells in the colonies were more hemoglobinized as estimated by a deeper red color compared to those colony cells from mock or dominant negative RAS1 gene transduced TF1 cells, suggesting increased erythroid differentiation of TF1 cells after transduction of wild type H-ras in vitro. The mRNA levels of beta- and gamma-, but not alpha-, globin genes were significantly higher in H-ras transduced TF1 cells than those in TF1 cells transduced with mock or dominant negative RAS1 gene. Moreover, a 4kb pre-mRNA of the Erythropoietin receptor (EpoR) was highly expressed only in H-ras transduced TF1 cells. Additionally, human umbilical cord blood (CB) CD34 cells which are highly enriched for hematopoietic stem/progenitor cells were transduced with the same retroviral vectors to evaluate in normal primary cells the activities of H-ras in erythroid differentiation. Increased numbers of erythroid cell containing colonies (BFU-E and CFU-GEMM) were observed in CD34 cells transduced with the H-ras cDNA, compared to that from mock transduced cells. These data suggest a possible role for ras in erythroid differentiation.

siRNA blocking the RAS signalling pathway and inhibits the growth of oesophageal squamous cell carcinoma in nude mice.

PubMed

Wang, Xinjie; Zheng, Yuling; Fan, Qingxia; Zhang, Xudong; Shi, Yonggang

2014-12-01

The aim of this study was to study RAS-siRNA blocking RAS pathway and suppressing cell growth in human oesophageal squamous cell carcinoma in nude mice. The methods in this study was to construct RAS-siRNA expression vector, establish 40 oesophageal squamous cell carcinoma xenograft animal models and divided them into five groups: control group, siRNA control group, RAS-siRNA group, paclitaxel group and RAS-siRNA and paclitaxel group. We observed tumour growth in nude mice, studied histology by HE staining, tumour growth inhibition by TUNEL assay and detected the RAS, MAPK and cyclin D1 protein expression by immunohistochemistry and western blot. We have obtained the following results: (i) successfully established animal models; (ii) nude mice in each group after treatment inhibited tumour volume was significantly reduced compared with the control group (p < 0.05); (iii) compared with the control group, the number of apoptotic cells were significantly increased in the siRNA control group and the RAS-siRNA group, and the number of apoptosis cells in the paclitaxel and RAS-siRNA group is significantly most than the paclitaxel group and RAS-siRNA group (p < 0.05); and (iv) after treatment, RAS, MAPK and cyclin D1 expression in five groups was decreasing gradually. After adding paclitaxel, the protein expression in the paclitaxel and RAS-siRNA group was significantly lower than that of paclitaxel group, negative control and paclitaxel group (p < 0.05). We therefore conclude that RAS-siRNA can block the RAS signal transduction pathway, reduce the activity of tumour cells, arrest tumour cell cycle, promote apoptosis, inhibit cell proliferation and increase tumour cell sensitivity to chemotherapeutic drugs. Copyright © 2014 John Wiley & Sons, Ltd.

Mucosal and salivary microbiota associated with recurrent aphthous stomatitis.

PubMed

Kim, Yun-Ji; Choi, Yun Sik; Baek, Keum Jin; Yoon, Seok-Hwan; Park, Hee Kyung; Choi, Youngnim

2016-04-01

Recurrent aphthous stomatitis (RAS) is a common oral mucosal disorder of unclear etiopathogenesis. Although recent studies of the oral microbiota by high-throughput sequencing of 16S rRNA genes have suggested that imbalances in the oral microbiota may contribute to the etiopathogenesis of RAS, no specific bacterial species associated with RAS have been identified. The present study aimed to characterize the microbiota in the oral mucosa and saliva of RAS patients in comparison with control subjects at the species level. The bacterial communities of the oral mucosa and saliva from RAS patients with active lesions (RAS, n = 18 for mucosa and n = 8 for saliva) and control subjects (n = 18 for mucosa and n = 7 for saliva) were analyzed by pyrosequencing of the 16S rRNA genes. There were no significant differences in the alpha diversity between the controls and the RAS, but the mucosal microbiota of the RAS patients showed increased inter-subject variability. A comparison of the relative abundance of each taxon revealed decreases in the members of healthy core microbiota but increases of rare species in the mucosal and salivary microbiota of RAS patients. Particularly, decreased Streptococcus salivarius and increased Acinetobacter johnsonii in the mucosa were associated with RAS risk. A dysbiosis index, which was developed using the relative abundance of A. johnsonii and S. salivarius and the regression coefficients, correctly predicted 83 % of the total cases for the absence or presence of RAS. Interestingly, A. johnsonii substantially inhibited the proliferation of gingival epithelial cells and showed greater cytotoxicity against the gingival epithelial cells than S. salivarius. RAS is associated with dysbiosis of the mucosal and salivary microbiota, and two species associated with RAS have been identified. This knowledge may provide a diagnostic tool and new targets for therapeutics for RAS.

Ha-ras(val12) induces HSP70b transcription via the HSE/HSF1 system, but HSP70b expression is suppressed in Ha-ras(val12)-transformed cells.

PubMed

Stanhill, A; Levin, V; Hendel, A; Shachar, I; Kazanov, D; Arber, N; Kaminski, N; Engelberg, D

2006-03-09

Heat shock proteins (Hsps) are overexpressed in many tumors, but are downregulated in some tumors. To check for a direct effect of Ha-Ras(val12) on HSP70 transcription, we transiently expressed the oncoprotein in Rat1 fibroblasts and monitored its effect on HSP70b promoter-driven reporter gene. We show that expression of Ha-Ras(val12) induced this promoter. Promoter analysis via systematic deletions and point mutations revealed that Ha-Ras(val12) induces HSP70b transcription via heat shock elements (HSEs). Also, Ha-Ras(val12) induction of HSE-mediated transcription was dramatically reduced in HSF1-/- cells. Yet, residual effect of Ha-Ras(val12) that was still measured in HSF1-/- cells suggests that some of the Ha-Ras(val12) effect is Hsf1-independent. When HSF1-/- cells, stably expressing Ha-Ras(val12), were grown on soft agar only small colonies were formed suggesting a role for heat shock factor 1 (Hsf1) in Ha-Ras(val12)-mediated transformation. Although Ha-ras(Val12) seems to be an inducer of HSP70's expression, we found that in Ha-ras(Val12-)transformed fibroblasts expression of this gene is suppressed. This suppression is correlated with higher sensitivity of Ha-ras(val12)-transformed cells to heat shock. We suggest that Ha-ras(Val12) is involved in Hsf1 activation, thereby inducing the cellular protective response. Cells that repress this response are perhaps those that acquire the capability to further proliferate and become transformed clones.

The Significance of Ras Activity in Pancreatic Cancer Initiation.

PubMed

Logsdon, Craig D; Lu, Weiqin

2016-01-01

The genetic landscape of pancreatic cancer shows nearly ubiquitous mutations of K-RAS. However, oncogenic K-Ras(mt) alone is not sufficient to lead to pancreatic ductal adenocarcinoma (PDAC) in either human or in genetically modified adult mouse models. Many stimulants, such as high fat diet, CCK, LPS, PGE2 and others, have physiological effects at low concentrations that are mediated in part through modest increases in K-Ras activity. However, at high concentrations, they induce inflammation that, in the presence of oncogenic K-Ras expression, substantially accelerates PDAC formation. The mechanism involves increased activity of oncogenic K-Ras(mt). Unlike what has been proposed in the standard paradigm for the role of Ras in oncogenesis, oncogenic K-Ras(mt) is now known to not be constitutively active. Rather, it can be activated by standard mechanisms similar to wild-type K-Ras, but its activity is sustained for a prolonged period. Furthermore, if the level of K-Ras activity exceeds a threshold at which it begins to generate its own activators, then a feed-forward loop is formed between K-Ras activity and inflammation and pathological processes including oncogenesis are initiated. Oncogenic K-Ras(mt) activation, a key event in PDAC initiation and development, is subject to complex regulatory mechanisms. Reagents which inhibit inflammation, such as the Cox2 inhibitor celecoxib, block the feed-forward loop and prevent induction of PDAC in models with endogenous oncogenic K-Ras(mt). Increased understanding of the role of activating and inhibitory mechanisms on oncogenic K-Ras(mt) activity is of paramount importance for the development of preventive and therapeutic strategies to fight against this lethal disease.

Exploiting the bad eating habits of Ras-driven cancers.

PubMed

White, Eileen

2013-10-01

Oncogenic Ras promotes glucose fermentation and glutamine use to supply central carbon metabolism, but how and why have only emerged recently. Ras-mediated metabolic reprogramming generates building blocks for growth and promotes antioxidant defense. To fuel metabolic pathways, Ras scavenges extracellular proteins and lipids. To bolster metabolism and mitigate stress, Ras activates cellular self-cannibalization and recycling of proteins and organelles by autophagy. Targeting these distinct features of Ras-driven cancers provides novel approaches to cancer therapy.

Rabex-5 ubiquitin ligase activity restricts Ras signaling to establish pathway homeostasis in Drosophila.

PubMed

Yan, Hua; Jahanshahi, Maryam; Horvath, Elizabeth A; Liu, Hsiu-Yu; Pfleger, Cathie M

2010-08-10

The Ras signaling pathway allows cells to translate external cues into diverse biological responses. Depending on context and the threshold reached, Ras signaling can promote growth, proliferation, differentiation, or cell survival. Failure to maintain precise control of Ras can have adverse physiological consequences. Indeed, excess Ras signaling disrupts developmental patterning and causes developmental disorders [1, 2], and in mature tissues, it can lead to cancer [3-5]. We identify Rabex-5 as a new component of Ras signaling crucial for achieving proper pathway outputs in multiple contexts in vivo. We show that Drosophila Rabex-5 restricts Ras signaling to establish organism size, wing vein pattern, and eye versus antennal fate. Rabex-5 has both Rab5 guanine nucleotide exchange factor (GEF) activity that regulates endocytic trafficking [6] and ubiquitin ligase activity [7, 8]. Surprisingly, overexpression studies demonstrate that Rabex-5 ubiquitin ligase activity, not its Rab5 GEF activity, is required to restrict wing vein specification and to suppress the eye phenotypes of oncogenic Ras expression. Furthermore, genetic interaction experiments indicate that Rabex-5 acts at the step of Ras, and tissue culture studies show that Rabex-5 promotes Ras ubiquitination. Together, these findings reveal a new mechanism for attenuating Ras signaling in vivo and suggest an important role for Rabex-5-mediated Ras ubiquitination in pathway homeostasis. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Molecular pathways: targeting RAC-p21-activated serine-threonine kinase signaling in RAS-driven cancers.

PubMed

Baker, Nicole M; Yee Chow, Hoi; Chernoff, Jonathan; Der, Channing J

2014-09-15

Cancers driven by oncogenic Ras proteins encompass some of the most deadly human cancer types, and there is a pressing need to develop therapies for these diseases. Although recent studies suggest that mutant Ras proteins may yet be druggable, the most promising and advanced efforts involve inhibitors of Ras effector signaling. Most efforts to target Ras signaling have been aimed at the ERK mitogen-activated protein kinase and the phosphoinositide 3-kinase signaling networks. However, to date, no inhibitors of these Ras effector pathways have been effective against RAS-mutant cancers. This ineffectiveness is due, in part, to the involvement of additional effectors in Ras-dependent cancer growth, such as the Rac small GTPase and the p21-activated serine-threonine kinases (PAK). PAK proteins are involved in many survival, cell motility, and proliferative pathways in the cell and may present a viable new target in Ras-driven cancers. In this review, we address the role and therapeutic potential of Rac and group I PAK proteins in driving mutant Ras cancers. ©2014 American Association for Cancer Research.

Caveolae regulate the nanoscale organization of the plasma membrane to remotely control Ras signaling

PubMed Central

Ariotti, Nicholas; Fernández-Rojo, Manuel A.; Zhou, Yong; Hill, Michelle M.; Rodkey, Travis L.; Inder, Kerry L.; Tanner, Lukas B.; Wenk, Markus R.

2014-01-01

The molecular mechanisms whereby caveolae exert control over cellular signaling have to date remained elusive. We have therefore explored the role caveolae play in modulating Ras signaling. Lipidomic and gene array analyses revealed that caveolin-1 (CAV1) deficiency results in altered cellular lipid composition, and plasma membrane (PM) phosphatidylserine distribution. These changes correlated with increased K-Ras expression and extensive isoform-specific perturbation of Ras spatial organization: in CAV1-deficient cells K-RasG12V nanoclustering and MAPK activation were enhanced, whereas GTP-dependent lateral segregation of H-Ras was abolished resulting in compromised signal output from H-RasG12V nanoclusters. These changes in Ras nanoclustering were phenocopied by the down-regulation of Cavin1, another crucial caveolar structural component, and by acute loss of caveolae in response to increased osmotic pressure. Thus, we postulate that caveolae remotely regulate Ras nanoclustering and signal transduction by controlling PM organization. Similarly, caveolae transduce mechanical stress into PM lipid alterations that, in turn, modulate Ras PM organization. PMID:24567358

Nitrative and oxidative DNA damage caused by K-ras mutation in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohnishi, Shiho; Saito, Hiromitsu; Suzuki, Noboru

2011-09-23

Highlights: {yields} Mutated K-ras in transgenic mice caused nitrative DNA damage, 8-nitroguanine. {yields} The mutagenic 8-nitroguanine seemed to be generated by iNOS via Ras-MAPK signal. {yields} Mutated K-ras produces additional mutagenic lesions, as a new oncogenic role. -- Abstract: Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-{kappa}B, IKK, MAPK, MEK,more » and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.« less

A New Strategy to Control and Eradicate “Undruggable” Oncogenic K-RAS-Driven Pancreatic Cancer: Molecular Insights and Core Principles Learned from Developmental and Evolutionary Biology

PubMed Central

Lee, Michael P.; Lee, Caroline Dasom; Lafever, Alex C.; Svyatova, Elizaveta; Kanda, Kevin; Collier, Amber L.; Siewertsz van Reesema, Lauren L.; Tang-Tan, Angela M.; Zheleva, Vasilena; Bwayi, Monicah N.; Bian, Minglei; Schmidt, Rebecca L.; Petersen, Gloria M.

2018-01-01

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely “undruggable”. Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future. PMID:29757973

Ras-related tumorigenesis is suppressed by BNIP3-mediated autophagy through inhibition of cell proliferation.

PubMed

Wu, Shan-Ying; Lan, Sheng-Hui; Cheng, Da-En; Chen, Wei-Kai; Shen, Cheng-Huang; Lee, Ying-Ray; Zuchini, Roberto; Liu, Hsiao-Sheng

2011-12-01

Autophagy plays diverse roles in Ras-related tumorigenesis. H-ras(val12) induces autophagy through multiple signaling pathways including Raf-1/ERK pathway, and various ERK downstream molecules of autophagy have been reported. In this study, Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) is identified as a downstream transducer of the Ras/Raf/ERK signaling pathway to induce autophagy. BNIP3 was upregulated by H-ras(val12) at the transcriptional level to compete with Beclin 1 for binding with Bcl-2. H-ras(val12)-induced autophagy suppresses cell proliferation demonstrated both in vitro and in vivo by expression of ectopic BNIP3, Atg5, or interference RNA of BNIP3 (siBNIP3) and Atg5 (shAtg5) using mouse NIH3T3 and embryo fibroblast cells. H-ras(val12) induces different autophagic responses depending on the duration of Ras overexpression. After a short time (48 hours) of Ras overexpression, autophagy inhibits cell proliferation. In contrast, a longer time (2 weeks) of Ras overexpression, cell proliferation was enhanced by autophagy. Furthermore, overexpression of mutant Ras, BNIP3, and LC3-II was detected in bladder cancer T24 cells and the tumor parts of 75% of bladder cancer specimens indicating a positive correlation between autophagy and tumorigenesis. Taken together, our mouse model demonstrates a balance between BNIP3-mediated autophagy and H-ras(val12)-induced tumor formation and reveals that H-ras(val12) induces autophagy in a BNIP3-dependent manner, and the threshold of autophagy plays a decisive role in H-ras(val12)-induced tumorigenesis. Our findings combined with others' reports suggest a new therapeutic strategy against Ras-related tumorigenesis by negative or positive regulation of autophagic activity, which is determined by the level of autophagy and tumor progression stages.

Inhibition of Fas (CD95) expression and Fas-mediated apoptosis by oncogenic Ras.

PubMed

Fenton, R G; Hixon, J A; Wright, P W; Brooks, A D; Sayers, T J

1998-08-01

The ras oncogene plays an important role in the multistep progression to cancer by activation of signal transduction pathways that contribute to aberrant growth regulation. Although many of these effects are cell autonomous, the ras oncogene also regulates the expression of genes that alter host/tumor interactions. We now extend the mechanisms through which ras promotes tumor survival by demonstrating that oncogenic Ras inhibits expression of the fas gene and renders Ras-transformed cells resistant to Fas-induced apoptosis. A panel of Ras-transformed clones exhibited a marked inhibition in fas mRNA and Fas cell surface expression as compared with untransformed parental cell lines. Fas expression was induced by culture in the presence of IFN-gamma + tumor necrosis factor alpha; however, the maximal level attained in Ras transformants was approximately 10-fold below the level of untransformed cells. Whereas untransformed cells were sensitive to apoptotic death induced by cross-linking surface Fas (especially after cytokine treatment), Ras-transformed cells were very resistant to Fas-induced death even under the most stringent assay conditions. To demonstrate that this resistance was mediated by oncogenic Ras and not secondary genetic events, pools of Ras-transformed cells were generated using a highly efficient retroviral transduction technique. Transformed pools were assayed 6 days after infection and demonstrated a marked decrease in fas gene expression and Fas-mediated apoptosis. Oncogenic Ras did not promote general resistance to apoptosis, because ectopic expression of a fas cDNA in Ras-transformed cells restored sensitivity to Fas-induced apoptosis. These data indicate that oncogenic Ras inhibits basal levels of expression of the fas gene, and although cytokine signal transduction pathways are functional in these cells, the level of surface Fas expression remains below the threshold required for induction of apoptosis. These data identify a mechanism by which Ras-transformed cells may escape from host-mediated immune destruction.

Ovarian expression of cellular Ki-ras p21 varies with physiological status.

PubMed Central

Palejwala, S; Goldsmith, L T

1992-01-01

To elucidate the potential role of the ras protooncogene proteins in a specific tissue, the present study determined the levels of individual c-ras-encoded p21 proteins in the rat ovary during various stages of physiological function. p21 protein was extracted from ovaries taken from immature normal female rats, mature nonpregnant animals in the metestrus stage of the estrus cycle, rats at various stages of pregnancy, and actively lactating animals. Levels of individual p21s were evaluated by immunoblot analysis with specific antibodies to the p21 proteins encoded by the Kirsten, Harvey, and neuroblastoma c-ras protooncogenes, c-Ki-ras, c-Ha-ras, and N-ras. Results showed that c-Ki-ras p21 is at its lowest level in the immature ovary and increases with development of the corpora lutea to its highest levels at day 16 of pregnancy, after which levels decline and then rise again during lactation. This pattern, which mimics that of circulating progesterone levels, suggests that ovarian c-Ki-ras p21 levels are regulated and that c-Ki-ras p21 plays a role in the differentiated function of the rat ovary, likely the luteal compartment. In contrast, levels of c-N-ras p21 did not appear to vary with changes in the physiological function of the ovary but appeared to be constitutive. A preferential role for the c-Ki-ras p21 may be due to the documented unique differences in the structure of the carboxyl terminus of this particular c-ras p21. Images PMID:1570348

Mitochondrial clearance by the STK38 kinase supports oncogenic Ras-induced cell transformation

PubMed Central

Bettoun, Audrey; Surdez, Didier; Vallerand, David; Gundogdu, Ramazan; Sharif, Ahmad A.D.; Gomez, Marta; Cascone, Ilaria; Meunier, Brigitte; White, Michael A.; Codogno, Patrice; Parrini, Maria Carla; Camonis, Jacques H.; Hergovich, Alexander

2016-01-01

Oncogenic Ras signalling occurs frequently in many human cancers. However, no effective targeted therapies are currently available to treat patients suffering from Ras-driven tumours. Therefore, it is imperative to identify downstream effectors of Ras signalling that potentially represent promising new therapeutic options. Particularly, considering that autophagy inhibition can impair the survival of Ras-transformed cells in tissue culture and mouse models, an understanding of factors regulating the balance between autophagy and apoptosis in Ras-transformed human cells is needed. Here, we report critical roles of the STK38 protein kinase in oncogenic Ras transformation. STK38 knockdown impaired anoikis resistance, anchorage-independent soft agar growth, and in vivo xenograft growth of Ras-transformed human cells. Mechanistically, STK38 supports Ras-driven transformation through promoting detachment-induced autophagy. Even more importantly, upon cell detachment STK38 is required to sustain the removal of damaged mitochondria by mitophagy, a selective autophagic process, to prevent excessive mitochondrial reactive oxygen species production that can negatively affect cancer cell survival. Significantly, knockdown of PINK1 or Parkin, two positive regulators of mitophagy, also impaired anoikis resistance and anchorage-independent growth of Ras-transformed human cells, while knockdown of USP30, a negative regulator of PINK1/Parkin-mediated mitophagy, restored anchorage-independent growth of STK38-depleted Ras-transformed human cells. Therefore, our findings collectively reveal novel molecular players that determine whether Ras-transformed human cells die or survive upon cell detachment, which potentially could be exploited for the development of novel strategies to target Ras-transformed cells. PMID:27283898

New KRAS Antibodies Available | Office of Cancer Clinical Proteomics Research

Cancer.gov

Researchers estimate that approximately 30% of all human cancers are driven by RAS oncogenes. Mutated RAS genes are responsible for making RAS proteins that support cancer development. While anti-RAS therapies may have potential clinical benefit, researchers yet do not understand how the four RAS protein isoforms, KRAS4A, KRAS4B, HRAS, and NRAS, drive malignant phenotypes. Well-characterized and defined reagents like antibodies are central to reproducibility in biomedical research and necessary for future RAS studies.

Molecular Dynamics Simulations and Dynamic Network Analysis Reveal the Allosteric Unbinding of Monobody to H-Ras Triggered by R135K Mutation.

PubMed

Ni, Duan; Song, Kun; Zhang, Jian; Lu, Shaoyong

2017-10-26

Ras proteins, as small GTPases, mediate cell proliferation, survival and differentiation. Ras mutations have been associated with a broad spectrum of human cancers and thus targeting Ras represents a potential way forward for cancer therapy. A recently reported monobody NS1 allosterically disrupts the Ras-mediated signaling pathway, but its efficacy is reduced by R135K mutation in H-Ras. However, the detailed mechanism is unresolved. Here, using molecular dynamics (MD) simulations and dynamic network analysis, we explored the molecular mechanism for the unbinding of NS1 to H-Ras and shed light on the underlying allosteric network in H-Ras. MD simulations revealed that the overall structures of the two complexes did not change significantly, but the H-Ras-NS1 interface underwent significant conformational alteration in the mutant Binding free energy analysis showed that NS1 binding was unfavored after R135K mutation, which resulted in the unfavorable binding of NS1. Furthermore, the critical residues on H-Ras responsible for the loss of binding of NS1 were identified. Importantly, the allosteric networks for these important residues were revealed, which yielded a novel insight into the allosteric regulatory mechanism of H-Ras.

An orthosteric inhibitor of the RAS-SOS interaction.

PubMed

Nickerson, Seth; Joy, Stephen T; Arora, Paramjit S; Bar-Sagi, Dafna

2013-01-01

Rat sarcoma (RAS) proteins are signaling nodes that transduce extracellular cues into precise alterations in cellular physiology by engaging effector pathways. RAS signaling thus regulates diverse cell processes including proliferation, migration, differentiation, and survival. Owing to this central role in governing mitogenic signals, RAS pathway components are often dysregulated in human diseases. Targeted therapy of RAS pathways has generally not been successful, largely because of the robust biochemistry of the targets and their multifaceted network of molecular regulators. The rate-limiting step of RAS activation is Son of Sevenless (SOS)-mediated nucleotide exchange involving a single evolutionarily conserved catalytic helix from SOS. Structure function data of this mechanism provided a strong platform to design an SOS-derived, helically constrained peptide mimic as an inhibitor of the RAS-SOS interaction. In this chapter, we review RAS-SOS signaling dynamics and present evidence supporting the novel paradigm of inhibiting their interaction as a therapeutic strategy. We then describe a method of generating helically constrained peptide mimics of protein surfaces, which we have employed to inhibit the RAS-SOS active site interaction. The biochemical and functional properties of this SOS mimic support the premise that inhibition of RAS-nucleotide exchange can effectively block RAS activation and downstream signaling. © 2013 Elsevier Inc. All rights reserved.

R-Ras Contributes to LTP and Contextual Discrimination

PubMed Central

Darcy, Michael J.; Jin, Shan-Xue; Feig, Larry A.

2014-01-01

The ability to discriminate between closely related contexts is a specific form of hippocampal-dependent learning that may be impaired in certain neurodegenerative disorders such as Alzheimer's and Down Syndrome. However, signaling pathways regulating this form of learning are poorly understood. Previous studies have shown that the calcium-dependent exchange factor Ras-GRF1, an activator of Rac, Ras and R-Ras GTPases, is important for this form of learning and memory. Moreover, the ability to discriminate contexts was linked to the ability of Ras-GRF1 to promote high-frequency stimulation (HFS)-LTP via the activation of p38 Map kinase. Here, we show that R-Ras is involved in this form of learning by using virally-delivered miRNAs targeting R-Ras into the CA1 region of dorsal hippocampus and observing impaired contextual discrimination. Like the loss of GRF1, knockdown of R-Ras in the CA1 also impairs the induction of HFS-LTP and p38 Map kinase. Nevertheless, experiments indicate that this involvement of R-Ras in HFS-LTP that is required for contextual discrimination is independent of Ras-GRF1. Thus, R-Ras is a novel regulator of a form of hippocampal-dependent LTP as well as learning and memory that is affected in certain forms of neurodegenerative diseases. PMID:25043327

K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya

Amino-acid mutations of Gly{sup 12} (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH{sub 2}) as a consensus sequence. KRpep-2 showedmore » more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. K{sub D} and IC{sub 50} values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH{sub 2}) that inhibited enzyme activity of K-Ras(G12D) with IC{sub 50} = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs. - Highlights: • The first K-Ras(G12D)-selective inhibitory peptides were generated. • These peptides showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D) in compared to wild type K-Ras. • The peptide KRpep-2d suppressed downstream signal of K-Ras(G12D) and cell proliferations of cancer cell line A427.« less

Subcellular Distribution of S-Nitrosylated H-Ras in Differentiated and Undifferentiated PC12 Cells during Hypoxia.

PubMed

Barbakadze, Tamar; Goloshvili, Galina; Narmania, Nana; Zhuravliova, Elene; Mikeladze, David

2017-10-01

Hypoxia or exposure to excessive reactive oxygen or nitrogen species could induce S-nitrosylation of various target proteins, including GTPases of the Ras-superfamily. Under hypoxic conditions, the Ras-protein is translocated to the cytosol and interacts with the Golgi complex, endoplasmic reticulum, mitochondria. The mobility/translocation of Ras depend on the cells oxidative status. However, the importance of relocated Snitrosylated- H-Ras (NO-H-Ras) in proliferation/differentiation processes is not completely understood. We have determined the content of soluble- and membrane-bound-NO-HRas in differentiated (D) and undifferentiated (ND) rat pheochromocytoma (PC12) cells under hypoxic and normoxic conditions. In our experimental study, we analyzed NO-H-Ras levels under hypoxic/normoxic conditions in membrane and soluble fractions of ND and D PC12 cells with/without nitric oxide donor, sodium nitroprusside (SNP) treatment. Cells were analyzed by the S-nitrosylated kit, immunoprecipitation, and Western blot. We assessed the action of NO-H-Ras on oxidative metabolism of isolated mitochondria by determining mitochondrial hydrogen peroxide generation via the scopoletin oxidation method and ATPproduction as estimated by the luminometric method. Hypoxia did not influence nitrosylation of soluble H-Ras in ND PC12 cells. Under hypoxic conditions, the nitrosylation of soluble-H-Ras greatly decreased in D PC12 cells. SNP didn't change the levels of nitrosylation of soluble-H-Ras, in either hypoxic or normoxic conditions. On the other hand, hypoxia, per se, did not affect the nitrosylation of membrane-bound-H-Ras in D and ND PC12 cells. SNP-dependent nitrosylation of membrane-bound-H-Ras greatly increased in D PC12 cells. Both unmodified normal and mutated H-Ras enhanced the mitochondrial synthesis of ATP, whereas the stimulatory effects on ATP synthesis were eliminated after S-nitrosylation of H-Ras. According to the results, it may be proposed that hypoxia can decrease S-nitrosylation of soluble-H-Ras in D PC12 cells and abolish the inhibitory effect of NO-HRas in mitochondrial oxidative metabolism. Copyright© by Royan Institute. All rights reserved.

Anti-tumour activity in RAS-driven tumours by blocking AKT and MEK

PubMed Central

Tolcher, Anthony W.; Khan, Khurum; Ong, Michael; Banerji, Udai; Papadimitrakopoulou, Vassiliki; Gandara, David R.; Patnaik, Amita; Baird, Richard D.; Olmos, David; Garrett, Christopher R.; Skolnik, Jeffrey M.; Rubin, Eric H.; Smith, Paul D.; Huang, Pearl; Learoyd, Maria; Shannon, Keith A.; Morosky, Anne; Tetteh, Ernestina; Jou, Ying-Ming; Papadopoulos, Kyriakos P.; Moreno, Victor; Kaiser, Brianne; Yap, Timothy A.; Yan, Li; de Bono, Johann S.

2014-01-01

Purpose KRAS is the most commonly mutated oncogene in human tumours. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies. Experimental Design We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumours. Recommended dosing schedules were defined as MK-2206 135 mg weekly and selumetinib 100 mg once-daily. Results Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhoea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug-drug interactions. Clinical anti-tumour activity included RECIST 1.0-confirmed partial responses in non-small cell lung cancer and low-grade ovarian carcinoma. Conclusion Responses in KRAS-mutant cancers were generally durable. Clinical co-targeting of MEK and AKT signalling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). PMID:25516890

Antitumor activity in RAS-driven tumors by blocking AKT and MEK.

PubMed

Tolcher, Anthony W; Khan, Khurum; Ong, Michael; Banerji, Udai; Papadimitrakopoulou, Vassiliki; Gandara, David R; Patnaik, Amita; Baird, Richard D; Olmos, David; Garrett, Christopher R; Skolnik, Jeffrey M; Rubin, Eric H; Smith, Paul D; Huang, Pearl; Learoyd, Maria; Shannon, Keith A; Morosky, Anne; Tetteh, Ernestina; Jou, Ying-Ming; Papadopoulos, Kyriakos P; Moreno, Victor; Kaiser, Brianne; Yap, Timothy A; Yan, Li; de Bono, Johann S

2015-02-15

KRAS is the most commonly mutated oncogene in human tumors. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies. We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumors. Recommended dosing schedules were defined as MK-2206 at 135 mg weekly and selumetinib at 100 mg once daily. Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug-drug interactions. Clinical antitumor activity included RECIST 1.0-confirmed partial responses in non-small cell lung cancer and low-grade ovarian carcinoma. Responses in KRAS-mutant cancers were generally durable. Clinical cotargeting of MEK and AKT signaling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). ©2014 American Association for Cancer Research.

K-Ras protein as a drug target.

PubMed

McCormick, Frank

2016-03-01

K-Ras proteins are major drivers of human cancers, playing a direct causal role in about one million cancer cases/year. In cancers driven by mutant K-Ras, the protein is locked in the active, GTP-bound state constitutively, through a defect in the off-switch mechanism. As such, the mutant protein resembles the normal K-Ras protein from a structural perspective, making therapeutic attack extremely challenging. K-Ras is a member of a large family of related proteins, which share very similar GDP/GTP-binding domains, making specific therapies more difficult. Furthermore, Ras proteins lack pockets to which small molecules can bind with high affinity, with a few interesting exceptions. However, new insights into the structure and function of K-Ras proteins reveal opportunities for intervention that were not appreciated many years ago, when efforts were launched to develop K-Ras therapies. Furthermore, K-Ras undergoes post-translational modification and interactions with cellular signaling proteins that present additional therapeutic opportunities, such as specific binding to calmodulin and regulation of non-canonical Wnt signaling.

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erickson, Keesha E.; Rukhlenko, Oleksii S.; Posner, Richard G.

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisitionmore » of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers.« less

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling

DOE PAGES

Erickson, Keesha E.; Rukhlenko, Oleksii S.; Posner, Richard G.; ...

2018-03-05

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisitionmore » of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers.« less

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling.

PubMed

Erickson, Keesha E; Rukhlenko, Oleksii S; Posner, Richard G; Hlavacek, William S; Kholodenko, Boris N

2018-03-05

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisition of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers. Copyright © 2018 Elsevier Ltd. All rights reserved.

SIRT2 and lysine fatty acylation regulate the transforming activity of K-Ras4a

PubMed Central

Wisner, Stephanie A; Chen, Xiao; Spiegelman, Nicole A; Linder, Maurine E

2017-01-01

Ras proteins play vital roles in numerous biological processes and Ras mutations are found in many human tumors. Understanding how Ras proteins are regulated is important for elucidating cell signaling pathways and identifying new targets for treating human diseases. Here we report that one of the K-Ras splice variants, K-Ras4a, is subject to lysine fatty acylation, a previously under-studied protein post-translational modification. Sirtuin 2 (SIRT2), one of the mammalian nicotinamide adenine dinucleotide (NAD)-dependent lysine deacylases, catalyzes the removal of fatty acylation from K-Ras4a. We further demonstrate that SIRT2-mediated lysine defatty-acylation promotes endomembrane localization of K-Ras4a, enhances its interaction with A-Raf, and thus promotes cellular transformation. Our study identifies lysine fatty acylation as a previously unknown regulatory mechanism for the Ras family of GTPases that is distinct from cysteine fatty acylation. These findings highlight the biological significance of lysine fatty acylation and sirtuin-catalyzed protein lysine defatty-acylation. PMID:29239724

Ras signaling in aging and metabolic regulation.

PubMed

Slack, Cathy

2017-12-07

Aberrant signal transduction downstream of the Ras GTPase has a well-established role in tumorigenesis. Mutations that result in hyperactivation of Ras are responsible for a third of all human cancers. Hence, small molecule inhibitors of the Ras signal transduction cascade have been under intense focus as potential cancer treatments. In both invertebrate and mammalian models, emerging evidence has also implicated components of the Ras signaling pathway in aging and metabolic regulation. Here, I review the current evidence for Ras signaling in these newly discovered roles highlighting the interactions between the Ras pathway and other longevity assurance mechanisms. Defining the role of Ras signaling in maintaining age-related health may have important implications for the development of interventions that could not only increase lifespan but also delay the onset and/or progression of age-related functional decline.

Ras inhibitors display an anti-metastatic effect by downregulation of lysyl oxidase through inhibition of the Ras-PI3K-Akt-HIF-1α pathway.

PubMed

Yoshikawa, Yoko; Takano, Osamu; Kato, Ichiro; Takahashi, Yoshihisa; Shima, Fumi; Kataoka, Tohru

2017-12-01

Metastasis stands as the major obstacle for the survival from cancers. Nonetheless most existing anti-cancer drugs inhibit only cell proliferation, and discovery of agents having both anti-proliferative and anti-metastatic properties would be more beneficial. We previously reported the discovery of small-molecule Ras inhibitors, represented by Kobe0065, that displayed anti-proliferative activity on xenografts of human colorectal cancer (CRC) cell line SW480 carrying the K-ras G12V gene. Here we show that treatment of cancer cells carrying the activated ras genes with Kobe0065 or a siRNA targeting Ras downregulates the expression of lysyl oxidase (LOX), which has been implicated in metastasis. LOX expression is enhanced by co-expression of Ras G12V through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and concomitant accumulation of hypoxia-inducible factor (HIF)-1α. Furthermore, Kobe0065 effectively inhibits not only migration and invasion of cancer cells carrying the activated ras genes but also lung metastasis of human CRC cell line SW620 carrying the K-ras G12V gene. Collectively, these results indicate that Kobe0065 prevents metastasis through inhibition of the Ras-PI3K-Akt-HIF-1α-LOX signaling and suggest that Ras inhibitors in general might exhibit both anti-proliferative and anti-metastatic properties toward cancer cells carrying the activated ras genes. Copyright © 2017 Elsevier B.V. All rights reserved.

Exploring environmental causes of altered ras effects: fragmentation plus integration?

PubMed

Porta, Miquel; Ayude, Daniel; Alguacil, Juan; Jariod, Manuel

2003-02-01

Mutations in ras genes are the most common abnormality of oncogenes in human cancer and a major example of activation by point mutation. Experimental and epidemiological studies support the notion that Ki-ras activation and expression may be chemically related. We discuss the potential role of several environmental compounds in the induction or promotion of ras mutations in humans, with a focus on exocrine pancreatic cancer, the human tumor with the highest prevalence at diagnosis of Ki-ras mutations. Organochlorine compounds, organic solvents, and coffee compounds may play an indirect role in causing Ki-ras mutations, rather than as direct inducers of the mutations. Although for some organochlorine compounds the induction of point mutations in ras oncogenes cannot be excluded, it seems more likely that the effects of these compounds are mediated through nongenomic or indirectly genotoxic mechanisms of action. Organic solvents also may act via enzymatic induction of ras mutagens or by providing a proliferation advantage to ras-mutated cell clones. In exocrine pancreatic cancer, caffeine, other coffee compounds, or other factors with which coffee drinking is associated could modulate Ki-ras activation by interfering with DNA repair, cell-cycle checkpoints, and apoptosis. Asbestos, cigarette smoking, and some dietary factors also may be involved in the initiation or the promotion of Ki-ras mutations in lung and colon cancers. Further development of the mechanistic scenarios proposed here could contribute to a meaningful integration of biological, clinical, and environmental knowledge on the causes of altered ras effects. Copyright 2003 Wiley-Liss, Inc.

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence*

PubMed Central

Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L.; Embrey, Kevin J.; Golovanov, Alexander P.

2016-01-01

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. PMID:26565026

Activation of RAS family genes in urothelial carcinoma.

PubMed

Boulalas, I; Zaravinos, A; Karyotis, I; Delakas, D; Spandidos, D A

2009-05-01

Bladder cancer is the fifth most common malignancy in men in Western society. We determined RAS codon 12 and 13 point mutations and evaluated mRNA expression levels in transitional cell carcinoma cases. Samples from 30 human bladder cancers and 30 normal tissues were analyzed by polymerase chain reaction/restriction fragment length polymorphism and direct sequencing to determine the occurrence of mutations in codons 12 and 13 of RAS family genes. Moreover, we used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profile of RAS genes in bladder cancer specimens compared to that in adjacent normal tissues. Overall H-RAS mutations in codon 12 were observed in 9 tumor samples (30%). Two of the 9 patients (22%) had invasive bladder cancer and 7 (77%) had noninvasive bladder cancer. One H-RAS mutation (11%) was homozygous and the remaining 89% were heterozygous. All samples were WT for K and N-RAS oncogenes. Moreover, 23 of 30 samples (77%) showed over expression in at least 1 RAS family gene compared to adjacent normal tissue. K and N-RAS had the highest levels of over expression in bladder cancer specimens (50%), whereas 27% of transitional cell carcinomas demonstrated H-RAS over expression relative to paired normal tissues. Our results underline the importance of H-RAS activation in human bladder cancer by codon 12 mutations. Moreover, they provide evidence that increased expression of all 3 RAS genes is a common event in bladder cancer that is associated with disease development.

Exploring the interactions of the RAS family in the human protein network and their potential implications in RAS-directed therapies

PubMed Central

Bueno, Anibal; Morilla, Ian; Diez, Diego; Moya-Garcia, Aurelio A.; Lozano, José; Ranea, Juan A.G.

2016-01-01

RAS proteins are the founding members of the RAS superfamily of GTPases. They are involved in key signaling pathways regulating essential cellular functions such as cell growth and differentiation. As a result, their deregulation by inactivating mutations often results in aberrant cell proliferation and cancer. With the exception of the relatively well-known KRAS, HRAS and NRAS proteins, little is known about how the interactions of the other RAS human paralogs affect cancer evolution and response to treatment. In this study we performed a comprehensive analysis of the relationship between the phylogeny of RAS proteins and their location in the protein interaction network. This analysis was integrated with the structural analysis of conserved positions in available 3D structures of RAS complexes. Our results show that many RAS proteins with divergent sequences are found close together in the human interactome. We found specific conserved amino acid positions in this group that map to the binding sites of RAS with many of their signaling effectors, suggesting that these pairs could share interacting partners. These results underscore the potential relevance of cross-talking in the RAS signaling network, which should be taken into account when considering the inhibitory activity of drugs targeting specific RAS oncoproteins. This study broadens our understanding of the human RAS signaling network and stresses the importance of considering its potential cross-talk in future therapies. PMID:27713118

The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

PubMed Central

Yamauchi, N; Kiessling, A A; Cooper, G M

1994-01-01

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos. Images PMID:7935384

Vibrational Stark effect spectroscopy reveals complementary electrostatic fields created by protein-protein binding at the interface of Ras and Ral.

PubMed

Walker, David M; Hayes, Ellen C; Webb, Lauren J

2013-08-07

Electrostatic fields at the interface of the GTPase H-Ras (Ras) docked with the Ras binding domain of the protein Ral guanine nucleoside dissociation stimulator (Ral) were measured with vibrational Stark effect (VSE) spectroscopy. Nine residues on the surface of Ras that participate in the protein-protein interface were systematically mutated to cysteine and subsequently converted to cyanocysteine in order to introduce a nitrile VSE probe into the protein-protein interface. The absorption energy of the nitrile was measured both on the surface of Ras in its monomeric state, then after incubation with the Ras binding domain of Ral to form the docked complex. Boltzmann-weighted structural snapshots of the nitrile-labeled Ras protein were generated both in monomeric and docked configurations from molecular dynamics simulations using enhanced sampling of the cyanocysteine side chain's χ2 dihedral angle. These snapshots were used to determine that on average, most of the nitrile probes were aligned along the Ras surface, parallel to the Ras-Ral interface. The average solvent-accessible surface areas (SASA) of the cyanocysteine side chain were found to be <60 Å(2) for all measured residues, and was not significantly different whether the nitrile was on the surface of the Ras monomer or immersed in the docked complex. Changes in the absorption energy of the nitrile probe at nine positions along the Ras-Ral interface were compared to results of a previous study examining this interface with Ral-based probes, and found a pattern of low electrostatic field in the core of the interface surrounded by a ring of high electrostatic field around the perimeter of the interface. These data are used to rationalize several puzzling features of the Ras-Ral interface.

Cost-effectiveness of RAS screening before monoclonal antibodies therapy in metastatic colorectal cancer based on FIRE3 Study

PubMed Central

Wen, Feng; Yang, Yu; Zhang, Pengfei; Zhang, Jian; Zhou, Jing; Tang, Ruilei; Chen, Hongdou; Zheng, Hanrui; Fu, Ping; Li, Qiu

2015-01-01

The surprising results published by FIRE-3 revealed that the overall survival (OS) of RAS wild-type metastatic colorectal cancer (mCRC) patients treated with Cetuximab(Cmab) and FOLFIRI combination was prolonged to 33.1 months. The substantial increase in testing and treatment costs, however, impose a considerable health burden on patients and society. Hence the study was aimed to assess the cost-effectiveness of RAS screening before monoclonal antibodies (mAbs) therapy based on FIRE-3 study. Four groups were analyzed: group 1, patients with KRAS testing treated with Cmab and FOLFIRI; group 2, patients with RAS testing treated with Cmab and FOLFIRI; group 3, patients with KRAS testing treated with bevacizumab(Bmab) and FOLFIRI; group 4, patients with RAS testing treated with Bmab and FOLFIRI. A Markov model comprising 3 health states (progression-free survival, progressive disease and death) was built. The costs were calculated from a Chinese payer perspective, and survival was reported in quality-adjusted life-months (QALMs). Average total lifetime costs ranged from $104,682.44 (RAS-Bmab) to $136,867.44 (RAS-Cmab), while the survival gained varied from 16.88 QALMs in RAS-Bmab to 21.85 QALMs in RAS-Cmab. The cost per QALM was $6,263.86 for RAS-Cmab, $6,145.84 for KRAS-Bmab, $6,201.57 for RAS-Bmab and $6,960.70 for KRAS-Cmab respectively. The KRAS-Cmab strategy was dominated by the other 3 groups. The first-treatment cost of RAS-Cmab was the most influential one to the model. In all, the RAS screening prior to Cmab treatment in mCRC seems to be a cost-effective strategy in the time of monoclonal antibodies (mAbs) therapy with the most gained QALMs. PMID:26418570

The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis.

PubMed

Tsujita, Maristela; Batista, Wagner L; Ogata, Fernando T; Stern, Arnold; Monteiro, Hugo P; Arai, Roberto J

2008-05-16

p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras(C118S)) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG.

The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsujita, Maristela; Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, SP; Batista, Wagner L.

2008-05-16

p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras{sup C118S}) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinasesmore » by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG.« less

Signaling added response-independent reinforcement to assess Pavlovian processes in resistance to change and relapse.

PubMed

Podlesnik, Christopher A; Fleet, James D

2014-09-01

Behavioral momentum theory asserts Pavlovian stimulus-reinforcer relations govern the persistence of operant behavior. Specifically, resistance to conditions of disruption (e.g., extinction, satiation) reflects the relation between discriminative stimuli and the prevailing reinforcement conditions. The present study assessed whether Pavlovian stimulus-reinforcer relations govern resistance to disruption in pigeons by arranging both response-dependent and -independent food reinforcers in two components of a multiple schedule. In one component, discrete-stimulus changes preceded response-independent reinforcers, paralleling methods that reduce Pavlovian conditioned responding to contextual stimuli. Compared to the control component with no added stimuli preceding response-independent reinforcement, response rates increased as discrete-stimulus duration increased (0, 5, 10, and 15 s) across conditions. Although resistance to extinction decreased as stimulus duration increased in the component with the added discrete stimulus, further tests revealed no effect of discrete stimuli, including other disrupters (presession food, intercomponent food, modified extinction) and reinstatement designed to control for generalization decrement. These findings call into question a straightforward conception that the stimulus-reinforcer relations governing resistance to disruption reflect the same processes as Pavlovian conditioning, as asserted by behavioral momentum theory. © Society for the Experimental Analysis of Behavior.

The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B

NASA Astrophysics Data System (ADS)

Lu, Shaoyong; Jang, Hyunbum; Nussinov, Ruth; Zhang, Jian

2016-02-01

Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4BWT-GTP/GDP) catalytic domain, the K-Ras4BWT-GTP-GAP complex, and the mutants (K-Ras4BG12C/G12D/G12V-GTP/GDP, K-Ras4BG13D-GTP/GDP, K-Ras4BQ61H-GTP/GDP) and their complexes with GAP. In addition, we simulated ‘exchanged’ nucleotide states. These comprehensive simulations reveal that in solution K-Ras4BWT-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4BG12C/G12D-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer.

H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation.

PubMed

Martín-Sánchez, Paloma; Luengo, Alicia; Griera, Mercedes; Orea, María Jesús; López-Olañeta, Marina; Chiloeches, Antonio; Lara-Pezzi, Enrique; de Frutos, Sergio; Rodríguez-Puyol, Manuel; Calleros, Laura; Rodríguez-Puyol, Diego

2018-02-01

Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H- ras gene deletion produces mice hypotension via a soluble guanylate cyclase-protein kinase G (PKG)-dependent mechanism. In this study, we analyzed the consequences of H- ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H -ras -/- ) and control wild-type (H -ras +/+ ) mice, as were extracellular matrix protein expression. Increased cardiac PKG-Iβ protein expression in H -ras -/- mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H -ras -/- mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3β-dependent activation of the transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iβ overexpression in H -ras -/- mouse embryonic fibroblasts. This study demonstrates that H- ras deletion protects against AngII-induced cardiac remodeling, possibly via a mechanism in which PKG-Iβ overexpression could play a partial role, and points to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.-Martín-Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López-Olañeta, M., Chiloeches, A., Lara-Pezzi, E., de Frutos, S., Rodríguez-Puyol, M., Calleros, L., Rodríguez-Puyol, D. H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation.

The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Han-Soo; Kim, Ju Won; Gang, Jingu

2006-09-15

LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21{sup CIP1/WAF1}, RhoB and EGFR, that can explain themore » differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21{sup CIP1/WAF1} and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21{sup CIP1/WAF1} and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells.« less

R-Ras contributes to LTP and contextual discrimination.

PubMed

Darcy, M J; Jin, S-X; Feig, L A

2014-09-26

The ability to discriminate between closely related contexts is a specific form of hippocampal-dependent learning that may be impaired in certain neurodegenerative disorders such as Alzheimer's and Down Syndrome. However, signaling pathways regulating this form of learning are poorly understood. Previous studies have shown that the calcium-dependent exchange factor Ras-GRF1, an activator of Rac, Ras and R-Ras GTPases, is important for this form of learning and memory. Moreover, the ability to discriminate contexts was linked to the ability of Ras-GRF1 to promote high-frequency stimulation long-term potentiation (HFS-LTP) via the activation of p38 Map kinase. Here, we show that R-Ras is involved in this form of learning by using virally-delivered miRNAs targeting R-Ras into the CA1 region of the dorsal hippocampus and observing impaired contextual discrimination. Like the loss of GRF1, knockdown of R-Ras in the CA1 also impairs the induction of HFS-LTP and p38 Map kinase. Nevertheless, experiments indicate that this involvement of R-Ras in HFS-LTP that is required for contextual discrimination is independent of Ras-GRF1. Thus, R-Ras is a novel regulator of a form of hippocampal-dependent LTP as well as learning and memory that is affected in certain forms of neurodegenerative diseases. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Normal Human Fibroblasts Are Resistant to RAS-Induced Senescence

PubMed Central

Benanti, Jennifer A.; Galloway, Denise A.

2004-01-01

Oncogenic stimuli are thought to induce senescence in normal cells in order to protect against transformation and to induce proliferation in cells with altered p53 and/or retinoblastoma (Rb) pathways. In human fibroblasts, RAS initiates senescence through upregulation of the cyclin-dependent kinase inhibitor p16INK4A. We show here that in contrast to cultured fibroblast strains, freshly isolated normal fibroblasts are resistant to RAS-induced senescence and instead show some characteristics of transformation. RAS did not induce growth arrest or expression of senescence-associated β-galactosidase, and Rb remained hyperphosphorylated despite elevated levels of p16. Instead, RAS promoted anchorage-independent growth of normal fibroblasts, although expression of hTert with RAS increased colony formation and allowed normal fibroblasts to bypass contact inhibition. To test the hypothesis that p16 levels determine how cells respond to RAS, we expressed RAS in freshly isolated fibroblasts that expressed very low levels of p16, in hTert-immortalized fibroblasts that had accumulated intermediate levels of p16, and in IMR90 fibroblasts with high levels of p16. RAS induced growth arrest in cells with higher p16 levels, and this effect was reversed by p16 knockdown in the hTert-immortalized fibroblasts. These findings indicate that culture-imposed stress sensitizes cells to RAS-induced arrest, whereas early passage cells do not arrest in response to RAS. PMID:15024073

Identification of Ras-degrading small molecules that inhibit the transformation of colorectal cancer cells independent of β-catenin signaling.

PubMed

Shin, Wookjin; Lee, Sang-Kyu; Hwang, Jeong-Ha; Park, Jong-Chan; Cho, Yong-Hee; Ro, Eun Ji; Song, Yeonhwa; Seo, Haeng Ran; Choi, Kang-Yell

2018-06-06

Although the development of drugs that control Ras is an emerging topic in cancer therapy, no clinically applicable drug is currently available. We have previously utilized knowledge of the Wnt/β-catenin signaling-dependent mechanism of Ras protein stability regulation to identify small molecules that inhibit the proliferation and transformation of various colorectal cancer (CRC) cells via degradation of both β-catenin and Ras. Due to the absence of Ras degradation in cells expressing a nondegradable mutant form of β-catenin and the need to determine an alternative mechanism of Ras degradation, we designed a cell-based system to screen compounds that degrade Ras independent of the Wnt/β-catenin signaling pathway. A cell-based high-content screening (HCS) system that monitors the levels of EGFP-K-Ras G12V was established using HCT-116 cells harboring a nondegradable mutant CTNNB1 (ΔS45). Through HCS of a chemical library composed of 10,000 compounds and subsequent characterization of hits, we identified several compounds that degrade Ras without affecting the β-catenin levels. KY7749, one of the most effective compounds, inhibited the proliferation and transformation of CRC cells, especially KRAS-mutant cells that are resistant to the EGFR monoclonal antibody cetuximab. Small molecules that degrade Ras independent of β-catenin may able to be used in treatments for cancers caused by aberrant EGFR and Ras.

High-throughput screening identifies small molecules that bind to the RAS:SOS:RAS complex and perturb RAS signaling.

PubMed

Burns, Michael C; Howes, Jennifer E; Sun, Qi; Little, Andrew J; Camper, DeMarco V; Abbott, Jason R; Phan, Jason; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2018-05-01

K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

DA-Raf, a dominant-negative antagonist of the Ras-ERK pathway, is a putative tumor suppressor.

PubMed

Kanno, Emiri; Kawasaki, Osamu; Takahashi, Kazuya; Takano, Kazunori; Endo, Takeshi

2018-01-01

Activating mutations of RAS genes, particularly KRAS, are detected with high frequency in human tumors. Mutated Ras proteins constitutively activate the ERK pathway (Raf-MEK-ERK phosphorylation cascade), leading to cellular transformation and tumorigenesis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain (RBD) but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative fashion and suppresses constitutively activated K-Ras-induced cellular transformation. Thus, we have addressed whether DA-Raf serves as a tumor suppressor of Ras-induced tumorigenesis. DA-Raf(R52Q), which is generated from a single nucleotide polymorphism (SNP) in the RBD, and DA-Raf(R52W), a mutant detected in a lung cancer, neither bound to active K-Ras nor interfered with the activation of the ERK pathway. They were incapable of suppressing activated K-Ras-induced cellular transformation and tumorigenesis in mice, in which K-Ras-transformed cells were transplanted. Furthermore, although DA-Raf was highly expressed in lung alveolar epithelial type 2 (AE2) cells, its expression was silenced in AE2-derived lung adenocarcinoma cell lines with oncogenic KRAS mutations. These results suggest that DA-Raf represents a tumor suppressor protein against Ras-induced tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Imaging of Ras/Raf activity induced by low energy laser irradiation in living cell using FRET

NASA Astrophysics Data System (ADS)

Wang, Fang; Chen, Tong-Sheng; Xing, Da

2005-01-01

Ras/Raf signaling pathway is an important signaling pathway that governs cell proliferation, differential and apoptosis. Low-energy laser irradiation (LELI) was found to modulate various processes. Generally, cell proliferation is induced by low doses LELI and apoptosis is induced by high doses LELI. Mechanism of biological effect of LELI has not been clear. Recently, activation of MEK (mitogen-activated protein kinase) and ERK (extracellular-signal-regulated kinase), which are downstream protein kinases of Ras/Raf, are observed during LELI-induced cell proliferation by immunoprecipitation and western blot analysis. RaichuRas reporter consisting of fusions of H-ras, the Ras-binding domain of Raf (RafRBD), a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP). Therefore, intramolecular binding of GTP-Ras to RafRBD brings CFP close to YFP and increases FRET between CFP and YFP. Human lung adenocarcinoma cell line (ASTC-a-1) was transfected with the plasmid (pRaichuRas) and then treated with LELI at dose of 60J/cm2. Effect of LELI on Ras/Raf in physiological condition of living cells was observed by fluorescence resonance energy transfer (FRET) technique during lung adenocarcinoma cell apoptosis induced by high dose (60J/cm2) LELI. Experimental results showed that after high dose LELI treatment, the binding of Ras and Raf decreases obviously, Ras/Raf signaling pathway deregulates and cell apoptosis occurs.

RAS - Target Identification - Informatics

Cancer.gov

The RAS Informatics lab group develops tools to track and analyze “big data” from the RAS Initiative, as well as analyzes data from external projects. By integrating internal and external data, this group helps improve understanding of RAS-driven cancers.

PI3K: A Crucial Piece in the RAS Signaling Puzzle.

PubMed

Krygowska, Agata Adelajda; Castellano, Esther

2018-06-01

RAS proteins are key signaling switches essential for control of proliferation, differentiation, and survival of eukaryotic cells. RAS proteins are mutated in 30% of human cancers. In addition, mutations in upstream or downstream signaling components also contribute to oncogenic activation of the pathway. RAS proteins exert their functions through activation of several signaling pathways and dissecting the contributions of these effectors in normal cells and in cancer is an ongoing challenge. In this review, we summarize our current knowledge about how RAS regulates type I phosphatidylinositol 3-kinase (PI3K), one of the main RAS effectors. RAS signaling through PI3K is necessary for normal lymphatic vasculature development and for RAS-induced transformation in vitro and in vivo, especially in lung cancer, where it is essential for tumor initiation and necessary for tumor maintenance. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Dictyostelium RasG Is Required for Normal Motility and Cytokinesis, But Not Growth

PubMed Central

Tuxworth, Richard I.; Cheetham, Janet L.; Machesky, Laura M.; Spiegelmann, George B.; Weeks, Gerald; Insall, Robert H.

1997-01-01

RasG is the most abundant Ras protein in growing Dictyostelium cells and the closest relative of mammalian Ras proteins. We have generated null mutants in which expression of RasG is completely abolished. Unexpectedly, RasG − cells are able to grow at nearly wild-type rates. However, they exhibit defective cell movement and a wide range of defects in the control of the actin cytoskeleton, including a loss of cell polarity, absence of normal lamellipodia, formation of unusual small, punctate polymerized actin structures, and a large number of abnormally long filopodia. Despite their lack of polarity and abnormal cytoskeleton, mutant cells perform normal chemotaxis. However, rasG − cells are unable to perform normal cytokinesis, becoming multinucleate when grown in suspension culture. Taken together, these data suggest a principal role for RasG in coordination of cell movement and control of the cytoskeleton. PMID:9245789

Drugging the undruggable Ras: mission possible?

PubMed Central

Cox, Adrienne D.; Fesik, Stephen W.; Kimmelman, Alec C.; Luo, Ji; Der, Channing J.

2015-01-01

Despite more than three decades of intensive effort, no effective pharmacologic inhibitors of the Ras oncoproteins have reached the clinic, prompting the widely held perception that Ras proteins are “undruggable”. However, there is renewed hope that this is not the case. In this review, we summarize the progress and promise of five key directions. First, we focus on the prospects of direct inhibitors of Ras. Second, we revisit the issue of whether blocking Ras membrane association is a viable approach. Third, we assess the status of targeting Ras downstream effector signalling, arguably the most favourable current direction. Fourth, we address whether the search for synthetic lethal interactors of mutant RAS still holds promise. Finally, Ras-mediated changes in cell metabolism have recently been described. Can these changes be exploited for new therapeutic directions? We conclude with perspectives on how additional complexities, not yet fully understood, may impact each of these approaches. PMID:25323927

Genetic analysis of Ras genes in epidermal development and tumorigenesis

PubMed Central

Drosten, Matthias; Lechuga, Carmen G; Barbacid, Mariano

2013-01-01

Proliferation and differentiation of epidermal keratinocytes are tightly controlled to ensure proper development and homeostasis of the epidermis. The Ras family of small GTPases has emerged as a central node in the coordination of cell proliferation in the epidermis. Recent genetic evidence from mouse models has revealed that the intensity of Ras signaling modulates the proliferative capacity of epidermal keratinocytes. Interfering with Ras signaling either by combined elimination of the 3 Ras genes from the basal layer of the epidermis or by overexpression of dominant-negative Ras isoforms caused epidermal thinning due to hypoproliferation of keratinocytes. In contrast, overexpression of oncogenic Ras mutants in different epidermal cell layers led to hyperproliferative phenotypes including the development of papillomas and squamous cell carcinomas. Here, we discuss the value of loss- and gain-of-function studies in mouse models to assess the role of Ras signaling in the control of epidermal proliferation. PMID:24150175

The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling

PubMed Central

Sandri, Chiara; Caccavari, Francesca; Valdembri, Donatella; Camillo, Chiara; Veltel, Stefan; Santambrogio, Martina; Lanzetti, Letizia; Bussolino, Federico; Ivaska, Johanna; Serini, Guido

2012-01-01

During developmental and tumor angiogenesis, semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases. R-Ras is mainly expressed in vascular cells, where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms. We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and -angiogenic activity of R-Ras. Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia. Upon binding, GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF) to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active β1 integrins and then causes the translocation of R-Ras to early endosomes. Here, the R-Ras/RIN2/Rab5 signaling module activates Rac1-dependent cell adhesion via TIAM1, a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate. In conclusion, the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active β1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Rac1. PMID:22825554

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

PubMed

Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L; Embrey, Kevin J; Golovanov, Alexander P

2016-01-22

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.

PubMed Central

Satoh, T; Fantl, W J; Escobedo, J A; Williams, L T; Kaziro, Y

1993-01-01

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types. Images PMID:8388543

The Intracrine Renin-Angiotensin System

PubMed Central

Kumar, Rajesh; Thomas, Candice M.; Yong, Qian Chen; Chen, Wen; Baker, Kenneth M.

2014-01-01

The renin-angiotensin system (RAS) is one of the earliest and most extensively studied hormonal systems. The RAS is an atypical hormonal system in several ways. The major bioactive peptide of the system, angiotensin (Ang) II, is neither synthesized in, nor targets one specific organ. New research has identified additional peptides with important physiological and pathological roles. More peptides also mean newer enzymatic cascades that generate these peptides and more receptors that mediate the function. In addition, completely different roles of components that constitute the RAS have been uncovered, such as that for prorenin via the prorenin receptor. Complexity of the RAS is further enhanced by the presence of sub-systems in tissues, which act in an autocrine/paracrine manner independent of the endocrine system. The RAS seems relevant at the cellular level, wherein individual cells have a complete system, termed the intracellular RAS. Thus, from cells to tissues to the entire organism, the RAS exhibits continuity while maintaining independent control at different levels. The intracellular RAS is a relatively new concept for the RAS. The current review presents a synopsis of the literature on this system in different tissues. PMID:22590974

Society News: PhD theses could win prizes; Last chance for IYA2009 grants; New Fellows; RAS Fellows win prizes; Need a job? Need staff? RAS Library Saturdays

NASA Astrophysics Data System (ADS)

2009-08-01

Fellows who are PhD student supervisors should be on the lookout for exceptionally good work from research students submitting their theses this year, for nomination for the RAS Michael Penston Astronomy Prize and the RAS Keith Runcorn Prize. The RAS is offering one last chance to apply for grants towards International Year of Astronomy activities, but you'll have to apply soon. The Society sends congratulations to Fellows of the RAS who have recently received prestigious awards for their work.

Intrinsic K-Ras dynamics: A novel molecular dynamics data analysis method shows causality between residue pair motions

NASA Astrophysics Data System (ADS)

Vatansever, Sezen; Gümüş, Zeynep H.; Erman, Burak

2016-11-01

K-Ras is the most frequently mutated oncogene in human cancers, but there are still no drugs that directly target it in the clinic. Recent studies utilizing dynamics information show promising results for selectively targeting mutant K-Ras. However, despite extensive characterization, the mechanisms by which K-Ras residue fluctuations transfer allosteric regulatory information remain unknown. Understanding the direction of information flow can provide new mechanistic insights for K-Ras targeting. Here, we present a novel approach -conditional time-delayed correlations (CTC) - using the motions of all residue pairs of a protein to predict directionality in the allosteric regulation of the protein fluctuations. Analyzing nucleotide-dependent intrinsic K-Ras motions with the new approach yields predictions that agree with the literature, showing that GTP-binding stabilizes K-Ras motions and leads to residue correlations with relatively long characteristic decay times. Furthermore, our study is the first to identify driver-follower relationships in correlated motions of K-Ras residue pairs, revealing the direction of information flow during allosteric modulation of its nucleotide-dependent intrinsic activity: active K-Ras Switch-II region motions drive Switch-I region motions, while α-helix-3L7 motions control both. Our results provide novel insights for strategies that directly target mutant K-Ras.

The cornerstone K-RAS mutation in pancreatic adenocarcinoma: From cell signaling network, target genes, biological processes to therapeutic targeting.

PubMed

Jonckheere, Nicolas; Vasseur, Romain; Van Seuningen, Isabelle

2017-03-01

RAS belongs to the super family of small G proteins and plays crucial roles in signal transduction from membrane receptors in the cell. Mutations of K-RAS oncogene lead to an accumulation of GTP-bound proteins that maintains an active conformation. In the pancreatic ductal adenocarcinoma (PDAC), one of the most deadly cancers in occidental countries, mutations of the K-RAS oncogene are nearly systematic (>90%). Moreover, K-RAS mutation is the earliest genetic alteration occurring during pancreatic carcinogenetic sequence. In this review, we discuss the central role of K-RAS mutations and their tremendous diversity of biological properties by the interconnected regulation of signaling pathways (MAPKs, NF-κB, PI3K, Ral…). In pancreatic ductal adenocarcinoma, transcriptome analysis and preclinical animal models showed that K-RAS mutation alters biological behavior of PDAC cells (promoting proliferation, migration and invasion, evading growth suppressors, regulating mucin pattern, and miRNA expression). K-RAS also impacts tumor microenvironment and PDAC metabolism reprogramming. Finally we discuss therapeutic targeting strategies of K-RAS that have been developed without significant clinical success so far. As K-RAS is considered as the undruggable target, targeting its multiple effectors and target genes should be considered as potential alternatives. Copyright © 2017 Elsevier B.V. All rights reserved.

The Bcr Kinase Downregulates Ras Signaling by Phosphorylating AF-6 and Binding to Its PDZ Domain

PubMed Central

Radziwill, G.; Erdmann, R. A.; Margelisch, U.; Moelling, K.

2003-01-01

The protein kinase Bcr is a negative regulator of cell proliferation and oncogenic transformation. We identified Bcr as a ligand for the PDZ domain of the cell junction and Ras-interacting protein AF-6. The Bcr kinase phosphorylates AF-6, which subsequently allows efficient binding of Bcr to AF-6, showing that the Bcr kinase is a regulator of the PDZ domain-ligand interaction. Bcr and AF-6 colocalize in epithelial cells at the plasma membrane. In addition, Bcr, AF-6, and Ras form a trimeric complex. Bcr increases the affinity of AF-6 to Ras, and a mutant of AF-6 that lacks a specific phosphorylation site for Bcr shows a reduced binding to Ras. Wild-type Bcr, but not Bcr mutants defective in binding to AF-6, interferes with the Ras-dependent stimulation of the Raf/MEK/ERK pathway. Since AF-6 binds to Bcr via its PDZ domain and to Ras via its Ras-binding domain, we propose that AF-6 functions as a scaffold-like protein that links Bcr and Ras to cellular junctions. We suggest that this trimeric complex is involved in downregulation of Ras-mediated signaling at sites of cell-cell contact to maintain cells in a nonproliferating state. PMID:12808105

Ras promotes cell survival by antagonizing both JNK and Hid signals in the Drosophila eye.

PubMed

Wu, Yue; Zhuang, Yuan; Han, Min; Xu, Tian; Deng, Kejing

2009-10-20

Programmed cell death, or apoptosis, is a fundamental physiological process during normal development or in pathological conditions. The activation of apoptosis can be elicited by numerous signalling pathways. Ras is known to mediate anti-apoptotic signals by inhibiting Hid activity in the Drosophila eye. Here we report the isolation of a new loss-of-function ras allele, rasKP, which causes excessive apoptosis in the Drosophila eye. This new function is likely to be mediated through the JNK pathway since the inhibition of JNK signalling can significantly suppress rasKP-induced apoptosis, whereas the removal of hid only weakly suppresses the phenotype. Furthermore, the reduction of JNK signalling together with the expression of the baculovirus caspase inhibitor p35, which blocks Hid activity, strongly suppresses the rasKP cell death. In addition, we find a strong correlation between rasKP-induced apoptosis in the eye disc and the activation of JNK signalling. In the Drosophila eye, Ras may protect cells from apoptosis by inhibiting both JNK and Hid activities. Surprisingly, reducing Ras activity in the wing, however, does not cause apoptosis but rather affects cell and organ size. Thus, in addition to its requirement for cell viability, Ras appears to mediate different biological roles depending on the developmental context and on the level of its expression.

Molecular Dynamics Simulations and Dynamic Network Analysis Reveal the Allosteric Unbinding of Monobody to H-Ras Triggered by R135K Mutation

PubMed Central

Song, Kun; Zhang, Jian; Lu, Shaoyong

2017-01-01

Ras proteins, as small GTPases, mediate cell proliferation, survival and differentiation. Ras mutations have been associated with a broad spectrum of human cancers and thus targeting Ras represents a potential way forward for cancer therapy. A recently reported monobody NS1 allosterically disrupts the Ras-mediated signaling pathway, but its efficacy is reduced by R135K mutation in H-Ras. However, the detailed mechanism is unresolved. Here, using molecular dynamics (MD) simulations and dynamic network analysis, we explored the molecular mechanism for the unbinding of NS1 to H-Ras and shed light on the underlying allosteric network in H-Ras. MD simulations revealed that the overall structures of the two complexes did not change significantly, but the H-Ras–NS1 interface underwent significant conformational alteration in the mutant Binding free energy analysis showed that NS1 binding was unfavored after R135K mutation, which resulted in the unfavorable binding of NS1. Furthermore, the critical residues on H-Ras responsible for the loss of binding of NS1 were identified. Importantly, the allosteric networks for these important residues were revealed, which yielded a novel insight into the allosteric regulatory mechanism of H-Ras. PMID:29072601

TAN-1813, a novel Ras-farnesyltransferase inhibitor produced by Phoma sp. taxonomy, fermentation, isolation and biological activities in vitro and in vivo.

PubMed

Ishii, T; Hayashi, K; Hida, T; Yamamoto, Y; Nozaki, Y

2000-08-01

A novel Ras-farnesyltransferase inhibitor designated TAN-1813 was isolated from the culture broth of a fungus strain, FL-41510, isolated as a plant endophyte. The producer was taxonomically characterized as Phoma sp. FL-41510. TAN-1813 inhibited rat brain farnesyltransferase and geranylgeranyltransferase I activity with IC50 values of 23 microg/ml and 47/microg/ml, respectively. TAN-1813 showed mixed-type inhibition with respect to farnesylpyrophosphate and noncompetitive inhibition with respect to a K-Ras C-terminal peptide. It also inhibited the in situ farnesylation of cellular Ras proteins in a K-ras transformant (NIH3T3/K-ras) of mouse embryonic fibroblast cell line NIH3T3. TAN- 1813 inhibited the proliferation of various human cancer cells, some of which harbor activated ras alleles, with IC50 values of 15 approximately 110 ng/ml as well as that of NIH3T3 and NIH3T3/K-ras cells with IC50S of 540 and 310 ng/ml, respectively. Flow cytometric analysis indicated that TAN-1813 arrests NIH3T3/K-ras cells at both G1 and G2/M phases of the cell cycle. In addition, TAN-1813 was found to induce morphological reversion of NIH3T3/K-ras cells from the transformed phenotype. Antitumor activity of TAN-1813 against human fibrosarcoma HT-1080 and NIH3T3/K-ras tumors in nude mice was also verified.

ACE insertion/deletion polymorphism (rs1799752) modifies the renoprotective effect of renin-angiotensin system blockade in patients with IgA nephropathy.

PubMed

Teranishi, Junya; Yamamoto, Ryohei; Nagasawa, Yasuyuki; Shoji, Tatsuya; Iwatani, Hirotsugu; Okada, Noriyuki; Moriyama, Toshiki; Yamauchi, Atsushi; Tsubakihara, Yoshiharu; Imai, Enyu; Rakugi, Hiromi; Isaka, Yoshitaka

2015-09-01

Little is known about genetic predictors that modify the renoprotective effect of renin-angiotensin system (RAS) blockade in IgA nephropathy (IgAN). The present multicenter retrospective observational study examined effect modification between RAS blockade and three RAS-related gene polymorphisms in 237 IgAN patients, including ACE I/D (rs1799752), AT1R A1166C (rs5186) and AGT T704C (rs699). During 9.9 ± 4.2 years of observation, 63 patients progressed to a 50% increase in serum creatinine level. Only ACE I/D predicted the outcome (ACE DD vs ID/II, hazard ratio 1.86 (95% confidence interval 1.03, 3.33)) and modified the renoprotective effect of RAS blockade (p for interaction between ACE DD and RAS blockade = 0.087). RAS blockade suppressed progression in ACE DD patients but not in ID/II patients (ACE ID/II with RAS blockade as a reference; ID/II without RAS blockade 1.45 (0.72, 2.92); DD without RAS blockade 3.06 (1.39, 6.73); DD with RAS blockade 1.51 (0.54, 4.19)), which was ascertained in a model with the outcome of slope of estimated glomerular filtration rate (p = 0.045 for interaction). ACE I/D predicted the IgAN progression and the renoprotective effect of RAS blockade in IgAN patients whereas neither AT1R A1166C nor AGT T704C did. © The Author(s) 2014.

Specific repression of mutant K-RAS by 10-23 DNAzyme: Sensitizing cancer cell to anti-cancer therapies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, S.-H.; Wang, T.-H.; Department of Medical Research and Education, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei 11227, Taiwan

2009-01-09

Point mutations of the Ras family are frequently found in human cancers at a prevalence rate of 30%. The most common mutation K-Ras(G12V), required for tumor proliferation, survival, and metastasis due to its constitutively active GTPase activity, has provided an ideal target for cancer therapy. 10-23 DNAzyme, an oligodeoxyribonucleotide-based ribonuclease consisting of a 15-nucleotide catalytical domain flanked by two target-specific complementary arms, has been shown to effectively cleave the target mRNA at purine-pyrimidine dinucleotide. Taking advantage of this specific property, 10-23 DNAzyme was designed to cleave mRNA of K-Ras(G12V)(GGU {yields} GUU) at the GU dinucleotide while left the wild-type (WT)more » K-Ras mRNA intact. The K-Ras(G12V)-specific 10-23 DNAzyme was able to reduce K-Ras(G12V) at both mRNA and protein levels in SW480 cell carrying homozygous K-Ras(G12V). No effect was observed on the WT K-Ras in HEK cells. Although K-Ras(G12V)-specific DNAzymes alone did not inhibit proliferation of SW480 or HEK cells, pre-treatment of this DNAzyme sensitized the K-Ras(G12V) mutant cells to anti-cancer agents such as doxorubicin and radiation. These results offer a potential of using allele-specific 10-23 DNAzyme in combination with other cancer therapies to achieve better effectiveness on cancer treatment.« less

Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buhrman, Greg; O; #8242

2012-09-17

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the 'off' and 'on' allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond themore » active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.« less

Balanced RAP/RAS mix design and performance evaluation for project - specific service conditions.

DOT National Transportation Integrated Search

2013-01-01

This presentation summarizes Projects 0-6092/0-6614. It includes accomplishments, best practices, field performance data of RAP/RAS test sections, balanced RAP/RAS mix design for project-specific conditions, and approaches for improving RAP/RAS mix p...

RAS mutation prevalence among patients with metastatic colorectal cancer: a meta-analysis of real-world data.

PubMed

Kafatos, George; Niepel, Daniela; Lowe, Kimberley; Jenkins-Anderson, Sophie; Westhead, Hal; Garawin, Tamer; Traugottová, Zuzana; Bilalis, Antonios; Molnar, Edit; Timar, Jozsef; Toth, Erika; Gouvas, Nikolaos; Papaxoinis, George; Murray, Samuel; Mokhtar, Nadia; Vosmikova, Hana; Fabian, Pavel; Skalova, Alena; Wójcik, Piotr; Tysarowski, Andrzej; Barugel, Mario; van Krieken, J Han; Trojan, Jörg

2017-07-27

A confirmed wild-type RAS tumor status is commonly required for prescribing anti-EGFR treatment for metastatic colorectal cancer. This noninterventional, observational research project estimated RAS mutation prevalence from real-world sources. Aggregate RAS mutation data were collected from 12 sources in three regions. Each source was analyzed separately; pooled prevalence estimates were then derived from meta-analyses. The pooled RAS mutation prevalence from 4431 tumor samples tested for RAS mutation status was estimated to be 43.6% (95% CI: 38.8-48.5%); ranging from 33.7% (95% CI: 28.4-39.3%) to 54.1% (95% CI: 51.7-56.5%) between sources. The RAS mutation prevalence estimates varied among sources. The reasons for this are not clear and highlight the need for further research.

Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture.

PubMed Central

Giannini, C D; Roth, W K; Piiper, A; Zeuzem, S

1999-01-01

Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki- ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki- ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene. PMID:10373591

K-RAS(V12) Induces Autocrine Production of EGFR Ligands and Mediates Radioresistance Through EGFR-Dependent Akt Signaling and Activation of DNA-PKcs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minjgee, Minjmaa; Toulany, Mahmoud; Kehlbach, Rainer

2011-12-01

Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)-transfected cells enhanced activation of the phosphatidylinositol-3-kinase-Akt pathway and increased postirradiation survival ofmore » wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)-neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor {alpha} was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR-phosphatidylinositol-3-kinase-Akt cascade.« less

The mucin MUC4 is a transcriptional and post-transcriptional target of K-ras oncogene in pancreatic cancer. Implication of MAPK/AP-1, NF-κB and RalB signaling pathways.

PubMed

Vasseur, Romain; Skrypek, Nicolas; Duchêne, Belinda; Renaud, Florence; Martínez-Maqueda, Daniel; Vincent, Audrey; Porchet, Nicole; Van Seuningen, Isabelle; Jonckheere, Nicolas

2015-12-01

The membrane-bound mucinMUC4 is a high molecularweight glycoprotein frequently deregulated in cancer. In pancreatic cancer, one of the most deadly cancers in occidental countries, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras remains clinically undruggable. Targeting early downstream K-ras signaling in cancer may thus appear as an interesting strategy and MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-rasG12D mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-κB signaling pathways. In vitro, transfection of constitutively activated K-rasG12V in pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was found to be mediated at the transcriptional level by AP-1 and NF-κB transcription factors via MAPK, JNK and NF-κB pathways and at the posttranscriptional level by a mechanism involving the RalB GTPase. Altogether, these results identify MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer.

RAS mutations predict radiologic and pathologic response in patients treated with chemotherapy prior to resection of colorectal liver metastases

PubMed Central

Mise, Yoshihiro; Kopetz, Scott; Loyer, Evelyne M.; Andreou, Andreas; Cooper, Amanda B.; Kaur, Harmeet; Aloia, Thomas A.; Maru, Dipen M.; Vauthey, Jean-Nicolas

2014-01-01

Purpose RAS mutations have been reported to be a potential prognostic factor in patients with colorectal liver metastases (CLM). However, the impact of RAS mutations on response to chemotherapy remains unclear. We sought to determine the association between RAS mutations and response to preoperative chemotherapy and their impact on survival in patients undergoing curative resection of CLM. Methods RAS mutational status was assessed and its relation to morphologic response and pathologic response was investigated in 184 patients meeting inclusion criteria. Predictors of survival were assessed. The prognostic impact of RAS mutational status was then analyzed using two different multivariate models including either radiologic morphologic response (model 1) or pathologic response (model 2). Results Optimal morphologic response and major pathologic response were more common in patients with wild-type RAS (32.9% and 58.9%, respectively) than in patients with RAS mutations (10.5% and 36.8%; P =.006 and .015, respectively). Multivariate analysis confirmed that wild-type RAS was a strong predictor of optimal morphologic response (odds ratio [OR], 4.38; 95% CI, 1.45-13.2) and major pathologic response (OR,2.79; 95% CI, 1.29-6.04). RAS mutations were independently correlated with both overall survival and recurrence free-survival (hazard ratios, 3.25 and 2.02, respectively, in model 1, and 3.19 and 2.23, respectively, in model 2). Subanalysis revealed that RAS mutational status clearly stratified prognosis in patients with inadequate response to preoperative chemotherapy. Conclusion RAS mutational status can be used to complement the current prognostic indicators for patients undergoing curative resection of CLM after preoperative modern chemotherapy. PMID:25227306

RAS mutations predict radiologic and pathologic response in patients treated with chemotherapy before resection of colorectal liver metastases.

PubMed

Zimmitti, Giuseppe; Shindoh, Junichi; Mise, Yoshihiro; Kopetz, Scott; Loyer, Evelyne M; Andreou, Andreas; Cooper, Amanda B; Kaur, Harmeet; Aloia, Thomas A; Maru, Dipen M; Vauthey, Jean-Nicolas

2015-03-01

RAS mutations have been reported to be a potential prognostic factor in patients with colorectal liver metastases (CLM). However, the impact of RAS mutations on response to chemotherapy remains unclear. The purpose of this study was to investigate the correlation between RAS mutations and response to preoperative chemotherapy and their impact on survival in patients undergoing curative resection of CLM. RAS mutational status was assessed and its relation to morphologic response and pathologic response was investigated in 184 patients meeting inclusion criteria. Predictors of survival were assessed. The prognostic impact of RAS mutational status was then analyzed using two different multivariate models, including either radiologic morphologic response (model 1) or pathologic response (model 2). Optimal morphologic response and major pathologic response were more common in patients with wild-type RAS (32.9 and 58.9%, respectively) than in patients with RAS mutations (10.5 and 36.8%; P = 0.006 and 0.015, respectively). Multivariate analysis confirmed that wild-type RAS was a strong predictor of optimal morphologic response [odds ratio (OR), 4.38; 95% CI 1.45-13.15] and major pathologic response (OR, 2.61; 95% CI 1.17-5.80). RAS mutations were independently correlated with both overall survival and recurrence free-survival (hazard ratios, 3.57 and 2.30, respectively, in model 1, and 3.19 and 2.09, respectively, in model 2). Subanalysis revealed that RAS mutational status clearly stratified survival in patients with inadequate response to preoperative chemotherapy. RAS mutational status can be used to complement the current prognostic indicators for patients undergoing curative resection of CLM after preoperative modern chemotherapy.

K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

PubMed

Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya; Yaguchi, Masahiro; Niida, Ayumu; Sasaki, Shigekazu; Miwa, Masanori; Ohkubo, Shoichi; Sakamoto, Jun-Ichi; Kamaura, Masahiro; Cho, Nobuo; Tani, Akiyoshi

2017-03-11

Amino-acid mutations of Gly 12 (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH 2 ) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. K D and IC 50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH 2 ) that inhibited enzyme activity of K-Ras(G12D) with IC 50  = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Naumov, Inna; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; Kazanov, Dina

2012-01-15

Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1more » and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.« less

Ras mutations are rare in solitary cold and toxic thyroid nodules.

PubMed

Krohn, K; Reske, A; Ackermann, F; Müller, A; Paschke, R

2001-08-01

Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site.

PubMed

Mazhab-Jafari, Mohammad T; Marshall, Christopher B; Smith, Matthew J; Gasmi-Seabrook, Geneviève M C; Stathopulos, Peter B; Inagaki, Fuyuhiko; Kay, Lewis E; Neel, Benjamin G; Ikura, Mitsuhiko

2015-05-26

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.

Impairment of K-Ras signaling networks and increased efficacy of epidermal growth factor receptor inhibitors by a novel synthetic miR-143.

PubMed

Akao, Yukihiro; Kumazaki, Minami; Shinohara, Haruka; Sugito, Nobuhiko; Kuranaga, Yuki; Tsujino, Takuya; Yoshikawa, Yuki; Kitade, Yukio

2018-05-01

Despite considerable research on K-Ras inhibitors, none had been established until now. We synthesized nuclease-resistant synthetic miR-143 (miR-143#12), which strongly silenced K-Ras, its effector signal molecules AKT and ERK, and the K-Ras activator Sos1. We examined the anti-proliferative effect of miR-143#12 and the mechanism in human colon cancer DLD-1 cell (G13D) and other cell types harboring K-Ras mutations. Cell growth was markedly suppressed in a concentration-dependent manner by miR-143#12 (IC 50 : 1.32 nmol L -1 ) with a decrease in the K-Ras mRNA level. Interestingly, this mRNA level was also downregulated by either a PI3K/AKT or MEK inhibitor, which indicates a positive circuit of K-Ras mRNA expression. MiR-143#12 silenced cytoplasmic K-Ras mRNA expression and impaired the positive circuit by directly targeting AKT and ERK mRNA. Combination treatment with miR-143#12 and a low-dose EGFR inhibitor induced a synergistic inhibition of growth with a marked inactivation of both PI3K/AKT and MAPK/ERK signaling pathways. However, silencing K-Ras by siR-KRas instead of miR-143#12 did not induce this synergism through the combined treatment with the EGFR inhibitor. Thus, miR-143#12 perturbed the K-Ras expression system and K-Ras activation by silencing Sos1 and, resultantly, restored the efficacy of the EGFR inhibitors. The in vivo results also supported those of the in vitro experiments. The extremely potent miR-143#12 enabled us to understand K-Ras signaling networks and shut them down by combination treatment with this miRNA and EGFR inhibitor in K-Ras-driven colon cancer cell lines. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Ras history

PubMed Central

2010-01-01

Although the roots of Ras sprouted from the rich history of retrovirus research, it was the discovery of mutationally activated RAS genes in human cancer in 1982 that stimulated an intensive research effort to understand Ras protein structure, biochemistry and biology. While the ultimate goal has been developing anti-Ras drugs for cancer treatment, discoveries from Ras have laid the foundation for three broad areas of science. First, they focused studies on the origins of cancer to the molecular level, with the subsequent discovery of genes mutated in cancer that now number in the thousands. Second, elucidation of the biochemical mechanisms by which Ras facilitates signal transduction established many of our fundamental concepts of how a normal cell orchestrates responses to extracellular cues. Third, Ras proteins are also founding members of a large superfamily of small GTPases that regulate all key cellular processes and established the versatile role of small GTP-binding proteins in biology. We highlight some of the key findings of the last 28 years. PMID:21686117

Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins.

PubMed

Martinko, Alexander J; Truillet, Charles; Julien, Olivier; Diaz, Juan E; Horlbeck, Max A; Whiteley, Gordon; Blonder, Josip; Weissman, Jonathan S; Bandyopadhyay, Sourav; Evans, Michael J; Wells, James A

2018-01-23

While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRAS G12V , and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS-induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations. In parallel, a cell-surface CRISPRi screen identified integrin and Wnt signaling proteins as critical to RAS-transformed cells. We show that antibodies targeting CDCP1, a protein common to our proteomics and CRISPRi datasets, can be leveraged to deliver cytotoxic and immunotherapeutic payloads to RAS-transformed cancer cells and report for RAS signaling status in vivo. Taken together, this work presents a technological platform for attacking RAS from outside the cell. © 2018, Martinko et al.

Deconstruction of the Ras switching cycle through saturation mutagenesis

PubMed Central

Bandaru, Pradeep; Shah, Neel H; Bhattacharyya, Moitrayee; Barton, John P; Kondo, Yasushi; Cofsky, Joshua C; Gee, Christine L; Chakraborty, Arup K; Kortemme, Tanja; Ranganathan, Rama; Kuriyan, John

2017-01-01

Ras proteins are highly conserved signaling molecules that exhibit regulated, nucleotide-dependent switching between active and inactive states. The high conservation of Ras requires mechanistic explanation, especially given the general mutational tolerance of proteins. Here, we use deep mutational scanning, biochemical analysis and molecular simulations to understand constraints on Ras sequence. Ras exhibits global sensitivity to mutation when regulated by a GTPase activating protein and a nucleotide exchange factor. Removing the regulators shifts the distribution of mutational effects to be largely neutral, and reveals hotspots of activating mutations in residues that restrain Ras dynamics and promote the inactive state. Evolutionary analysis, combined with structural and mutational data, argue that Ras has co-evolved with its regulators in the vertebrate lineage. Overall, our results show that sequence conservation in Ras depends strongly on the biochemical network in which it operates, providing a framework for understanding the origin of global selection pressures on proteins. DOI: http://dx.doi.org/10.7554/eLife.27810.001 PMID:28686159

Frequencies and prognostic impact of RAS mutations in MLL-rearranged acute lymphoblastic leukemia in infants

PubMed Central

Driessen, Emma M.C.; van Roon, Eddy H.J.; Spijkers-Hagelstein, Jill A.P.; Schneider, Pauline; de Lorenzo, Paola; Valsecchi, Maria Grazia; Pieters, Rob; Stam, Ronald W.

2013-01-01

Acute lymphoblastic leukemia in infants represents an aggressive malignancy associated with a high incidence (approx. 80%) of translocations involving the Mixed Lineage Leukemia (MLL) gene. Attempts to mimic Mixed Lineage Leukemia fusion driven leukemogenesis in mice raised the question whether these fusion proteins require secondary hits. RAS mutations are suggested as candidates. Earlier results on the incidence of RAS mutations in Mixed Lineage Leukemia-rearranged acute lymphoblastic leukemia are inconclusive. Therefore, we studied frequencies and relation with clinical parameters of RAS mutations in a large cohort of infant acute lymphoblastic leukemia patients. Using conventional sequencing analysis, we screened neuroblastoma RAS viral (v-ras) oncogene homolog gene (NRAS), v-Ki-ras Kirsten rat sarcoma viral oncogene homolog gene (KRAS), and v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) for mutations in a large cohort (n=109) of infant acute lymphoblastic leukemia patients and studied the mutations in relation to several clinical parameters, and in relation to Homeobox gene A9 expression and the presence of ALL1 fused gene 4-Mixed Lineage Leukemia (AF4-MLL). Mutations were detected in approximately 14% of all cases, with a higher frequency of approximately 24% in t(4;11)-positive patients (P=0.04). Furthermore, we identified RAS mutations as an independent predictor (P=0.019) for poor outcome in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia, with a hazard ratio of 3.194 (95% confidence interval (CI):1.211–8.429). Also, RAS-mutated infants have higher white blood cell counts at diagnosis (P=0.013), and are more resistant to glucocorticoids in vitro (P<0.05). Finally, we demonstrate that RAS mutations, and not the lack of Homeobox gene A9 expression nor the expression of AF4-MLL are associated with poor outcome in t(4;11)-rearranged infants. We conclude that the presence of RAS mutations in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia is an independent predictor for a poor outcome. Therefore, future risk-stratification based on abnormal RAS-pathway activation and RAS-pathway inhibition could be beneficial in RAS-mutated infant acute lymphoblastic leukemia patients. PMID:23403319

Synthetic lipopeptide inhibitors of RAS oncoproteins | NCI Technology Transfer Center | TTC

Cancer.gov

It is well known that overactive Ras signaling is linked to many forms of cancer, and despite intensive efforts worldwide to develop effective inhibitors of Ras, to date there is no anti-Ras inhibitor in clinical use. Researchers at the NCI’s Cancer and Inflammation Program, in collaboration with scientists at Vanderbilt University and the University of Illinois in Chicago, have identified a number of small peptidomimetic compounds that bind to Ras proteins with nanomolar affinity. NCI’s Cancer and Inflammation Program seeks partners interested in licensing or co-development of synthetic, highly potent cell-permeable inhibitors of Ras that bind to the protein directly.

Computational representation and hemodynamic characterization of in vivo acquired severe stenotic renal artery geometries using turbulence modeling.

PubMed

Kagadis, George C; Skouras, Eugene D; Bourantas, George C; Paraskeva, Christakis A; Katsanos, Konstantinos; Karnabatidis, Dimitris; Nikiforidis, George C

2008-06-01

The present study reports on computational fluid dynamics in the case of severe renal artery stenosis (RAS). An anatomically realistic model of a renal artery was reconstructed from CT scans, and used to conduct CFD simulations of blood flow across RAS. The recently developed shear stress transport (SST) turbulence model was pivotally applied in the simulation of blood flow in the region of interest. Blood flow was studied in vivo under the presence of RAS and subsequently in simulated cases before the development of RAS, and after endovascular stent implantation. The pressure gradients in the RAS case were many orders of magnitude larger than in the healthy case. The presence of RAS increased flow resistance, which led to considerably lower blood flow rates. A simulated stent in place of the RAS decreased the flow resistance at levels proportional to, and even lower than, the simulated healthy case without the RAS. The wall shear stresses, differential pressure profiles, and net forces exerted on the surface of the atherosclerotic plaque at peak pulse were shown to be of relevant high distinctiveness, so as to be considered potential indicators of hemodynamically significant RAS.

Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia

PubMed Central

Oshima, Koichi; Khiabanian, Hossein; da Silva-Almeida, Ana C.; Tzoneva, Gannie; Abate, Francesco; Ambesi-Impiombato, Alberto; Sanchez-Martin, Marta; Carpenter, Zachary; Penson, Alex; Perez-Garcia, Arianne; Eckert, Cornelia; Nicolas, Concepción; Balbin, Milagros; Sulis, Maria Luisa; Kato, Motohiro; Koh, Katsuyoshi; Paganin, Maddalena; Basso, Giuseppe; Gastier-Foster, Julie M.; Devidas, Meenakshi; Loh, Mignon L.; Kirschner-Schwabe, Renate; Palomero, Teresa; Rabadan, Raul; Ferrando, Adolfo A.

2016-01-01

Although multiagent combination chemotherapy is curative in a significant fraction of childhood acute lymphoblastic leukemia (ALL) patients, 20% of cases relapse and most die because of chemorefractory disease. Here we used whole-exome and whole-genome sequencing to analyze the mutational landscape at relapse in pediatric ALL cases. These analyses identified numerous relapse-associated mutated genes intertwined in chemotherapy resistance-related protein complexes. In this context, RAS-MAPK pathway-activating mutations in the neuroblastoma RAS viral oncogene homolog (NRAS), kirsten rat sarcoma viral oncogene homolog (KRAS), and protein tyrosine phosphatase, nonreceptor type 11 (PTPN11) genes were present in 24 of 55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, whereas in others RAS mutant clones present at diagnosis were replaced by RAS wild-type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Consistently, functional dissection of mouse and human wild-type and mutant RAS isogenic leukemia cells demonstrated induction of methotrexate resistance but also improved the response to vincristine in mutant RAS-expressing lymphoblasts. These results highlight the central role of chemotherapy-driven selection as a central mechanism of leukemia clonal evolution in relapsed ALL, and demonstrate a previously unrecognized dual role of RAS mutations as drivers of both sensitivity and resistance to chemotherapy. PMID:27655895

[Expressions of Ras and Sos1 in epithelial ovarian cancer tissues and their clinical significance].

PubMed

Xiao, Zheng-Hua; Linghu, Hua; Liu, Qian-Fen

2016-11-20

To detect the expressions of Ras and Sos1 proteins in human epithelial ovarian cancer (EOC) tissues and explore their correlation with the clinicopathological features of the patients. The expressions of Ras and Sos1 proteins were detected immunohistochemically in 62 EOC tissues, 5 borderline ovarian cancer tissues, 15 benign epithelial ovarian neoplasm tissues, and 18 normal ovarian tissues. The EOC tissues showed significantly higher expression levels of both Ras and Sos1 than the other tissues tested (P<0.05). In EOC tissues, Ras and Sos1 proteins were expressed mostly on the cell membrane and in the cytoplasm. The expression level of Ras was correlated with pathological types of the tumor (P<0.05) and was the highest in serous cystadenomcarcinoma; Sos1 expression did not show significant correlation with the clinicopathological indexes of the patients. High expressions of both Ras and Sos1 proteins were associated with shorter progression-free survival of the patients, but this association was not statistically significant. Ras and Sos1 protein may participate in in the occurrence and development of EOC. The tissue-specific variation of Ras expression can lend support to a specific diagnosis of ovarian serous adenocarcinoma. The association of Ras and Sos1 protein expression with the tumor-free survival time of the patients awaits further investigation with a larger sample size.

Temporal dissection of K-ras(G12D) mutant in vitro and in vivo using a regulatable K-ras(G12D) mouse allele.

PubMed

Wang, Zuoyun; Feng, Yan; Bardeesy, Nabeel; Bardessy, Nabeel; Wong, Kwok-Kin; Liu, Xin-Yuan; Ji, Hongbin

2012-01-01

Animal models which allow the temporal regulation of gene activities are valuable for dissecting gene function in tumorigenesis. Here we have constructed a conditional inducible estrogen receptor-K-ras(G12D) (ER-K-ras(G12D)) knock-in mice allele that allows us to temporally switch on or off the activity of K-ras oncogenic mutant through tamoxifen administration. In vitro studies using mice embryonic fibroblast (MEF) showed that a dose of tamoxifen at 0.05 µM works optimally for activation of ER-K-ras(G12D) independent of the gender status. Furthermore, tamoxifen-inducible activation of K-ras(G12D) promotes cell proliferation, anchor-independent growth, transformation as well as invasion, potentially via activation of downstream MAPK pathway and cell cycle progression. Continuous activation of K-ras(G12D) in vivo by tamoxifen treatment is sufficient to drive the neoplastic transformation of normal lung epithelial cells in mice. Tamoxifen withdrawal after the tumor formation results in apoptosis and tumor regression in mouse lungs. Taken together, these data have convincingly demonstrated that K-ras mutant is essential for neoplastic transformation and this animal model may provide an ideal platform for further detailed characterization of the role of K-ras oncogenic mutant during different stages of lung tumorigenesis.

Ras mutation cooperates with β-catenin activation to drive bladder tumourigenesis.

PubMed

Ahmad, I; Patel, R; Liu, Y; Singh, L B; Taketo, M M; Wu, X-R; Leung, H Y; Sansom, O J

2011-03-03

Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels. The Wnt signalling pathway is deregulated in approximately 25% of UCC, so we examined if this correlated with the activation of MAPK signalling in human UCC and found a significant correlation. To test the functional significance of this association we examined the impact of combining Ras mutation (H-Ras(Q61L) or K-Ras(G12D)) with an activating β-catenin mutation within the mouse bladder using Cre-LoxP technology. Although alone, neither Ras mutation nor β-catenin activation led to UCC (within 12 months), mice carrying both mutations rapidly developed UCC. Mechanistically this was associated with reduced levels of p21 with dependence on the MAPK signalling pathway. Moreover, tumours from these mice were sensitive to MEK inhibition. Importantly, in human UCC there was a negative correlation between levels of p-ERK and p21 suggesting that p21 accumulation may block tumour progression following Ras mutation. Taken together these data definitively show Ras pathway activation strongly cooperates with Wnt signalling to drive UCC in vivo.

Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

DOE PAGES

Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; ...

2015-06-16

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referredmore » to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas G12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas G12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.« less

Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

PubMed Central

Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li-Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

2015-01-01

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors. PMID:26080442

Reptile-associated salmonellosis in children aged under 5 years in South West England.

PubMed

Murphy, Dan; Oshin, Femi

2015-04-01

To determine the proportion of Salmonella cases in children aged <5 years that were reptile-associated salmonellosis (RAS) and to compare the severity of illness. To analyse all cases of salmonellosis reported to public health authorities in children aged under 5 years in the South West of the UK from January 2010 to December 2013 for reptile exposure, age, serotype, hospitalisation and invasive disease. 48 of 175 (27%) Salmonella cases had exposure to reptiles. The median age of RAS cases was significantly lower than non-RAS cases (0.5 vs 1.0 year). RAS cases were 2.5 times more likely to be hospitalised (23/48) compared with non-RAS cases (25/127; p=0.0002). This trend continued in cases aged under 12 months, with significantly more RAS cases hospitalised (19/38) than non-RAS cases (8/42; p=0.003). Significantly more RAS cases had invasive disease (8/48: 5 bacteraemia, 2 meningitis, 1 colitis) than non-RAS cases (4/127: 3 bacteraemia, 1 meningitis). Reptile exposure was found in over a quarter of all reported Salmonella cases in children under 5 years of age. RAS is associated with young age, hospitalisation and invasive disease. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

The RasGAP Gene, RASAL2, is a Tumor and Metastasis Suppressor

PubMed Central

McLaughlin, Sara Koenig; Olsen, Sarah Naomi; Dake, Benjamin; De Raedt, Thomas; Lim, Elgene; Bronson, Roderick Terry; Beroukhim, Rameen; Polyak, Kornelia; Brown, Myles; Kuperwasser, Charlotte; Cichowski, Karen

2013-01-01

SUMMARY RAS genes are commonly mutated in cancer; however, RAS mutations are rare in breast cancer, despite the fact that Ras and ERK are frequently hyperactivated. Here we report that the RasGAP gene, RASAL2, functions as a tumor and metastasis suppressor. RASAL2 is mutated or suppressed in human breast cancer and RASAL2 ablation promotes tumor growth, progression, and metastasis in mouse models. In human breast cancer RASAL2-loss is associated with metastatic disease, low RASAL2 levels correlate with recurrence of luminal B tumors, and RASAL2 ablation promotes metastasis of luminal mouse tumors. Additional data reveal a broader role for RASAL2 inactivation in other tumor-types. These studies highlight the expanding role of RasGAPs and reveal an alternative mechanism of activating Ras in cancer. PMID:24029233

Quantitative two-dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes.

PubMed

Miller, M J; Maher, V M; McCormick, J J

1992-11-01

Quantitative two-dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin-derived human fibroblasts cell line (LG1) and three immortal derivatives of LG1. One derivative, designated MSU-1.1 VO, was selected for its ability to grow in the absence of serum and is non-tumorigenic in athymic mice. The other two strains were selected for focus-formation following transfection with either Ha-ras or N-ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two-dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU-1.1 VO, and the ras transformed cell strains. The MSU-1.1 VO cells were more closely related to the parental LG1 than to the ras-transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P < 0.01, T test, mean ratio of intensity > 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras-transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha-ras and N-ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha-ras transformed cells than the N-ras.(ABSTRACT TRUNCATED AT 250 WORDS)

Risk factors for invasive reptile-associated salmonellosis in children.

PubMed

Meyer Sauteur, Patrick M; Relly, Christa; Hug, Martina; Wittenbrink, Max M; Berger, Christoph

2013-06-01

Reptile-associated salmonellosis (RAS) in children has been reported primarily due to direct contact with turtles, but recently also due to indirect contact with more exotic reptiles, causing disease in infants. To evaluate risk factors for RAS, we reviewed the RAS cases published in the literature since 1965. A case was defined as a child ≤18 years of age with an epidemiological link by identification of Salmonella enterica in cultures from both the affected child and the exposed reptile. We identified a total of 177 otherwise healthy children (median age 1.0 years, range 2 days to 17.0 years). RAS manifested mainly with gastrointestinal disease, but 15% presented with invasive RAS, including septicemia, meningitis, and bone and joint infection. The children with invasive RAS were significantly younger than children with noninvasive disease (median age 0.17 and 2.0 years, p<0.0001). RAS is most frequently seen after exposure to turtles (42%). However, children with invasive RAS had been exposed more often (p≤0.001) to reptiles other than turtles, including iguanas, bearded dragons, snakes, chameleons, and geckos. Children exposed to those latter reptiles usually kept indoors were younger than children exposed to turtles mostly kept outdoors (p<0.0001). RAS in children is significantly associated with invasive disease at young age, in particular infants <6 months of age. Exposure to reptiles, other than turtles, kept indoors is associated with RAS at younger age and more invasive disease. This finding is helpful for recognizing or even preventing invasive RAS in young infants that are at highest risk.

Mixing and compaction recommendations for warm mix asphalt (WMA) with recycled asphalt shingles (RAS) : final report.

DOT National Transportation Integrated Search

2017-05-01

The use of recycled asphalt shingles (RAS) is an attractive option for asphalt mixture producers due to the : high amount of recycled asphalt binder available in RAS. By weight, RAS contains 10 to 25% asphalt by total : weight of the shingle. The asp...

Developing Sediment Transport and Dredging Prediction Model of Ohio River at Olmsted Locks and Dams Area using HEC-RAS (1D/2D)By Ganesh Raj Ghimire1 and Bruce A. Devantier 2

NASA Astrophysics Data System (ADS)

Ghimire, G. R.

2015-12-01

Sediment deposition is a serious issue in the construction and operation of large reservoir and inland navigation projects in the United States and around the world. Olmsted Locks and Dams in the Ohio River navigation system is facing similar challenges of huge sediment deposition during the ongoing in-wet construction methodology since 1993. HEC-RAS 5.0 integrated with ArcGIS, will be used to yield unsteady 2D hydrodynamic model of Ohio River at Olmsted area. Velocity, suspended sediment, bed sediment and hydrographic survey data acquired from public archives of USGS and USACE Louisville District will be input into the model. Calibration and validation of model will be performed against the measured stage, flow and velocity data. It will be subjected to completely unsteady 1D sediment transport modeling new to HEC-RAS 5.0 which incorporates sediment load and bed gradation via a DSS file, commercial dredging and BSTEM model. Sediment model will be calibrated to replicate the historical bed volume changes. Excavated cross-sections at Olmsted area will also be used to predict the sediment volume trapped inside the ditch over the period between excavations and placement of dam shells at site. Model will attempt to replicate historical dredging volume data and compare with the deposition volume from simulation model to formulate the dredging prediction model. Hence, the results of this research will generate a model that can form a basis for scheduling the dredging event prior to the placement of off-shore cast shells replacing the current as and when required approach of dredging plan. 1 Graduate Student, Department of Civil Engineering, Southern Illinois University Carbondale Carbondale, Illinois, 62901-6603 2 Professor, Department of Civil Engineering, Southern Illinois University Carbondale Carbondale, Illinois, 62901-6603

Regulating the Regulator: Post-Translational Modification of Ras

PubMed Central

Ahearn, Ian M.; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R.

2013-01-01

Ras proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on Ras is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which regulate the activation state of Ras without covalently modifying it. In contrast, post-translational modifications (PTMs) of Ras proteins direct them to various cellular membranes and, in some cases, modulate GTP–GDP exchange. Important Ras PTMs include the constitutive and irreversible remodelling of its C-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications including phosphorylation, peptidyl-proly isomerisation, mono- and di-ubiquitination, nitrosylation, ADP ribosylation and glucosylation. PMID:22189424

The possible mechanism of enhanced carcinogenesis induced by genotoxic carcinogens in rasH2 mice.

PubMed

Okamura, Miwa; Unami, Akira; Moto, Mitsuyoshi; Muguruma, Masako; Ito, Tadashi; Jin, Meilan; Oishi, Yuji; Kashida, Yoko; Mitsumori, Kunitoshi

2007-01-08

Microarray and RT-PCR analyses were performed for the transgene and Ras-related genes in forestomach squamous cell carcinomas (SCCs) induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rasH2 mice; these results were compared with our previous molecular data of N-ethyl-N-nitrosourea-induced forestomach SCCs and urethane-induced lung adenomas in rasH2 mice. Overexpression of the transgene was detected in the DMBA-induced SCCs, suggesting that the transgene plays an important role in enhanced carcinogenesis in rasH2 mice. In addition, the mouse endogenous ras genes were up-regulated in the DMBA-induced SCCs, and are probably involved in the tumorigenesis of forestomach SCCs. Genes such as osteopontin, Cks1b, Tpm1, Reck, gelsolin, and amphiregulin that were commonly altered in these three different carcinogen-induced tumors may contribute to the development of tumors in rasH2 mice.

Oncogenic Ras induces inflammatory cytokine production by up-regulating the squamous cell carcinoma antigens SerpinB3/B4

PubMed Central

Pan, Ji-An; Sun, Yu; Shi, Chanjuan; Li, Jinyu; Powers, R. Scott; Crawford, Howard C.; Zong, Wei-Xing

2014-01-01

Mounting evidence indicates that oncogenic Ras can modulate cell autonomous inflammatory cytokine production, although the underlying mechanism remains unclear. Here we show that squamous cell carcinoma antigens 1 and 2 (SCCA1/2), members of the Serpin family of serine/cysteine protease inhibitors, are transcriptionally up-regulated by oncogenic Ras via MAPK and the ETS family transcription factor PEA3. Increased SCCA expression leads to inhibition of protein turnover, unfolded protein response, activation of NF-κB, and is essential for Ras-mediated cytokine production and tumor growth. Analysis of human colorectal and pancreatic tumor samples reveals a positive correlation between Ras mutation, enhanced SCCA expression, and IL-6 expression. These results indicate that SCCA is a Ras-responsive factor that has a role in Ras-associated cytokine production and tumorigenesis. PMID:24759783

Absence of ras-gene hot-spot mutations in canine fibrosarcomas and melanomas.

PubMed

Murua Escobar, Hugo; Günther, Kathrin; Richter, Andreas; Soller, Jan T; Winkler, Susanne; Nolte, Ingo; Bullerdiek, Jörn

2004-01-01

Point mutations within ras proto-oncogenes, particularly within the mutational hot-spot codons 12, 13 and 61, are frequently detected in human malignancies and in different types of experimentally-induced tumours in animals. So far little is known about ras mutations in naturally occurring canine fibrosarcomas or K-ras mutations in canine melanomas. To elucidate whether ras mutations exist in these naturally occurring tumours in dogs, in the present study we screened 13 canine fibrosarcomas, 2 feline fibrosarcomas and 11 canine melanomas for point mutations, particularly within the mutational hot-spots, making this the first study to investigate a large number of canine fibrosarcomas. None of the samples showed a K- or N-ras hot spot mutation. Thus, our data strongly suggest that ras mutations at the hot-spot loci are very rare and do not play a major role in the pathogenesis of the spontaneously occurring canine tumours investigated.

EGFR and Ras regulate DDX59 during lung cancer development.

PubMed

Yang, Lin; Zhang, Hanyin; Chen, Dan; Ding, Peikun; Yuan, Yunchang; Zhang, Yandong

2018-02-05

Oncogenes EGFR and ras are frequently mutated and activated in human lung cancers. In this report, we found that both EGFR and Ras signaling can upregulate RNA helicase DDX59 in lung cancer cells. DDX59 can be induced through the mitogen activated protein kinase (MAPK) pathway after EGFR or Ras activation. Inhibitors for Ras/Raf/MAP pathway significantly decreased DDX59 expression at both protein and mRNA levels. Through immunohistochemistry, we found that DDX59 protein expression correlated with Ras and EGFR mutation status in human lung adenocarcinoma. Finally, through a xenograft nude mice model, we demonstrated that DDX59 is pivotal for EGFR mutated lung cancer cell growth in vivo. Our study identified a novel protein downstream of Ras and EGFR, which may serve as a potential therapeutic drug target for lung cancer patients. Copyright © 2017 Elsevier B.V. All rights reserved.

The renin-angiotensin system in thyroid disorders and its role in cardiovascular and renal manifestations.

PubMed

Vargas, Félix; Rodríguez-Gómez, Isabel; Vargas-Tendero, Pablo; Jimenez, Eugenio; Montiel, Mercedes

2012-04-01

Thyroid disorders are among the most common endocrine diseases and affect virtually all physiological systems, with an especially marked impact on cardiovascular and renal systems. This review summarizes the effects of thyroid hormones on the renin-angiotensin system (RAS) and the participation of the RAS in the cardiovascular and renal manifestations of thyroid disorders. Thyroid hormones are important regulators of cardiac and renal mass, vascular function, renal sodium handling, and consequently blood pressure (BP). The RAS acts globally to control cardiovascular and renal functions, while RAS components act systemically and locally in individual organs. Various authors have implicated the systemic and local RAS in the mediation of functional and structural changes in cardiovascular and renal tissues due to abnormal thyroid hormone levels. This review analyzes the influence of thyroid hormones on RAS components and discusses the role of the RAS in BP, cardiac mass, vascular function, and renal abnormalities in thyroid disorders.

Subcellular characteristics of functional intracellular renin–angiotensin systems☆

PubMed Central

Abadir, Peter M.; Walston, Jeremy D.; Carey, Robert M.

2013-01-01

The renin–angio tensin system (RAS) is now regarded as an integral component in not only the development of hypertension, but also in physiologic and pathophysiologic mechanisms in multiple tissues and chronic disease states. While many of the endocrine (circulating), paracrine (cell-to-different cell) and autacrine (cell-to-same cell) effects of the RAS are believed to be mediated through the canonical extracellular RAS, a complete, independent and differentially regulated intracellular RAS (iRAS) has also been proposed. Angiotensinogen, the enzymes renin and angiotensin-converting enzyme (ACE) and the angiotensin peptides can all be synthesized and retained intracellularly. Angiotensin receptors (types I and 2) are also abundant intracellularly mainly at the nuclear and mitochondrial levels. The aim of this review is to focus on the most recent information concerning the subcellular localization, distribution and functions of the iRAS and to discuss the potential consequences of activation of the subcellular RAS on different organ systems. PMID:23032352

Field performance of RAS test sections and laboratory investigation of impact of rejuvenators on engineering properties of RAP/RAS mixes.

DOT National Transportation Integrated Search

2014-04-01

In the last several years, recycled asphalt shingles (RAS), in addition to reclaimed asphalt pavement (RAP), : have been widely used in Texas. The use of RAS can significantly reduce the cost of asphalt mixtures, conserve : energy, and protect the en...

The Yeast Saccharomyces cerevisiae as a Model for Understanding RAS Proteins and Their Role in Human Tumorigenesis

PubMed Central

Cazzanelli, Giulia; Francisco, Rita; Azevedo, Luísa; Carvalho, Patrícia Dias; Almeida, Ana; Côrte-Real, Manuela; Oliveira, Maria José; Lucas, Cândida; Sousa, Maria João

2018-01-01

The exploitation of the yeast Saccharomyces cerevisiae as a biological model for the investigation of complex molecular processes conserved in multicellular organisms, such as humans, has allowed fundamental biological discoveries. When comparing yeast and human proteins, it is clear that both amino acid sequences and protein functions are often very well conserved. One example of the high degree of conservation between human and yeast proteins is highlighted by the members of the RAS family. Indeed, the study of the signaling pathways regulated by RAS in yeast cells led to the discovery of properties that were often found interchangeable with RAS proto-oncogenes in human pathways, and vice versa. In this work, we performed an updated critical literature review on human and yeast RAS pathways, specifically highlighting the similarities and differences between them. Moreover, we emphasized the contribution of studying yeast RAS pathways for the understanding of human RAS and how this model organism can contribute to unveil the roles of RAS oncoproteins in the regulation of mechanisms important in the tumorigenic process, like autophagy. PMID:29463063

Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras.

PubMed

Xu, Shenyuan; Long, Brian N; Boris, Gabriel H; Chen, Anqi; Ni, Shuisong; Kennedy, Michael A

2017-12-01

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras-GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.

Role of the renin-angiotensin system in hepatic fibrosis and portal hypertension.

PubMed

Shim, Kwang Yong; Eom, Young Woo; Kim, Moon Young; Kang, Seong Hee; Baik, Soon Koo

2018-05-01

The renin-angiotensin system (RAS) is an important regulator of cirrhosis and portal hypertension. As hepatic fibrosis progresses, levels of the RAS components angiotensin (Ang) II, Ang-(1-7), angiotensin-converting enzyme (ACE), and Ang II type 1 receptor (AT1R) are increased. The primary effector Ang II regulates vasoconstriction, sodium homoeostasis, fibrosis, cell proliferation, and inflammation in various diseases, including liver cirrhosis, through the ACE/Ang II/AT1R axis in the classical RAS. The ACE2/Ang-(1-7)/Mas receptor and ACE2/Ang-(1-9)/AT2R axes make up the alternative RAS and promote vasodilation, antigrowth, proapoptotic, and anti-inflammatory effects; thus, countering the effects of the classical RAS axis to reduce hepatic fibrogenesis and portal hypertension. Patients with portal hypertension have been treated with RAS antagonists such as ACE inhibitors, Ang receptor blockers, and aldosterone antagonists, with very promising hemodynamic results. In this review, we examine the RAS, its roles in hepatic fibrosis and portal hypertension, and current therapeutic approaches based on the use of RAS antagonists in patients with portal hypertension.

RAS and sex differences in diabetic nephropathy.

PubMed

Clotet, Sergi; Riera, Marta; Pascual, Julio; Soler, Maria José

2016-03-09

The incidence and progression of kidney diseases are influenced by sex. The renin-angiotensin system (RAS) is an important regulator of cardiovascular and renal function. Sex differences in the renal response to RAS blockade have been demonstrated. Circulating and renal RAS has been shown to be altered in type 1 and type 2 diabetes; this enzymatic cascade plays a critical role in the development of diabetic nephropathy (DN). Angiotensin converting enzyme (ACE) and ACE2 are differentially regulated depending on its localization within the diabetic kidney. Furthermore, clinical and experimental studies have shown that circulating levels of sex hormones are clearly modulated in the context of diabetes, suggesting that sex-dependent RAS regulation may be also be affected in these individuals. The effect of sex hormones on circulating and renal RAS may be involved in the sex differences observed in DN progression. In this paper we will review the influence of sex hormones on RAS expression and its relation to diabetic kidney disease. A better understanding of the sex dimorphism on RAS might provide a new approach for diabetic kidney disease treatment. Copyright © 2015, American Journal of Physiology - Renal Physiology.

Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis

PubMed Central

Bunda, Severa; Burrell, Kelly; Heir, Pardeep; Zeng, Lifan; Alamsahebpour, Amir; Kano, Yoshihito; Raught, Brian; Zhang, Zhong-Yin; Zadeh, Gelareh; Ohh, Michael

2015-01-01

Ras is phosphorylated on a conserved tyrosine at position 32 within the switch I region via Src kinase. This phosphorylation inhibits the binding of effector Raf while promoting the engagement of GTPase-activating protein (GAP) and GTP hydrolysis. Here we identify SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signalling. In comparison to normal astrocytes, SHP2 activity is elevated in astrocytes isolated from glioblastoma multiforme (GBM)-prone H-Ras(12V) knock-in mice as well as in glioma cell lines and patient-derived GBM specimens exhibiting hyperactive Ras. Pharmacologic inhibition of SHP2 activity attenuates cell proliferation, soft-agar colony formation and orthotopic GBM growth in NOD/SCID mice and decelerates the progression of low-grade astrocytoma to GBM in a spontaneous transgenic glioma mouse model. These results identify SHP2 as a direct activator of Ras and a potential therapeutic target for cancers driven by a previously ‘undruggable' oncogenic or hyperactive Ras. PMID:26617336

Trabecular meshwork ECM remodeling in glaucoma: could RAS be a target?

PubMed

Agarwal, Puneet; Agarwal, Renu

2018-06-14

Disturbances of extracellular matrix (ECM) homeostasis in trabecular meshwork (TM) cause increased aqueous outflow resistance leading to elevated intraocular pressure (IOP) in glaucomatous eyes. Therefore, restoration of ECM homeostasis is a rational approach to prevent disease progression. Since renin-angiotensin system (RAS) inhibition positively alters ECM homeostasis in cardiovascular pathologies involving pressure and volume overload, it is likely that RAS inhibitors reduce IOP primarily by restoring ECM homeostasis. Areas covered: Current evidence showing the presence of RAS components in ocular tissue and its role in regulating aqueous humor dynamics is briefly summarized. The role of RAS in ECM remodeling is discussed both in terms of its effects on ECM synthesis and its breakdown. The mechanisms of ECM remodeling involving interactions of RAS with transforming growth factor-β, Wnt/β-catenin signaling, bone morphogenic proteins, connective tissue growth factor, and matrix metalloproteinases in ocular tissue are discussed. Expert opinion: Current literature strongly indicates a significant role of RAS in ECM remodeling in TM of hypertensive eyes. Hence, IOP-lowering effect of RAS inhibitors may primarily be attributed to restoration of ECM homeostasis in aqueous outflow pathways rather than its vascular effects. However, the mechanistic targets for RAS inhibitors have much wider distribution and consequences, which remain relatively unexplored in TM.

Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS.

PubMed

Yao, Zhan; Yaeger, Rona; Rodrik-Outmezguine, Vanessa S; Tao, Anthony; Torres, Neilawattie M; Chang, Matthew T; Drosten, Matthias; Zhao, Huiyong; Cecchi, Fabiola; Hembrough, Todd; Michels, Judith; Baumert, Hervé; Miles, Linde; Campbell, Naomi M; de Stanchina, Elisa; Solit, David B; Barbacid, Mariano; Taylor, Barry S; Rosen, Neal

2017-08-10

Approximately 200 BRAF mutant alleles have been identified in human tumours. Activating BRAF mutants cause feedback inhibition of GTP-bound RAS, are RAS-independent and signal either as active monomers (class 1) or constitutively active dimers (class 2). Here we characterize a third class of BRAF mutants-those that have impaired kinase activity or are kinase-dead. These mutants are sensitive to ERK-mediated feedback and their activation of signalling is RAS-dependent. The mutants bind more tightly than wild-type BRAF to RAS-GTP, and their binding to and activation of wild-type CRAF is enhanced, leading to increased ERK signalling. The model suggests that dysregulation of signalling by these mutants in tumours requires coexistent mechanisms for maintaining RAS activation despite ERK-dependent feedback. Consistent with this hypothesis, melanomas with these class 3 BRAF mutations also harbour RAS mutations or NF1 deletions. By contrast, in lung and colorectal cancers with class 3 BRAF mutants, RAS is typically activated by receptor tyrosine kinase signalling. These tumours are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. We have thus defined three distinct functional classes of BRAF mutants in human tumours. The mutants activate ERK signalling by different mechanisms that dictate their sensitivity to therapeutic inhibitors of the pathway.

p120Ras-GAP binds the DLC1 Rho-GAP tumor suppressor protein and inhibits its RhoA GTPase and growth-suppressing activities.

PubMed

Yang, X-Y; Guan, M; Vigil, D; Der, C J; Lowy, D R; Popescu, N C

2009-03-19

DLC1 (deleted in liver cancer 1), which encodes a Rho GTPase-activating protein (Rho-GAP), is a potent tumor suppressor gene that is frequently inactivated in several human cancers. DLC1 is a multidomain protein that has been shown previously to bind members of the tensin gene family. Here we show that p120Ras-GAP (Ras-GAP; also known as RASA1) interacts and extensively colocalizes with DLC1 in focal adhesions. The binding was mapped to the SH3 domain located in the N terminus of Ras-GAP and to the Rho-GAP catalytic domain located in the C terminus of the DLC1. In vitro analyses with purified proteins determined that the isolated Ras-GAP SH3 domain inhibits DLC1 Rho-GAP activity, suggesting that Ras-GAP is a negative regulator of DLC1 Rho-GAP activity. Consistent with this possibility, we found that ectopic overexpression of Ras-GAP in a Ras-GAP-insensitive tumor line impaired the growth-suppressing activity of DLC1 and increased RhoA activity in vivo. Our observations expand the complexity of proteins that regulate DLC1 function and define a novel mechanism of the cross talk between Ras and Rho GTPases.1R01CA129610

Coupled excitable Ras and F-actin activation mediates spontaneous pseudopod formation and directed cell movement

PubMed Central

van Haastert, Peter J. M.; Keizer-Gunnink, Ineke; Kortholt, Arjan

2017-01-01

Many eukaryotic cells regulate their mobility by external cues. Genetic studies have identified >100 components that participate in chemotaxis, which hinders the identification of the conceptual framework of how cells sense and respond to shallow chemical gradients. The activation of Ras occurs during basal locomotion and is an essential connector between receptor and cytoskeleton during chemotaxis. Using a sensitive assay for activated Ras, we show here that activation of Ras and F-actin forms two excitable systems that are coupled through mutual positive feedback and memory. This coupled excitable system leads to short-lived patches of activated Ras and associated F-actin that precede the extension of protrusions. In buffer, excitability starts frequently with Ras activation in the back/side of the cell or with F-actin in the front of the cell. In a shallow gradient of chemoattractant, local Ras activation triggers full excitation of Ras and subsequently F-actin at the side of the cell facing the chemoattractant, leading to directed pseudopod extension and chemotaxis. A computational model shows that the coupled excitable Ras/F-actin system forms the driving heart for the ordered-stochastic extension of pseudopods in buffer and for efficient directional extension of pseudopods in chemotactic gradients. PMID:28148648

RasGRP1 regulates antigen-induced developmental programming by naive CD8 T cells.

PubMed

Priatel, John J; Chen, Xiaoxi; Huang, Yu-Hsuan; Chow, Michael T; Zenewicz, Lauren A; Coughlin, Jason J; Shen, Hao; Stone, James C; Tan, Rusung; Teh, Hung Sia

2010-01-15

Ag encounter by naive CD8 T cells initiates a developmental program consisting of cellular proliferation, changes in gene expression, and the formation of effector and memory T cells. The strength and duration of TCR signaling are known to be important parameters regulating the differentiation of naive CD8 T cells, although the molecular signals arbitrating these processes remain poorly defined. The Ras-guanyl nucleotide exchange factor RasGRP1 has been shown to transduce TCR-mediated signals critically required for the maturation of developing thymocytes. To elucidate the role of RasGRP1 in CD8 T cell differentiation, in vitro and in vivo experiments were performed with 2C TCR transgenic CD8 T cells lacking RasGRP1. In this study, we report that RasGRP1 regulates the threshold of T cell activation and Ag-induced expansion, at least in part, through the regulation of IL-2 production. Moreover, RasGRP1(-/-) 2C CD8 T cells exhibit an anergic phenotype in response to cognate Ag stimulation that is partially reversible upon the addition of exogenous IL-2. By contrast, the capacity of IL-2/IL-2R interactions to mediate Ras activation and CD8 T cell expansion and differentiation appears to be largely RasGRP1-independent. Collectively, our results demonstrate that RasGRP1 plays a selective role in T cell signaling, controlling the initiation and duration of CD8 T cell immune responses.

Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution.

PubMed

Coyle, Scott M; Lim, Wendell A

2016-01-14

The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras's ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease.

Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Audemard, Eric

2014-08-22

Highlights: • Oncogenic H-ras stimulates emission of extracellular vesicles containing double-stranded DNA. • Vesicle-associated extracellular DNA contains mutant N-ras sequences. • Vesicles mediate intercellular transfer of mutant H-ras DNA to normal fibroblasts where it remains for several weeks. • Fibroblasts exposed to vesicles containing H-ras DNA exhibit increased proliferation. - Abstract: Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellularmore » vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30 days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells.« less

Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk signaling pathway

PubMed Central

Drosten, Matthias; Sum, Eleanor Y. M.; Lechuga, Carmen G.; Simón-Carrasco, Lucía; Jacob, Harrys K. C.; García-Medina, Raquel; Huang, Sidong; Beijersbergen, Roderick L.; Bernards, Rene; Barbacid, Mariano

2014-01-01

The Ras family of small GTPases constitutes a central node in the transmission of mitogenic stimuli to the cell cycle machinery. The ultimate receptor of these mitogenic signals is the retinoblastoma (Rb) family of pocket proteins, whose inactivation is a required step to license cell proliferation. However, little is known regarding the molecular events that connect Ras signaling with the cell cycle. Here, we provide genetic evidence to illustrate that the p53/p21 Cdk-interacting protein 1 (Cip1)/Rb axis is an essential component of the Ras signaling pathway. Indeed, knockdown of p53, p21Cip1, or Rb restores proliferative properties in cells arrested by ablation of the three Ras loci, H-, N- and K-Ras. Ras signaling selectively inactivates p53-mediated induction of p21Cip1 expression by inhibiting acetylation of specific lysine residues in the p53 DNA binding domain. Proliferation of cells lacking both Ras proteins and p53 can be prevented by reexpression of the human p53 ortholog, provided that it retains an active DNA binding domain and an intact lysine residue at position 164. These results unveil a previously unidentified role for p53 in preventing cell proliferation under unfavorable mitogenic conditions. Moreover, we provide evidence that cells lacking Ras and p53 proteins owe their proliferative properties to the unexpected retroactivation of the Raf/Mek/Erk cascade by a Ras-independent mechanism. PMID:25288756

Endometrial carcinomas with significant mucinous differentiation associated with higher frequency of k-ras mutations: a morphologic and molecular correlation study.

PubMed

Xiong, Jinjun; He, Mai; Jackson, Cynthia; Ou, Joyce J; Sung, C James; Breese, Virgina; Steinhoff, Margaret M; Quddus, M Ruhul; Tejada-Berges, Trevor; Lawrence, W Dwayne

2013-09-01

K-ras gene product in the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway is critical in the development of certain types of malignancies. K-ras mutation-associated pancreatic and ovarian carcinomas often display mucinous differentiation. Previous studies have shown that k-ras mutation is found in 10% to 30% of endometrial carcinomas. We investigated k-ras mutations in several morphologic subtypes of endometrial carcinomas with particular emphasis on various degrees of mucinous differentiation. Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue sections. Polymerase chain reaction amplification for k-ras codons 12 and 13 were performed, followed by sequencing using capillary electrophoresis. The Fisher exact test is used to compare the prevalent difference of k-ras mutation among the groups. P < 0.05 was considered significant. K-ras mutations were detected in 8 (80%) of 10 mucinous carcinomas, 12 (67%) of 18 endometrioid carcinomas (ECs) with significant mucinous differentiation (ECMD), 4 (25%) of 16 ECs, and 1 (9%) of 11 serous carcinomas. The differences were statistically significant between mucinous carcinomas versus EC (P < 0.01) and ECMD versus EC (P < 0.05). The findings suggest that mucinous carcinoma and endometrioid carcinoma with significant mucinous component are more likely to be associated with k-ras mutation. Potential clinical implications of k-ras mutation lies in the management of recurrent or higher-stage endometrial mucinous tumors, which would not be responsive to treatment protocols containing epidermal growth factor receptor inhibitors.

Mechanism study of low-energy laser irradiation-induced lung adenocarcinoma cell proliferation by FRET in living cell

NASA Astrophysics Data System (ADS)

Wang, Fang; Chen, Xiao-Chuan; Xing, Da

2004-07-01

Low-energy laser irradiation (LELI) has been shown to promote cell proliferation in various cell types, yet the mechanism of which has not been fully clarified. The Ras/Raf/MEK (mitogen-activated protein kinase)ERK kinase)/ERK (extracellular-signal-regulated kinase) signaling pathway is a network that govern proliferation, differentiation and cell survival. Recent studies suggested that Ras/Raf/MEK/ERK pathway is involved in the LELI-induced cell proliferation. Here, we utilized fluorescence resonance energy transfer (FRET) technique to investigate the effect of LELI on Ras/Raf signaling pathway in living cells. Raichu-Ras reporter plasmid was utilized which consisted of fusions of H-ras, the Ras-binding domain of Raf(RafRBD), a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP), so that intramolecular binding of GTP-Ras to RafRBD brings CFP close to YFP and increases FRET between CFP and YFP. Human lung adenocarcinoma cell line (ASTC-a-1) were transfected with the plasmid (pRaichu-Ras) and then were treated by LELI. The living cell imaging showed the increase of FRET at different time points after LELI at the dose of 1.8 J/cm2, which corresponds to the Ras/Raf activation assayed by Western Blotting. Furthermore, this dose of LELI enhanced the proliferation of ASTC-a-1 cells. Taken together, these in vivo imaging data provide direct evidences with temporal or spatial resolution that Ras/Raf/MEK/ pathway plays an important role in LELI-promoted cell proliferation.

Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice.

PubMed

Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y

2014-05-22

Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR-Ras-Raf-MEK-ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [(3)H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras-MAPK activity could be important in its anticancer activity.

Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

PubMed Central

Pathan, Akbar Ali Khan; Panthi, Bhavana; Khan, Zahid; Koppula, Purushotham Reddy; Alanazi, Mohammed Saud; Sachchidanand; Parine, Narasimha Reddy; Chourasia, Mukesh

2016-01-01

Objective Kirsten rat sarcoma (K-Ras) protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results Interestingly, the designed compounds exhibit a binding preference for the “off” state over “on” state conformation of K-Ras protein. Moreover, the designed compounds’ interactions are similar to guanosine diphosphate and, thus, could presumably act as a potential lead for K-Ras. The predicted drug-likeness properties of these compounds suggest that these compounds follow the Lipinski’s rule of five and have tolerable absorption, distribution, metabolism, excretion and toxicity values. Conclusion Thus, through the current study, we propose targeting only “off” state conformations as a promising strategy for the design of reversible inhibitors to pharmacologically inhibit distinct conformations of K-Ras protein. PMID:27217775

Conserved electrostatic fields at the Ras-effector interface measured through vibrational Stark effect spectroscopy explain the difference in tilt angle in the Ras binding domains of Raf and RalGDS.

PubMed

Walker, David M; Wang, Ruifei; Webb, Lauren J

2014-10-07

Vibrational Stark effect (VSE) spectroscopy was used to measure the electrostatic fields present at the interface of the human guanosine triphosphatase (GTPase) Ras docked with the Ras binding domain (RBD) of the protein kinase Raf. Nine amino acids located on the surface of Raf were selected for labeling with a nitrile vibrational probe. Eight of the probe locations were situated along the interface of Ras and Raf, and one probe was 2 nm away on the opposite side of Raf. Vibrational frequencies of the nine Raf nitrile probes were compared both in the monomeric, solvated protein and when docked with wild-type (WT) Ras to construct a comprehensive VSE map of the Ras-Raf interface. Molecular dynamics (MD) simulations employing an umbrella sampling strategy were used to generate a Boltzmann-weighted ensemble of nitrile positions in both the monomeric and docked complexes to determine the effect that docking has on probe location and orientation and to aid in the interpretation of VSE results. These results were compared to an identical study that was previously conducted on nine nitrile probes on the RBD of Ral guanidine dissociation stimulator (RalGDS) to make comparisons between the docked complexes formed when either of the two effectors bind to WT Ras. This comparison finds that there are three regions of conserved electrostatic fields that are formed upon docking of WT Ras with both downstream effectors. Conservation of this pattern in the docked complex then results in different binding orientations observed in otherwise structurally similar proteins. This work supports an electrostatic cause of the known binding tilt angle between the Ras-Raf and Ras-RalGDS complexes.

The Use of Footstep Sounds as Rhythmic Auditory Stimulation for Gait Rehabilitation in Parkinson's Disease: A Randomized Controlled Trial.

PubMed

Murgia, Mauro; Pili, Roberta; Corona, Federica; Sors, Fabrizio; Agostini, Tiziano A; Bernardis, Paolo; Casula, Carlo; Cossu, Giovanni; Guicciardi, Marco; Pau, Massimiliano

2018-01-01

The use of rhythmic auditory stimulation (RAS) has been proven useful in the management of gait disturbances associated with Parkinson's disease (PD). Typically, the RAS consists of metronome or music-based sounds (artificial RAS), while ecological footstep sounds (ecological RAS) have never been used for rehabilitation programs. The aim of this study was to compare the effects of a rehabilitation program integrated either with ecological or with artificial RAS. An observer-blind, randomized controlled trial was conducted to investigate the effects of 5 weeks of supervised rehabilitation integrated with RAS. Thirty-eight individuals affected by PD were randomly assigned to one of the two conditions (ecological vs. artificial RAS); thirty-two of them (age 68.2 ± 10.5, Hoehn and Yahr 1.5-3) concluded all phases of the study. Spatio-temporal parameters of gait and clinical variables were assessed before the rehabilitation period, at its end, and after a 3-month follow-up. Thirty-two participants were analyzed. The results revealed that both groups improved in the majority of biomechanical and clinical measures, independently of the type of sound. Moreover, exploratory analyses for separate groups were conducted, revealing improvements on spatio-temporal parameters only in the ecological RAS group. Overall, our results suggest that ecological RAS is equally effective compared to artificial RAS. Future studies should further investigate the role of ecological RAS, on the basis of information revealed by our exploratory analyses. Theoretical, methodological, and practical issues concerning the implementation of ecological sounds in the rehabilitation of PD patients are discussed. www.ClinicalTrials.gov, identifier NCT03228888.

Differential Expression of IL-17, 22 and 23 in the Progression of Colorectal Cancer in Patients with K-ras Mutation: Ras Signal Inhibition and Crosstalk with GM-CSF and IFN-γ

PubMed Central

Petanidis, Savvas; Anestakis, Doxakis; Argyraki, Maria; Hadzopoulou-Cladaras, Margarita; Salifoglou, Athanasios

2013-01-01

Recent studies have suggested that aberrant K-ras signaling is responsible for triggering immunological responses and inflammation-driven tumorigenesis. Interleukins IL-17, IL-22, and IL-23 have been reported in various types of malignancies, but the exact mechanistic role of these molecules remains to be elucidated. Given the role of K-ras and the involvement of interleukins in colorectal tumorigenesis, research efforts are reported for the first time, showing that differentially expressed interleukin IL-17, IL-22, and IL-23 levels are associated with K-ras in a stage-specific fashion along colorectal cancer progression. Specifically, a) the effect of K-ras signaling was investigated in the overall expression of interleukins in patients with colorectal cancer and healthy controls, and b) an association was established between mutant K-ras and cytokines GM-CSF and IFN-γ. The results indicate that specific interleukins are differentially expressed in K-ras positive patients and the use of K-ras inhibitor Manumycin A decreases both interleukin levels and apoptosis in Caco-2 cells by inhibiting cell viability. Finally, inflammation-driven GM-CSF and IFN-γ levels are modulated through interleukin expression in tumor patients, with interleukin expression in the intestinal lumen and cancerous tissue mediated by aberrant K-ras signaling. Collectively, the findings a) indicate that interleukin expression is influenced by ras signaling and specific interleukins play an oncogenic promoter role in colorectal cancer, highlighting the molecular link between inflammation and tumorigenesis, and b) accentuate the interwoven molecular correlations as leads to new therapeutic approaches in the future. PMID:24040001

Identification of Differentially Expressed K-Ras Transcript Variants in Patients With Leiomyoma.

PubMed

Zolfaghari, Nooshin; Shahbazi, Shirin; Torfeh, Mahnaz; Khorasani, Maryam; Hashemi, Mehrdad; Mahdian, Reza

2017-10-01

Molecular studies have demonstrated a wide range of gene expression variations in uterine leiomyoma. The rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase (RAS/RAF/MAPK) is the crucial cellular pathway in transmitting external signals into nucleus. Deregulation of this pathway contributes to excessive cell proliferation and tumorigenesis. The present study aims to investigate the expression profile of the K-Ras transcripts in tissue samples from patients with leiomyoma. The patients were leiomyoma cases who had no mutation in mediator complex subunit 12 ( MED12) gene. A quantitative approach has been applied to determine the difference in the expression of the 2 main K-Ras messenger RNA (mRNA) variants. The comparison between gene expression levels in leiomyoma and normal myometrium group was performed using relative expression software tool. The expression of K-Ras4B gene was upregulated in leiomyoma group ( P = .016), suggesting the involvement of K-Ras4B in the disease pathogenesis. Pairwise comparison of the K-Ras4B expression between each leiomyoma tissue and its matched adjacent normal myometrium revealed gene upregulation in 68% of the cases. The expression of K-Ras4A mRNA was relatively upregulated in leiomyoma group ( P = .030). In addition, the mean expression of K-Ras4A gene in leiomyoma tissues relative to normal samples was 4.475 (95% confidence interval: 0.10-20.42; standard error: 0.53-12.67). In total, 58% of the cases showed more than 2-fold increase in K-Ras4A gene expression. Our results demonstrated increased expression of both K-Ras mRNA splicing variants in leiomyoma tissue. However, the ultimate result of KRAS expression on leiomyoma development depends on the overall KRAS isoform balance and, consequently, on activated signaling pathways.

R-Ras Regulates Migration through an Interaction with Filamin A in Melanoma Cells

PubMed Central

Gawecka, Joanna E.; Griffiths, Genevieve S.; Ek-Rylander, Barbro; Ramos, Joe W.; Matter, Michelle L.

2010-01-01

Background Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins. Methods and Findings We identified Filamin A (FLNa) as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin β1, β2 and β7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaΔ3) abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaΔ3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly. Conclusions These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration. PMID:20585650

RAS mutation status predicts survival and patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases.

PubMed

Vauthey, Jean-Nicolas; Zimmitti, Giuseppe; Kopetz, Scott E; Shindoh, Junichi; Chen, Su S; Andreou, Andreas; Curley, Steven A; Aloia, Thomas A; Maru, Dipen M

2013-10-01

To determine the impact of RAS mutation status on survival and patterns of recurrence in patients undergoing curative resection of colorectal liver metastases (CLM) after preoperative modern chemotherapy. RAS mutation has been reported to be associated with aggressive tumor biology. However, the effect of RAS mutation on survival and patterns of recurrence after resection of CLM remains unclear. Somatic mutations were analyzed using mass spectroscopy in 193 patients who underwent single-regimen modern chemotherapy before resection of CLM. The relationship between RAS mutation status and survival outcomes was investigated. Detected somatic mutations included RAS (KRAS/NRAS) in 34 (18%), PIK3CA in 13 (7%), and BRAF in 2 (1%) patients. At a median follow-up of 33 months, 3-year overall survival (OS) rates were 81% in patients with wild-type versus 52.2% in patients with mutant RAS (P = 0.002); 3-year recurrence-free survival (RFS) rates were 33.5% with wild-type versus 13.5% with mutant RAS (P = 0.001). Liver and lung recurrences were observed in 89 and 83 patients, respectively. Patients with RAS mutation had a lower 3-year lung RFS rate (34.6% vs 59.3%, P < 0.001) but not a lower 3-year liver RFS rate (43.8% vs 50.2%, P = 0.181). In multivariate analyses, RAS mutation predicted worse OS [hazard ratio (HR) = 2.3, P = 0.002), overall RFS (HR = 1.9, P = 0.005), and lung RFS (HR = 2.0, P = 0.01), but not liver RFS (P = 0.181). RAS mutation predicts early lung recurrence and worse survival after curative resection of CLM. This information may be used to individualize systemic and local tumor-directed therapies and follow-up strategies.

RAS mutation status predicts survival and patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases

PubMed Central

Vauthey, Jean-Nicolas; Zimmitti, Giuseppe; Kopetz, Scott E.; Shindoh, Junichi; Chen, Su S.; Andreou, Andreas; Curley, Steven A.; Aloia, Thomas A.; Maru, Dipen M.

2013-01-01

Objective To determine the impact of RAS mutation status on survival and patterns of recurrence in patients undergoing curative resection of colorectal liver metastases (CLM) after preoperative modern chemotherapy. Summary Background Data RAS mutation has been reported to be associated with aggressive tumor biology. However, the effect of RAS mutation on survival and patterns of recurrence after resection of CLM remains unclear. Methods Somatic mutations were analyzed using mass spectroscopy in 193 patients who underwent single-regimen modern chemotherapy before resection of CLM. The relationship between RAS mutation status and survival outcomes was investigated. Results Detected somatic mutations included RAS (KRAS/NRAS) in 34 patients (18%), PIK3CA in 13 (7%), and BRAF in 2 (1%). At a median follow-up of 33 months, 3-year overall survival (OS) rates were 81% in patients with wild-type vs 52.2% in patients with mutant RAS (P=0.002); 3-year recurrence-free survival (RFS) rates were 33.5% with wild-type vs 13.5% with mutant RAS (P=0.001). Liver and lung recurrences were observed in 89 and 83 patients, respectively. Patients with RAS mutation had a lower 3-year lung RFS rate (34.6% vs 59.3%, P<0.001), but not a lower 3-year liver RFS rate (43.8% vs 50.2%, P=0.181). In multivariate analyses, RAS mutation predicted worse OS (hazard ratio [HR] 2.3, P=0.002), overall RFS (HR 1.9, P=0.005), and lung RFS (HR 2.0, P=0.01), but not liver RFS (P=0.181). Conclusions RAS mutation predicts early lung recurrence and worse survival after curative resection of CLM. This information may be used to individualize systemic and local tumor-directed therapies and follow-up strategies. PMID:24018645

Differential requirement of RasGRP1 for γδ T cell development and activation

PubMed Central

Chen, Yong; Ci, Xinxin; Gorentla, Balachandra; Sullivan, Sarah A.; Stone, James C.; Zhang, Weiguo; Pereira, Pablo; Lu, Jianxin; Zhong, Xiao-Ping

2012-01-01

γδ T cells (γδT) belong to a distinct T cell lineage that performs immune functions different from αβ T cells (αβT). Previous studies have established that Erk1/2 MAPKs are critical for positive selection of αβT cells. Additional evidence also suggests that elevated Erk1/2 activity promotes γδT cell generation. RasGRP1, a guanine nucleotide releasing factor for Ras, plays an important role in positive selection of αβT cells by activating the Ras-Erk1/2 pathway. In this report, we demonstrate that RasGRP1 is critical for TCR-induced Erk1/2 activation in γδT cells but exerts different roles for γδT cell generation and activation. Deficiency of RasGRP1 does not obviously affect γδT cell numbers in the thymus but leads to increased γδT cells, particularly CD4−CD8+ γδT cells, in the peripheral lymphoid organs. The virtually unhindered γδT cell development in the RasGRP1−/− thymus proved to be cell intrinsic, while the increase in CD8+ γδT cells is caused by non-cell-intrinsic mechanisms. Our data provides genetic evidence that decreased Erk1/2 activation in the absence of RasGRP1 is compatible for γδT cell generation. Although RasGRP1 is dispensable for γδT cell generation, RasGRP1-deficient γδT cells are defective in proliferation following TCR stimulation. Additionally, RasGRP1-deficient γδT cells are impaired to produce IL-17 but not IFNγ. Together, these observations have revealed that RasGRP1 plays differential roles for γδ and αβ T cell development but is critical for γδT cell proliferation and production of IL-17. PMID:22623331

Early superoxide scavenging accelerates renal microvascular rarefaction and damage in the stenotic kidney.

PubMed

Kelsen, Silvia; He, Xiaochen; Chade, Alejandro R

2012-08-15

Renal artery stenosis (RAS), the main cause of chronic renovascular disease (RVD), is associated with significant oxidative stress. Chronic RVD induces renal injury partly by promoting renal microvascular (MV) damage and blunting MV repair in the stenotic kidney. We tested the hypothesis that superoxide anion plays a pivotal role in MV dysfunction, reduction of MV density, and progression of renal injury in the stenotic kidney. RAS was induced in 14 domestic pigs and observed for 6 wk. Seven RAS pigs were chronically treated with the superoxide dismutase mimetic tempol (RAS+T) to reduce oxidative stress. Single-kidney hemodynamics and function were quantified in vivo using multidetector computer tomography (CT) and renal MV density was quantified ex vivo using micro-CT. Expression of angiogenic, inflammatory, and apoptotic factors was measured in renal tissue, and renal apoptosis and fibrosis were quantified in tissue sections. The degree of RAS and blood pressure were similarly increased in RAS and RAS+T. Renal blood flow (RBF) and glomerular filtration rate (GFR) were reduced in the stenotic kidney (280.1 ± 36.8 and 34.2 ± 3.1 ml/min, P < 0.05 vs. control). RAS+T kidneys showed preserved GFR (58.5 ± 6.3 ml/min, P = not significant vs. control) but a similar decreases in RBF (293.6 ± 85.2 ml/min) and further decreases in MV density compared with RAS. These changes were accompanied by blunted angiogenic signaling and increased apoptosis and fibrosis in the stenotic kidney of RAS+T compared with RAS. The current study shows that tempol administration provided limited protection to the stenotic kidney. Despite preserved GFR, renal perfusion was not improved by tempol, and MV density was further reduced compared with untreated RAS, associated with increased renal apoptosis and fibrosis. These results suggest that a tight balance of the renal redox status is necessary for a normal MV repair response to injury, at least at the early stage of RVD, and raise caution regarding antioxidant strategies in RAS.

Phosphatidylinositol 3-kinase, Cdc42, and Rac1 act downstream of Ras in integrin-dependent neurite outgrowth in N1E-115 neuroblastoma cells.

PubMed

Sarner, S; Kozma, R; Ahmed, S; Lim, L

2000-01-01

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A Ras(H40C;G12V) double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated Ras(G12V)-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42(G12V) was Rac1 dependent. Cdc42(G12V)-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42(G12V)-induced outgrowth did not need Ras or PI 3-kinase activity. Active Rho(G14V) reduced outgrowth promoted by Ras(G12V). Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells.

Phosphatidylinositol 3-Kinase, Cdc42, and Rac1 Act Downstream of Ras in Integrin-Dependent Neurite Outgrowth in N1E-115 Neuroblastoma Cells

PubMed Central

Sarner, Shula; Kozma, Robert; Ahmed, Sohail; Lim, Louis

2000-01-01

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A RasH40C;G12V double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated RasG12V-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42G12V was Rac1 dependent. Cdc42G12V-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42G12V-induced outgrowth did not need Ras or PI 3-kinase activity. Active RhoG14V reduced outgrowth promoted by RasG12V. Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells. PMID:10594018

Early superoxide scavenging accelerates renal microvascular rarefaction and damage in the stenotic kidney

PubMed Central

Kelsen, Silvia; He, Xiaochen

2012-01-01

Renal artery stenosis (RAS), the main cause of chronic renovascular disease (RVD), is associated with significant oxidative stress. Chronic RVD induces renal injury partly by promoting renal microvascular (MV) damage and blunting MV repair in the stenotic kidney. We tested the hypothesis that superoxide anion plays a pivotal role in MV dysfunction, reduction of MV density, and progression of renal injury in the stenotic kidney. RAS was induced in 14 domestic pigs and observed for 6 wk. Seven RAS pigs were chronically treated with the superoxide dismutase mimetic tempol (RAS+T) to reduce oxidative stress. Single-kidney hemodynamics and function were quantified in vivo using multidetector computer tomography (CT) and renal MV density was quantified ex vivo using micro-CT. Expression of angiogenic, inflammatory, and apoptotic factors was measured in renal tissue, and renal apoptosis and fibrosis were quantified in tissue sections. The degree of RAS and blood pressure were similarly increased in RAS and RAS+T. Renal blood flow (RBF) and glomerular filtration rate (GFR) were reduced in the stenotic kidney (280.1 ± 36.8 and 34.2 ± 3.1 ml/min, P < 0.05 vs. control). RAS+T kidneys showed preserved GFR (58.5 ± 6.3 ml/min, P = not significant vs. control) but a similar decreases in RBF (293.6 ± 85.2 ml/min) and further decreases in MV density compared with RAS. These changes were accompanied by blunted angiogenic signaling and increased apoptosis and fibrosis in the stenotic kidney of RAS+T compared with RAS. The current study shows that tempol administration provided limited protection to the stenotic kidney. Despite preserved GFR, renal perfusion was not improved by tempol, and MV density was further reduced compared with untreated RAS, associated with increased renal apoptosis and fibrosis. These results suggest that a tight balance of the renal redox status is necessary for a normal MV repair response to injury, at least at the early stage of RVD, and raise caution regarding antioxidant strategies in RAS. PMID:22622460

Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor.

PubMed

Tu, Zheng; Gui, Liming; Wang, Jianliu; Li, Xiaoping; Sun, Pengming; Wei, Lihui

2006-05-01

To investigate the tumorigenesis of mutant [12Asp]-K-ras in endometrial carcinoma and its relationship with ER. We constructed pcDI-[12Asp]K-ras4B by inserting full-length [12Asp]K-ras4B from human endometrial carcinoma Hec-1A cells, into pcDI vector. Cell proliferation of NIH3T3 after transfection with pcDI-[12Asp]K-ras4B was measured by MTT assay. The cell transformation was determined by colony formation and tumor nodule development. [12Asp]-K-ras4B-NIH3T3 cells were transfected with constitutively active pCMV-RafCAAX and dominant-negative pCMV-RafS621A. Cell growth was measured by MTT assay and [3H]thymidine incorporation. After transfected with pcDI-[12Asp]K-ras4B or pCMV-RafS621A, the cells were harvested for Western blot and reporter assay to determine the expression and transcriptional activity of ERalpha and ERbeta, respectively. [12Asp]-K-ras4B enhanced NIH3T3 cells proliferation after 48 h post-transfection (P < 0.05). More colonies were grown 10 days after incubating pcDI-[12Asp]-K-ras4B-NIH3T3 cells (13.48%) than pcDI-NIH3T3 (4.26%) or untreated NIH3T3 (2.33%). The pcDI-[12Asp]-K-ras4B-NIH3T3 cells injected to the nude mice Balb/C developed tumor nodules with poor-differentiated cells after 12 days. An increase of ERalpha and ERbeta was observed in pcDI-[12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERalpha and ERbeta expression. Estrogen induced the ER transcriptional activity by 5-fold in pcDI-NIH3T3 cells, 13-fold in pcDI-[12Asp]K-ras4B-NIH3T3 and 19-fold in HEC-1A. RafS621A suppressed the ER transcriptional activity. K-ras mutation induces tumorigenesis in endometrium, and this malignant transformation involves Raf signaling pathway and ER.

Economic evaluation study (CHEER-compliant): Cost-effectiveness analysis of RAS screening for treatment of metastatic colorectal cancer based on the CALGB 80405 trial.

PubMed

Zhou, Jing; Zhao, Rongce; Wen, Feng; Zhang, Pengfei; Tang, Ruilei; Chen, Hongdou; Zhang, Jian; Li, Qiu

2016-07-01

Cetuximab (Cetux)/Bevacizumab (Bev) treatments have shown considerably survival benefits for patients with metastatic colorectal cancer (mCRC) in the last decade. But they are costly. Currently, no data is available on the health economic implications of testing for extended RAS wild-type (wt) prior to Cetux/Bev treatments of patients with mCRC. This paper aimed to evaluate the cost-effectiveness of predictive testing for extended RAS-wt status in mCRC in the context of targeting the use of Cetux/Bev.Markov model 1 was conducted to provide evidence evaluating the cost-effectiveness of predictive testing for KRAS-wt or extended RAS-wt status based on treatments of chemotherapy plus Cetux/Bev. Markov model 2 assessed the cost-effectiveness of FOLFOX plus Cetux/Bev or FOLFIRI plus Cetux/Bev in extended RAS-wt population. Primary base case data were identified from the CALGB 80405 trial and the literatures. Costs were estimated from West China Hospital, Sichuan University, China. Survival benefits were reported in quality-adjusted life-years (QALYs). The incremental cost-effectiveness ratio (ICER) was calculated.In analysis 1, the cost per QALY was $88,394.09 for KRAS-Cetux, $80,797.82 for KRAS-Bev, $82,590.72 for RAS-Cetux, and $75,358.42 for RAS-Bev. The ICER for RAS-Cetux versus RAS-Bev was $420,700.50 per QALY gained. In analysis 2, the cost per QALY was $81,572.61, $80,856.50, $80,592.22, and $66,794.96 for FOLFOX-Cetux, FOLFOX-Bev, FOLFIRI-Cetux, and FOLFIRI-Bev, respectively. The analyses showed that the extended RAS-wt testing was less costly and more effective versus KRAS-wt testing before chemotherapy plus Cetux/Bev. Furthermore, FOLFIRI plus Bev was the most cost-effective strategy compared with others in extended RAS-wt population.It was economically favorable to identify patients with extended RAS-wt status. Furthermore, FOLFIRI plus Bev was the preferred strategy in extended RAS-wt patients.

Treatment of Ras-induced cancers by the F-actin cappers tensin and chaetoglobosin K, in combination with the caspase-1 inhibitor N1445.

PubMed

Tikoo, A; Cutler, H; Lo, S H; Chen, L B; Maruta, H

1999-01-01

For transforming normal fibroblasts to malignant cells, oncogenic Ras mutants such as v-Ha-ras require Rho family GTPases (Rho, Rac, and CDC42) that are responsible for controlling actin-cytoskeleton organization. Ras activates Rac through a PI-3 kinase-mediated pathway. Rac causes uncapping of actin filaments (F-actin) at the plus-ends, through phosphatidylinositol 4,5 bisphosphate (PIP2), and eventually induces membrane ruffling. Several distinct F-actin/PIP2-binding proteins, such as gelsolin, which severs and caps the plus-ends of actin filaments, or HS1, which cross-links actin filaments, have been shown to suppress v-Ha-Ras-induced malignant transformation when they are overexpressed. Interestingly, an F-actin cross-linking drug (photosensitizer) called MKT-077 suppresses Ras transformation. Thus, an F-actin capping/severing drug might also have an anticancer potential. This study was conducted to determine first whether Ras-induced malignant phenotype (anchorage-independent growth) is suppressed by overexpression of the gene encoding a large plus-end F-actin capping protein called tensin and second to test the anti-Ras potential of a unique fungal antibiotic (small compound) called chaetoglobosin K (CK) that also caps the plus-ends of actin filaments. DNA transfection with a retroviral vector carrying the tensin cDNA was used to overexpress tensin in v-Ha-Ras-transformed NIH 3T3 cells. All stable tensin transfectants rarely formed colonies in soft agar, indicating that tensin suppresses the anchorage-independent growth. The anti-Ras action of CK was determined by incubating the Ras-transformants in the presence of CK in soft agar. Two microM CK almost completely inhibited their colony formation, indicating that CK also suppresses the malignant phenotype. However, unlike tensin, CK causes an apoptosis of Ras-transformed NIH 3T3 cells and, less effectively, of normal NIH 3T3 cells, indicating that CK has an F-actin capping-independent side effect(s). CK-induced apoptosis is at least in part caused by CK-induced inhibition of the kinase PKB/AKT. However, a specific ICE/caspase-1 inhibitor called N1445 completely abolished the CK-induced apoptosis by reactivating PKB, but without affecting the CK-induced suppression of Ras transformation. Like the F-actin cross-linking drug MKT-077, the F-actin capping drug CK may be useful for the treatment of Ras-associated cancers if it is combined with the ICE inhibitor N1445, which abolishes the side effect of CK. Our observations that two distinct F-actin capping molecules (i.e., tensin and CK) suppress Ras-induced malignant phenotype strongly suggest, if not prove, that capping of actin filaments at the plus-ends alone is sufficient to block one of the Ras signaling pathways essential for its oncogenicity. This notion is compatible with the fact that Ras induces the uncapping of actin filaments at the plus-ends through the Rac/PIP2 pathway.

The potential of targeting Ras proteins in lung cancer.

PubMed

McCormick, Frank

2015-04-01

The Ras pathway is a major driver in lung adenocarcinoma: over 75% of all cases harbor mutations that activate this pathway. While spectacular clinical successes have been achieved by targeting activated receptor tyrosine kinases in this pathway, little, if any, significant progress has been achieved targeting Ras proteins themselves or cancers driven by oncogenic Ras mutants. New approaches to drug discovery, new insights into Ras function, new ways of attacking undruggable proteins through RNA interference and new ways of harnessing the immune system could change this landscape in the relatively near future.

Mind the GAP: A Novel Tumor-Promoting Mechanism | Center for Cancer Research

Cancer.gov

RAS proteins, like light switches, toggle between an “on” conformation where they promote cell growth, survival, and/or the formation of blood vessels (known as angiogenesis) and an “off” conformation in which they are unable to stimulate their target effector proteins. Nearly one-third of human tumors express a mutated RAS gene, which encodes a protein locked permanently in the active state. Other tumors, including liver hepatocellular carcinomas (HCCs), display aberrant RAS pathway signaling but lack RAS gene mutations, suggesting alternative mechanisms for this excessive RAS activity.

Ras trafficking, localization and compartmentalized signalling

PubMed Central

Prior, Ian A.; Hancock, John F.

2012-01-01

Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes. PMID:21924373

Assertiveness expectancies: how hard people push depends on the consequences they predict.

PubMed

Ames, Daniel R

2008-12-01

The present article seeks to explain varying levels of assertiveness in interpersonal conflict and negotiations with assertiveness expectancies, idiosyncratic predictions people make about the social and instrumental consequences of assertive behavior. This account complements motivation-based models of assertiveness and competitiveness, suggesting that individuals may possess the same social values (e.g., concern for relationships) but show dramatically different assertiveness due to different assumptions about behavioral consequences. Results clarify the form of assertiveness expectancies, namely that most people assume increasing assertiveness can yield positive social and instrumental benefits up to a point, beyond which benefits decline. However, people vary in how assertive this perceived optimal point is. These individual differences in expectancies are linked in 4 studies to assertiveness, including self-reported assertiveness, rated behavioral preferences in assorted interpersonal conflict scenarios, partner ratings of participants' behavior in a face-to-face dyadic negotiation, and work colleague ratings of participants' assertiveness in the workplace. In each case, the link between expectancies and behavior remained after controlling for values. The results suggest a place for expectancies alongside values in psychological models of interpersonal assertiveness.

RAP/RAS workshop.

DOT National Transportation Integrated Search

2013-01-01

: RAP & RAS increases mix stiffness : : Most WMA additives decrease stiffness : : Tear-Off shingles are stiffer than Man-waste shingles : : Using multiple recycled bins improves consistency : : Finer RAS material improves consiste...

RAS Initiative - Events

Cancer.gov

The NCI RAS Initiative has organized multiple events with outside experts to discuss how the latest scientific and technological breakthroughs can be applied to discover vulnerabilities in RAS-driven cancers.

Telecommuting's differential impact on work-family conflict: is there no place like home?

PubMed

Golden, Timothy D; Veiga, John F; Simsek, Zeki

2006-11-01

The literature on the impact of telecommuting on work-family conflict has been equivocal, asserting that telecommuting enhances work-life balance and reduces conflict, or countering that it increases conflict as more time and emotional energy are allocated to family. Surveying 454 professional-level employees who split their work time between an office and home, the authors examined how extensively working in this mode impacts work-to-family conflict and family-to-work conflict, as well as the contextual impact of job autonomy, scheduling flexibility, and household size. As hypothesized, the findings suggest that telecommuting has a differential impact on work-family conflict, such that the more extensively individuals work in this mode, the lower their work-to-family conflict, but the higher their family-to-work conflict. Additionally, job autonomy and scheduling flexibility were found to positively moderate telecommuting's impact on work-to-family conflict, but household size was found to negatively moderate telecommuting's impact on family-to-work conflict, suggesting that contextual factors may be domain specific. (c) 2006 APA, all rights reserved

Characterization of c-Ki-ras and N-ras oncogenes in aflatoxin B sub 1 -induced rat liver tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

McMahon, G.; Davis, E.F.; Huber, L.J.

c-Ki-ras and N-ras oncogenes have been characterized in aflatoxin B{sub 1}-induced hepatocellular carcinomas. Detection of different protooncogene and oncogene sequences and estimation of their frequency distribution were accomplished by polymerase chain reaction, cloning, and plaque screening methods. Two c-Ki-ras oncogene sequences were identified in DNA from liver tumors that contained nucleotide changes absent in DNA from livers of untreated control rats. Sequence changes involving G{center dot}C to T{center dot}A or G{center dot}C to A{center dot}T nucleotide substitutions in codon 12 were scored in three of eight tumor-bearing animals. Distributions of c-Ki-ras sequences in tumors and normal liver DNA indicated thatmore » the observed nucleotide changes were consistent with those expected to result from direct mutagenesis of the germ-line protooncogene by aflatoxin B{sub 1}. N-ras oncogene sequences were identified in DNA from two of eight tumors. Three N-ras gene regions were identified, one of which was shown to be associated with an oncogene containing a putative activating amino acid residing at codon 13. All three N-ras sequences, including the region detected in N-ras oncogenes, were present at similar frequencies in DNA samples from control livers as well as liver tumors. The presence of a potential germ-line oncogene may be related to the sensitivity of the Fischer rat strain to liver carcinogenesis by aflatoxin B{sub 1} and other chemical carcinogens.« less

Secreted Glioblastoma Nanovesicles Contain Intracellular Signaling Proteins and Active Ras Incorporated in a Farnesylation-dependent Manner*

PubMed Central

Luhtala, Natalie; Aslanian, Aaron; Yates, John R.; Hunter, Tony

2017-01-01

Glioblastomas (GBMs) are malignant brain tumors with a median survival of less than 18 months. Redundancy of signaling pathways represented within GBMs contributes to their therapeutic resistance. Exosomes are extracellular nanovesicles released from cells and present in human biofluids that represent a possible biomarker of tumor signaling state that could aid in personalized treatment. Herein, we demonstrate that mouse GBM cell-derived extracellular nanovesicles resembling exosomes from an H-RasV12 myr-Akt mouse model for GBM are enriched for intracellular signaling cascade proteins (GO: 0007242) and Ras protein signal transduction (GO: 0007265), and contain active Ras. Active Ras isolated from human and mouse GBM extracellular nanovesicles lysates using the Ras-binding domain of Raf also coprecipitates with ESCRT (endosomal sorting complex required for transport)-associated exosome proteins Vps4a and Alix. Although we initially hypothesized a role for active Ras protein signaling in exosome biogenesis, we found that GTP binding of K-Ras was dispensable for its packaging within extracellular nanovesicles and for the release of Alix. By contrast, farnesylation of K-Ras was required for its packaging within extracellular nanovesicles, yet expressing a K-Ras farnesylation mutant did not decrease the number of nanovesicles or the amount of Alix protein released per cell. Overall, these results emphasize the primary importance of membrane association in packaging of extracellular nanovesicle factors and indicate that screening nanovesicles within human fluids could provide insight into tissue origin and the wiring of signaling proteins at membranes to predict onset and behavior of cancer and other diseases linked to deregulated membrane signaling states. PMID:27909058

Association of p21ras with phosphatidylinositol 3-kinase.

PubMed Central

Sjölander, A; Yamamoto, K; Huber, B E; Lapetina, E G

1991-01-01

In mammalian cells, ras genes code for 21-kDa GTP-binding proteins. Increased expression and mutations in specific amino acids have been closely linked to alterations of normal cell morphology, growth, and differentiation and, in particular, to neoplastic transformation. The signal transduction induced by these p21ras proteins is largely unknown; however, the signaling pathways of several growth factors have been reported to involve phosphatidylinositol (PtdIns) 3-kinase. In the present study of a Ha-ras-transformed epithelial cell line, we demonstrated increased PtdIns 3-kinase activity in anti-phosphotyrosine and anti-receptor (insulin and hybrid insulin-like growth factor I) immunoprecipitates of cells that had been stimulated with insulin or insulin-like growth factor I. The PtdIns 3-kinase activity was also immunoprecipitated in these experiments by the anti-Ras monoclonal antibody Y13-259. The specificity of this association with p21ras was ascertained by the neutralizing effect of the antigen peptide and the absence of PtdIns 3-kinase activity in Y13-259 immunoprecipitates from cells in which the ras gene was turned off. These data indicate that PtdIns 3-kinase activity is an important step in the cascade of reactions in p21ras signal transduction, suggesting that the alterations of the cytoskeleton and growth in ras-transformed cells could be mediated by PtdIns 3-kinase activity. Images PMID:1716764

K-RasG12D–induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to γ-secretase inhibitors

PubMed Central

Cornejo, Melanie G.; Scholl, Claudia; Liu, Jianing; Leeman, Dena S.; Haydu, J. Erika; Fröhling, Stefan; Lee, Benjamin H.; Gilliland, D. Gary

2008-01-01

To study the impact of oncogenic K-Ras on T-cell leukemia/lymphoma development and progression, we made use of a conditional K-RasG12D murine knockin model, in which oncogenic K-Ras is expressed from its endogenous promoter. Transplantation of whole bone marrow cells that express oncogenic K-Ras into wild-type recipient mice resulted in a highly penetrant, aggressive T-cell leukemia/lymphoma. The lymphoblasts were composed of a CD4/CD8 double-positive population that aberrantly expressed CD44. Thymi of primary donor mice showed reduced cellularity, and immunophenotypic analysis demonstrated a block in differentiation at the double-negative 1 stage. With progression of disease, approximately 50% of mice acquired Notch1 mutations within the PEST domain. Of note, primary lymphoblasts were hypersensitive to γ-secretase inhibitor treatment, which is known to impair Notch signaling. This inhibition was Notch-specific as assessed by down-regulation of Notch1 target genes and intracellular cleaved Notch. We also observed that the oncogenic K-Ras-induced T-cell disease was responsive to rapamycin and inhibitors of the RAS/MAPK pathway. These data indicate that patients with T-cell leukemia with K-Ras mutations may benefit from therapies that target the NOTCH pathway alone or in combination with inhibition of the PI3K/AKT/MTOR and RAS/MAPK pathways. PMID:18663146

Effect of Angiotensin II and Small GTPase Ras Signaling Pathway Inhibition on Early Renal Changes in a Murine Model of Obstructive Nephropathy

PubMed Central

Rodríguez-Peña, Ana B.; Fuentes-Calvo, Isabel; Docherty, Neil G.; Arévalo, Miguel; Grande, María T.; Eleno, Nélida; Pérez-Barriocanal, Fernando; López-Novoa, José M.

2014-01-01

Tubulointerstitial fibrosis is a major feature of chronic kidney disease. Unilateral ureteral obstruction (UUO) in rodents leads to the development of renal tubulointerstitial fibrosis consistent with histopathological changes observed in advanced chronic kidney disease in humans. The purpose of this study was to assess the effect of inhibiting angiotensin II receptors or Ras activation on early renal fibrotic changes induced by UUO. Animals either received angiotensin II or underwent UUO. UUO animals received either losartan, atorvastatin, and farnesyl transferase inhibitor (FTI) L-744,832, or chaetomellic acid A (ChA). Levels of activated Ras, phospho-ERK1/2, phospho-Akt, fibronectin, and α-smooth muscle actin were subsequently quantified in renal tissue by ELISA, Western blot, and/or immunohistochemistry. Our results demonstrate that administration of angiotensin II induces activation of the small GTPase Ras/Erk/Akt signaling system, suggesting an involvement of angiotensin II in the early obstruction-induced activation of renal Ras. Furthermore, upstream inhibition of Ras signalling by blocking either angiotensin AT1 type receptor or by inhibiting Ras prenylation (atorvastatin, FTI o ChA) reduced the activation of the Ras/Erk/Akt signaling system and decreased the early fibrotic response in the obstructed kidney. This study points out that pharmacological inhibition of Ras activation may hold promise as a future strategy in the prevention of renal fibrosis. PMID:25101263

Effect of angiotensin II and small GTPase Ras signaling pathway inhibition on early renal changes in a murine model of obstructive nephropathy.

PubMed

Rodríguez-Peña, Ana B; Fuentes-Calvo, Isabel; Docherty, Neil G; Arévalo, Miguel; Grande, María T; Eleno, Nélida; Pérez-Barriocanal, Fernando; López-Novoa, José M

2014-01-01

Tubulointerstitial fibrosis is a major feature of chronic kidney disease. Unilateral ureteral obstruction (UUO) in rodents leads to the development of renal tubulointerstitial fibrosis consistent with histopathological changes observed in advanced chronic kidney disease in humans. The purpose of this study was to assess the effect of inhibiting angiotensin II receptors or Ras activation on early renal fibrotic changes induced by UUO. Animals either received angiotensin II or underwent UUO. UUO animals received either losartan, atorvastatin, and farnesyl transferase inhibitor (FTI) L-744,832, or chaetomellic acid A (ChA). Levels of activated Ras, phospho-ERK1/2, phospho-Akt, fibronectin, and α-smooth muscle actin were subsequently quantified in renal tissue by ELISA, Western blot, and/or immunohistochemistry. Our results demonstrate that administration of angiotensin II induces activation of the small GTPase Ras/Erk/Akt signaling system, suggesting an involvement of angiotensin II in the early obstruction-induced activation of renal Ras. Furthermore, upstream inhibition of Ras signalling by blocking either angiotensin AT1 type receptor or by inhibiting Ras prenylation (atorvastatin, FTI o ChA) reduced the activation of the Ras/Erk/Akt signaling system and decreased the early fibrotic response in the obstructed kidney. This study points out that pharmacological inhibition of Ras activation may hold promise as a future strategy in the prevention of renal fibrosis.

The effect of renin-angiotensin system blockade on renal protection in chronic kidney disease patients with hyperkalemia.

PubMed

Lee, Ju-Hyun; Kwon, Young Eun; Park, Jung Tak; Lee, Mi Jung; Oh, Hyung Jung; Han, Seung Hyeok; Kang, Shin-Wook; Choi, Kyu Hun; Yoo, Tae-Hyun

2014-12-01

The aim of this study was to determine the effects of renin-angiotensin system (RAS) blockade maintenance on renal protection in chronic kidney disease (CKD) patients with hyperkalemia occurring during treatment with RAS blockade. CKD III or IV patients, who were prescribed with RAS blockers and also had hyperkalemia, were included. The study population was divided into two groups based on maintenance or withdrawal of RAS blocker. Renal outcomes (doubling of creatinine or end-stage renal disease) and incidence of hyperkalemia were compared between the two groups. Out of 258 subjects who developed hyperkalemia during treatment with RAS blockers, 150 (58.1%) patients continued on RAS blockades, while RAS blockades were discontinued for more than 3 months in the remaining 108 patients. Renal event-free survival was significantly higher in the maintenance group compared with the withdrawal group. Cox proportional hazard ratio for renal outcomes was 1.35 (95% CI: 1.08-1.92, p=0.04) in the withdrawal group compared with the maintenance group. However, the incidence of hyperkalemia and hyperkalemia-related hospitalization or mortality did not differ between the two groups. This study demonstrated that the maintenance of RAS blockade is beneficial for the preservation of renal function and relatively tolerable in patients with CKD and hyperkalemia occurring during treatment with RAS blockade. © The Author(s) 2014.

K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions

PubMed Central

Ostrem, Jonathan M.; Peters, Ulf; Sos, Martin L.; Wells, James A.; Shokat, Kevan M.

2014-01-01

Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies1–3. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP4 and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis5,6. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP7 and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner. PMID:24256730

Dual knockdown of N-ras and epiregulin synergistically suppressed the growth of human hepatoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Meng; He, Hong-wei; Sun, Huan-xing

2009-09-18

Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, wemore » evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.« less

Social Desirability Responding in the Measurement of Assertive Behavior.

ERIC Educational Resources Information Center

Kiecolt, Janice; McGrath, Ellen

1979-01-01

Women completed behavioral measures of assertion and anxiety before and after assertiveness training. High social desirability scorers described themselves as more assertive and less anxious, but were behaviorally less assertive than low scorers. Although all scorers improved their assertion skills, high scorers did not appear less anxious after…

Genetic and pharmacological suppression of oncogenic mutations in RAS genes of yeast and humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schafer, W.R.; Sterne, R.; Thorner, J.

1989-07-28

The activity of an oncoprotein and the secretion of a pheromone can be affected by an unusual protein modification. Specifically, posttranslational modification of yeast-a-factor and Ras protein requires an intermediate of the cholesterol biosynthetic pathway. This modification is apparently essential for biological activity. Studies of yeast mutants blocked in sterol biosynthesis demonstrated that the membrane association and biological activation of the yeast Ras2 protein require mevalonate, a precursor of sterols and other isoprenes such as farnesyl pyrophosphate. Furthermore, drugs that inhibit mevalonate biosynthesis blocked the in vivo action of oncogenic derivatives of human Ras protein in the Xenopus oocyte assay.more » The same drugs and mutations also prevented the posttranslational processing and secretion of yeast a-factor, a peptide that is farnesylated. Thus, the mevalonate requirement for Ras activation may indicate that attachment of a mevalonate-derived (isoprenoid) moiety to Ras proteins is necessary for membrane association and biological function. These observations establish a connection between the cholesterol biosynthetic pathway and transformation by the ras oncogene and offer a novel pharmacological approach to investigating, and possibly controlling, ras-mediated malignant transformations. 50 refs., 3 figs., 3 tabs.« less

Introducing risk adjustment and free health plan choice in employer-based health insurance: Evidence from Germany.

PubMed

Pilny, Adam; Wübker, Ansgar; Ziebarth, Nicolas R

2017-12-01

To equalize differences in health plan premiums due to differences in risk pools, the German legislature introduced a simple Risk Adjustment Scheme (RAS) based on age, gender and disability status in 1994. In addition, effective 1996, consumers gained the freedom to choose among hundreds of existing health plans, across employers and state-borders. This paper (a) estimates RAS pass-through rates on premiums, financial reserves, and expenditures and assesses the overall RAS impact on market price dispersion. Moreover, it (b) characterizes health plan switchers and investigates their annual and cumulative switching rates over time. Our main findings are based on representative enrollee panel data linked to administrative RAS and health plan data. We show that sickness funds with bad risk pools and high pre-RAS premiums lowered their total premiums by 42 cents per additional euro allocated by the RAS. Consequently, post-RAS, health plan prices converged but not fully. Because switchers are more likely to be white collar, young and healthy, the new consumer choice resulted in more risk segregation and the amount of money redistributed by the RAS increased over time. Copyright © 2017 Elsevier B.V. All rights reserved.

NMR 1H,13C, 15N backbone and 13C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP.

PubMed

Sharma, Alok K; Lee, Seung-Joo; Rigby, Alan C; Townson, Sharon A

2018-05-02

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here 1 H N, 15 N, and 13 C resonance assignments for the 19.3 kDa (aa 1-169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS G12C-GDP ), using heteronuclear, multidimensional NMR spectroscopy. Backbone 1 H- 15 N correlations have been assigned for all non-proline residues, except for the first methionine residue.

Monoclonal antibodies of predefined specificity detect activated ras gene expression in human mammary and colon carcinomas.

PubMed Central

Hand, P H; Thor, A; Wunderlich, D; Muraro, R; Caruso, A; Schlom, J

1984-01-01

Monoclonal antibodies (MAbs) of predefined specificity have been generated by utilizing a synthetic peptide reflecting amino acid positions 10-17 of the Hu-rasT24 gene product as immunogen. These MAbs, designated RAP-1 through RAP-5 (RA, ras; P, peptide), have been shown to react with the ras gene product p21. Since the Hu-ras reactive determinants (positions 10-17) have been predicted to be within the tertiary structure of the p21 molecule, it was not unexpected that denaturation of cell extracts or tissue sections with Formalin or glutaraldehyde enhanced binding of the RAP MAbs. When paraffin-embedded Formalin-fixed tissue sections and the avidin-biotin complex immunoperoxidase method were used, the RAP MAbs clearly defined enhanced ras p21 expression in the majority of human colon and mammary carcinomas. The majority of all abnormal ducts and lobules from fibroadenoma and fibrocystic disease patients were negative, as were all normal mammary and colonic epithelia examined. The findings reported here form the basis for quantitative radioimmunoassays for a ras translational product and provide a means to evaluate ras p21 expression within individual cells of normal tissues and benign, "premalignant," and malignant lesions. Images PMID:6382261

Lin28-let7 Modulates Radiosensitivity of Human Cancer Cells With Activation of K-Ras

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Jee-Sun.; Kim, Jae-Jin; Byun, Ju-Yeon

2010-01-15

Purpose: To evaluate the potential of targeting Lin28-let7 microRNA regulatory network for overcoming the radioresistance of cancer cells having activated K-Ras signaling. Methods and Materials: A549 lung carcinoma cells and ASPC1 pancreatic cancer cells possessing K-RAS mutation were transfected with pre-let7a microRNA or Lin28 siRNA, respectively. Clonogenic assay, quantitative reverse transcription polymerase chain reaction, and Western analysis were performed. The effects of Lin28 on SQ20B cells having wild-type K-RAS, and a normal fibroblast were also assessed. Results: The overexpression of let-7a decreased expression of K-Ras and radiosensitized A549 cells. Inhibition of Lin28, a repressor of let-7, attenuated K-Ras expression andmore » radiosensitized A549 and ASPC1 cells. Neither SQ20B cells expressing wild-type K-RAS nor HDF, the normal human fibroblasts, were radiosensitized by this approach. Conclusions: The Lin28-let7 regulatory network may be a potentially useful therapeutic target for overcoming the radioresistance of human cancers having activated K-Ras signaling.« less

RasGRP3 limits Toll-like receptor-triggered inflammatory response in macrophages by activating Rap1 small GTPase.

PubMed

Tang, Songqing; Chen, Taoyong; Yu, Zhou; Zhu, Xuhui; Yang, Mingjin; Xie, Bin; Li, Nan; Cao, Xuetao; Wang, Jianli

2014-08-14

Host immune cells can detect and destruct invading pathogens via pattern-recognition receptors. Small Rap GTPases act as conserved molecular switches coupling extracellular signals to various cellular responses, but their roles as regulators in Toll-like receptor (TLR) signalling have not been fully elucidated. Here we report that Ras guanine nucleotide-releasing protein 3 (RasGRP3), a guanine nucleotide-exchange factor activating Ras and Rap1, limits production of proinflammatory cytokines (especially IL-6) in macrophages by activating Rap1 on activation by low levels of TLR agonists. We demonstrate that RasGRP3, a dominant member of RasGRPs in macrophages, impairs TLR3/4/9-induced IL-6 production and relieves dextrane sulphate sodium-induced colitis and collagen-induced arthritis. In RasGRP3-deficient RAW264.7 cells obtained by CRISPR-Cas9 genome editing, TLR3/4/9-induced activation of Rap1 was inhibited while ERK1/2 activation was enhanced. Our study suggests that RasGRP3 limits inflammatory response by activating Rap1 on low-intensity pathogen infection, setting a threshold for preventing excessive inflammatory response.

Clinical and functional outcome of assertive outreach for patients with schizophrenic disorder: Results of a quasi-experimental controlled trial.

PubMed

Kästner, D; Büchtemann, D; Warnke, I; Radisch, J; Baumgardt, J; Giersberg, S; Kopke, K; Moock, J; Kawohl, W; Rössler, W

2015-09-01

The majority of studies support modern assertive health service models. However, the evidence is limited for parts of continental Europe, as well as for the pharmacological adherence outcome parameter. We conducted a quasi-experimental controlled trial including adult patients with a schizophreniform disorder and a maximum of 60 points on the Global Assessment of Functioning Scale (GAF). Interventions (n=176) and controls (TAU, n=142) were assessed every six-month within one year in 17 study practices in rural areas. Mental and functional state were rated using the Brief Psychiatric Rating Scale (BPRS) and the GAF. Functional limitations and pharmacological adherence were patient-rated using the WHO-Disability Assessment Schedule II (WHODAS-II) and the Medication Adherence Report Scale (MARS). We computed multilevel mixed models. The GAF and BPRS of both groups improved significantly, yet the increase in the intervention group was significantly higher. In contrast, patient-rated variables - WHODAS-II and MARS - neither showed a stable temporal improvement nor a difference between groups. Our findings only partly support the investigated AO intervention, because of conflicting results between clinician- and patient-ratings. Accordingly, the benefits of AO need to be further evaluated. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Suppression of survivin expression in glioblastoma cells by the Ras inhibitor farnesylthiosalicylic acid promotes caspase-dependent apoptosis.

PubMed

Blum, Roy; Jacob-Hirsch, Jasmine; Rechavi, Gideon; Kloog, Yoel

2006-09-01

The Ras inhibitor farnesylthiosalicylic acid (FTS) has been shown to induce apoptosis in glioblastoma multiforme, but its mechanism of action was unknown. We show that FTS or dominant-negative Ras, by deregulating extracellular signal-regulated kinase and Akt signaling, decreases survivin gene transcripts in U87 glioblastoma multiforme, leading to disappearance of survivin protein and cell death. FTS affected both Ras-controlled regulators of survivin transcription and Ras-regulated survival signals. Thus, Ras inhibition by FTS resulted in release of the survivin "brake" on apoptosis and in activation of the mitochondrial apoptotic pathway: dephosphorylation of Bad, activation of Bax, release of cytochrome c, and caspase activation. FTS-induced apoptosis of U87 cells was strongly attenuated by forced expression of survivin or by caspase inhibitors. These results show that resistance to apoptosis in glioblastoma multiforme can be abolished by a single Ras inhibitor, which targets both survivin, a critical inhibitor of apoptosis, and the intrinsic mitochondrial apoptotic machinery.

About the RAS Initiative

Cancer.gov

The RAS Initiative, a "hub and spoke" model, connects researchers to better understand and target the more than 30% of cancers driven by mutations in RAS genes. Includes oversight and contact information.

Evaluation of transplant renal artery blood flow by Doppler sound-spectrum analysis.

PubMed

Reinitz, E R; Goldman, M H; Sais, J; Rittgers, S E; Lee, H M; Mendez-Picon, G; Muakkassa, W F; Barnes, R W

1983-04-01

Doppler ultrasonography sound-spectrum analysis (SSA) was used to evaluate blood flow in the transplanted kidney and its renal artery. Seven patients with posttransplant hypertension and a bruit over the transplanted kidney were screened for renal artery stenosis (RAS). In five patients, RAS was diagnosed by SSA, and in two it was not. These findings were confirmed by subsequent angiography in all patients. Three patients studied after surgical correction of their RAS had improvement in their SSA patterns. Fourteen hypertensive patients with a cause other than RAS were evaluated by SSA. None of them had SSA findings suggestive of RAS. Doppler ultrasonography with SSA is an effective, noninvasive technique of monitoring transplant renal blood flow, especially in the screening of hypertensive transplant recipients for transplant RAS.

[miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

PubMed

Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

2016-12-23

Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples ( n =5) and cervical intraepithelial neoplasia tissue samples ( n =5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P <0.05). The MTT assay revealed that the cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group ( P <0.05), and the cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group ( P <0.05) but higher than the miR-143 mimic group significantly ( P <0.05). The expression levels of K-ras protein in the miR-143 mimic group, the negative control group and the miR-143 mimic+ K-ras group were lowest, moderate, and highest, respectively (115.27±34.08, 521.36±41.89, and 706.52±89.44, all P <0.05). In the tissue samples, the miR-143 expression in the cervical cancer group was significantly lower than that of the cervical intraepithelial neoplasia group (0.32±0.06 vs. 0.93±0.17, P <0.05); whereas the K-ras protein expression in the cervical cancer group was significantly higher than that in the cervical intraepithelial neoplasia group (584.39±72.34 vs. 114.23±25.82, P <0.05). Conclusions: In vitro, miR-143 can inhibit the proliferative activity of HeLa cells through targeted regulating the expression of K-ras gene. In human cervical cancer tissues of a small sample set, the expression of miR-143 is downregulated, and the expression of K-ras is upregulated.

Ras plasma membrane signalling platforms

PubMed Central

2005-01-01

The plasma membrane is a complex, dynamic structure that provides platforms for the assembly of many signal transduction pathways. These platforms have the capacity to impose an additional level of regulation on cell signalling networks. In this review, we will consider specifically how Ras proteins interact with the plasma membrane. The focus will be on recent studies that provide novel spatial and dynamic insights into the micro-environments that different Ras proteins utilize for signal transduction. We will correlate these recent studies suggesting Ras proteins might operate within a heterogeneous plasma membrane with earlier biochemical work on Ras signal transduction. PMID:15954863

Assertive communication in condom negotiation: Insights from late adolescent couples’ subjective ratings of self and partner

PubMed Central

Schmid, Amy; Leonard, Noelle R.; Ritchie, Amanda S.; Gwadz, Marya V.

2015-01-01

Purpose Assertive communication has been associated with higher levels of condom use among youth using self-report survey methodology. The purpose of this study was to examine the subjective ratings of assertiveness among young, romantically-involved couples in the context of a condom negotiation task. Methods Using an innovative video-recall procedure, 32 couples (64 youth) engaged in a videotaped condom negotiation task and then rated self and partners’ level of assertiveness. Both individual ratings of assertiveness and couple-level assertiveness were assessed using dyadic hierarchical linear modeling. Results Individuals’ assertiveness was positively associated with condom use. Unexpectedly, the overall level of assertiveness in couples showed a curvilinear association with condom use. Very high and very low assertiveness was associated with lower condom use, while moderate levels of assertiveness were associated with higher condom use. Conclusions Moderate levels of assertiveness during condom negotiation may facilitate condom use in young couples. Increasing condom use among romantic partners may require developing interventions that strengthen youths’ ability to engage in assertive communication strategies that balance emotional intimacy with self-advocacy. PMID:25937470

A Perspective for Assertiveness Training for Blacks

ERIC Educational Resources Information Center

Minor, Billy Joe

1978-01-01

This article discussess assertiveness in the Black-White encounter. The author delineates the assertive options available to Blacks, distortions in interpretation of assertive messages, and strategies for more effective and efficient assertions. (Author)

Celebrating 50 years of the laser(Joint scientific session of the Physical Sciences Division of the Russian Academy of Sciences and of the Scientific Councils of the P N Lebedev Physical Institute, RAS and the A M Prokhorov General Physics Institute, RAS, 21 April 2010)

NASA Astrophysics Data System (ADS)

2011-01-01

A joint scientific session of the Physical Sciences Division of the Russian Academy of Sciences (RAS) and the scientific councils of the P N Lebedev Physical Institute, RAS and the A M Prokhorov General Physics Institute, RAS dedicated to the 50th anniversary of the advent of the laser was held in the conference hall of the Lebedev Physical Institute on 21 April 2010. The following reports were put on the session's agenda posted on the website www.gpad.ac.ru of the Physical Sciences Division, RAS: (1) Alferov Zh I (A F Ioffe Physical-Technical Institute RAS, St. Petersburg) "Semiconductor heterostructure lasers"; (2) Bagaev S N (Institute of Laser Physics, Siberian Branch, RAS, Novosibirsk) "Ultrahigh-resolution spectra and their fundamental application"; (3) Masalov A V (P N Lebedev Physical Institute, RAS, Moscow) "Optical Department of the Lebedev Physical Institute: early work on lasers"; (4) Garnov S V, Shcherbakov I A (A M Prokhorov General Physics Institute, RAS, Moscow) "Laser sources of megavolt terahertz pulses"; (5) Sergeev A M, Khazanov E A (Institute of Applied Physics, RAS, Nizhny Novgorod) "Structural functions of a developed turbulence"; (6) Popov Yu M (P N Lebedev Physical Institute, RAS, Moscow) "The early history of semiconductor lasers"; (7) Manenkov A A (A M Prokhorov General Physics Institute, RAS, Moscow) "Self-focusing laser pulses: current state and future prospects". The papers written on the basis of reports 3, 4, 6, and 7 are published below. A comprehensive version of report 5 prepared in the form of a review paper is published in this issue of Physics-Uspekhi on p. 9. • Optical Department of the Lebedev Physical Institute: early work on lasers, A V Masalov Physics-Uspekhi, 2011, Volume 54, Number 1, Pages 87-91 • Laser methods for generating megavolt terahertz pulses, S V Garnov, I A Shcherbakov Physics-Uspekhi, 2011, Volume 54, Number 1, Pages 91-96 • The early history of the injection laser, Yu M Popov Physics-Uspekhi, 2011, Volume 54, Number 1, Pages 96-100 • Self-focusing of laser pulses: current state and future prospects, A A Manenkov Physics-Uspekhi, 2011, Volume 54, Number 1, Pages 100-104

Detection of K-ras gene mutations in feces by magnetic nanoprobe in patients with pancreatic cancer: A preliminary study.

PubMed

Wang, Xiaoguang; Wang, Jingshuai; Chen, Fei; Zhong, Zhengxiang; Qi, Lifeng

2018-01-01

The present study aimed to investigate the feasibility and effectiveness of detecting K-ras mutation by using magnetic nanoparticles in fecal samples of patients with pancreatic cancer at different stages. The novel methodology of K-ras mutation detection was compared to the existing methodology of cancer antigen (CA)19-9 examination. Patients with pancreatic cancer (n=88), pancreatic benign diseases who displayed chronic pancreatitis (n=35), pancreatic mucinous cyst neoplasms (n=10) and pancreatic serous cyst (n=9) admitted to the Department of Surgery, Jiaxing Second Hospital were enrolled in the present study. Fecal samples were collected from all patients, DNA was extracted and magnetic nanoprobe was then used to detect K-ras mutation. The results obtained using the novel magnetic nanoprobe detection technique showed a K-ras mutation rate of 81.8% (72/88) in the patients with pancreatic cancer and 18.5% (10/54) in patients with pancreatic benign diseases. In patients with pancreatic cancer, the K-ras mutation rate was comparable in stages I + IIA and IIB + III + IV (78.9 vs. 84.0%; P>0.05). The sensitivity and specificity of K-ras mutation for detection of pancreatic cancer was 81.8 and 81.5%, respectively. Sixty-eight pancreatic cancer patients had >37 U/ml CA99 with a sensitivity and specificity for pancreatic cancer detection of 77.3 and 77.8%, which was not significantly lower than detection by the fecal K-ras mutations (P>0.05). Combinational detection of fecal K-ras mutations and serum CA19-9 significantly increased the sensitivity regarding pancreatic cancer detection to 97.7% (P<0.05), while the specificity was not enhanced (80.9%; P>0.05) compared with fecal K-ras mutations or CA19-9 alone. The findings showed that the magnetic nanoprobe is able to detect fecal K-ras mutations in different stages of pancreatic cancer, with comparable sensitivity and specificity to CA19-9 examination for differentiating pancreatic cancer. Furthermore, combined detection of CA19-9 and K-ras mutations has enhanced sensitivity compared with CA19-9 alone.

Executable assertions and flight software

NASA Technical Reports Server (NTRS)

Mahmood, A.; Andrews, D. M.; Mccluskey, E. J.

1984-01-01

Executable assertions are used to test flight control software. The techniques used for testing flight software; however, are different from the techniques used to test other kinds of software. This is because of the redundant nature of flight software. An experimental setup for testing flight software using executable assertions is described. Techniques for writing and using executable assertions to test flight software are presented. The error detection capability of assertions is studied and many examples of assertions are given. The issues of placement and complexity of assertions and the language features to support efficient use of assertions are discussed.

Involvement of Prolonged Ras Activation in Thrombopoietin-Induced Megakaryocytic Differentiation of a Human Factor-Dependent Hematopoietic Cell Line

PubMed Central

Matsumura, Itaru; Nakajima, Koichi; Wakao, Hiroshi; Hattori, Seisuke; Hashimoto, Koji; Sugahara, Hiroyuki; Kato, Takashi; Miyazaki, Hiroshi; Hirano, Toshio; Kanakura, Yuzuru

1998-01-01

Thrombopoietin (TPO) is a hematopoietic growth factor that plays fundamental roles is both megakaryopoiesis and thrombopoiesis through binding to its receptor, c-mpl. Although TPO has been shown to activate various types of intracellular signaling molecules, such as the Janus family of protein tyrosine kinases, signal transducers and activators of transcription (STATs), and ras, the precise mechanisms underlying TPO-induced proliferation and differentiation remain unknown. In an effort to clarify the mechanisms of TPO-induced proliferation and differentiation, c-mpl was introduced into F-36P, a human interleukin-3 (IL-3)-dependent erythroleukemia cell line, and the effects of TPO on the c-mpl-transfected F-36P (F-36P-mpl) cells were investigated. F-36P-mpl cells were found to proliferate and differentiate at a high rate into mature megakaryocytes in response to TPO. Dominant-negative (dn) forms of STAT1, STAT3, STAT5, and ras were inducibly expressed in F-36P-mpl cells, and their effects on TPO-induced proliferation and megakaryocytic differentiation were analyzed. Among these dn molecules, both dn ras and dn STAT5 reduced TPO- or IL-3-induced proliferation of F-36P-mpl cells by ∼30%, and only dn ras could inhibit TPO-induced megakaryocytic differentiation. In accord with this result, overexpression of activated ras (H-rasG12V) for 5 days led to megakaryocytic differentiation of F-36P-mpl cells. In a time course analysis on H-rasG12V-induced differentiation, activation of the ras pathway for 24 to 28 h was required and sufficient to induce megakaryocytic differentiation. Consistent with this result, the treatment of F-36P-mpl cells with TPO was able to induce prolonged activation of ras for more than 24 h, whereas IL-3 had only a transient effect. These results suggest that prolonged ras activation may be involved in TPO-induced megakaryocytic differentiation. PMID:9632812

Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression.

PubMed

DeSmet, Marsha L; Fleet, James C

2017-10-01

High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH) 2 D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH) 2 D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH) 2 D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (-300bp), an enhancer region at -6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH) 2 D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differentiated State of Initiating Tumor Cells Is Key to Distinctive Immune Responses Seen in H-RasG12V-Induced Squamous Tumors.

PubMed

Podolsky, Michael A; Bailey, Jacob T; Gunderson, Andrew J; Oakes, Carrie J; Breech, Kyle; Glick, Adam B

2017-03-01

Heterogeneity in tumor immune responses is a poorly understood yet critical parameter for successful immunotherapy. In two doxycycline-inducible models where oncogenic H-Ras G12V is targeted either to the epidermal basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or committed progenitor/suprabasal cells with an Involucrin-tTA transgene (InvRas), we observed strikingly distinct tumor immune responses. On threshold doxycycline levels yielding similar Ras expression, tumor latency, and numbers, tumors from K14Ras mice had an immunosuppressed microenvironment, whereas InvRas tumors had a proinflammatory microenvironment. On a Rag1 -/- background, InvRas mice developed fewer and smaller tumors that regressed over time, whereas K14Ras mice developed more tumors with shorter latency than Rag1 +/+ controls. Adoptive transfer and depletion studies revealed that B-cell and CD4 T-cell cooperation was critical for tumor yield, lymphocyte polarization, and tumor immune phenotype in Rag1 +/+ mice of both models. Coculture of tumor-conditioned B cells with CD4 T cells implicated direct contact for Th1 and regulatory T cell (Treg) polarization, and CD40-CD40L for Th1, Th2, and Treg generation, a response not observed from splenic B cells. Anti-CD40L caused regression of InvRas tumors but enhanced growth in K14Ras, whereas a CD40 agonist mAb had opposite effects in each tumor model. These data show that position of tumor-initiating cells within a stratified squamous epithelial tissue provokes distinct B- and CD4 T-cell interactions, which establish unique tumor microenvironments that regulate tumor development and response to immunotherapy. Cancer Immunol Res; 5(3); 198-210. ©2017 AACR . ©2017 American Association for Cancer Research.

RAS Initiative - Community Outreach

Cancer.gov

Through community and technical collaborations, workshops and symposia, and the distribution of reference reagents, the RAS Initiative seeks to increase the sharing of knowledge and resources essential to defeating cancers caused by mutant RAS genes.

Relationship Between the Children's Assertiveness Inventory (CAI) and the Revised Behavioral Assertiveness Test for Children (BAT-CR)

ERIC Educational Resources Information Center

Ollendick, Thomas H.; And Others

1986-01-01

The relationship between a self-report measure of assertive behavior in children and a role-play measure was examined in 69 elementary school children. Self-report of positive assertion related more closely to role-play measures and expert ratings of assertiveness than did self-report of negative assertion. (Author/LHW)

Assertiveness: making yourself heard in district nursing.

PubMed

Lawton, Sally; Stewart, Fiona

2005-06-01

Being assertive is not the same as being aggressive. Assertiveness is a tool for expressing ourselves confidently, and a way of saying 'yes' and 'no' in an appropriate way. This article explores issues concerned with assertiveness in district nurse settings. It outlines helpful techniques to develop assertiveness, such as the broken record, fogging, negative assertion and negative inquiry.

RAS mutations affect pattern of metastatic spread and increase propensity for brain metastasis in colorectal cancer.

PubMed

Yaeger, Rona; Cowell, Elizabeth; Chou, Joanne F; Gewirtz, Alexandra N; Borsu, Laetitia; Vakiani, Efsevia; Solit, David B; Rosen, Neal; Capanu, Marinela; Ladanyi, Marc; Kemeny, Nancy

2015-04-15

RAS and PIK3CA mutations in metastatic colorectal cancer (mCRC) have been associated with worse survival. We sought to evaluate the impact of RAS and PIK3CA mutations on cumulative incidence of metastasis to potentially curable sites of liver and lung and other sites such as bone and brain. We performed a computerized search of the electronic medical record of our institution for mCRC cases genotyped for RAS or PIK3CA mutations from 2008 to 2012. Cases were reviewed for patient characteristics, survival, and site-specific metastasis. Among the 918 patients identified, 477 cases were RAS wild type, and 441 cases had a RAS mutation (394 at KRAS exon 2, 29 at KRAS exon 3 or 4, and 18 in NRAS). RAS mutation was significantly associated with shorter median overall survival (OS) and on multivariate analysis independently predicted worse OS (HR, 1.6; P < .01). RAS mutant mCRC exhibited a significantly higher cumulative incidence of lung, bone, and brain metastasis and on multivariate analysis was an independent predictor of involvement of these sites (HR, 1.5, 1.6, and 3.7, respectively). PIK3CA mutations occurred in 10% of the 786 cases genotyped, did not predict for worse survival, and did not exhibit a site-specific pattern of metastatic spread. The metastatic potential of CRC varies with the presence of RAS mutation. RAS mutation is associated with worse OS and increased incidence of lung, bone, and brain metastasis. An understanding of this site-specific pattern of spread may help to inform physicians' assessment of symptoms in patients with mCRC. © 2014 American Cancer Society.

Ras-dva Is a Novel Pit-1- and Glucocorticoid-Regulated Gene in the Embryonic Anterior Pituitary Gland

PubMed Central

Ellestad, Laura E.

2013-01-01

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5′-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5′-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland. PMID:23161868

Ras-dva is a novel Pit-1- and glucocorticoid-regulated gene in the embryonic anterior pituitary gland.

PubMed

Ellestad, Laura E; Porter, Tom E

2013-01-01

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5'-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5'-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland.

Interaction between Wnt/β-catenin and RAS-ERK pathways and an anti-cancer strategy via degradations of β-catenin and RAS by targeting the Wnt/β-catenin pathway.

PubMed

Jeong, Woo-Jeong; Ro, Eun Ji; Choi, Kang-Yell

2018-01-01

Aberrant activation of the Wnt/β-catenin and RAS-extracellular signal-regulated kinase (ERK) pathways play important roles in the tumorigenesis of many different types of cancer, most notably colorectal cancer (CRC). Genes for these two pathways, such as adenomatous polyposis coli ( APC ) and KRAS are frequently mutated in human CRC, and involved in the initiation and progression of the tumorigenesis, respectively. Moreover, recent studies revealed interaction of APC and KRAS mutations in the various stages of colorectal tumorigenesis and even in metastasis accompanying activation of the cancer stem cells (CSCs). A key event in the synergistic cooperation between Wnt/β-catenin and RAS-ERK pathways is a stabilization of both β-catenin and RAS especially mutant KRAS by APC loss, and pathological significance of this was indicated by correlation of increased β-catenin and RAS levels in human CRC where APC mutations occur as high as 90% of CRC patients. Together with the notion of the protein activity reduction by lowering its level, inhibition of both β-catenin and RAS especially by degradation could be a new ideal strategy for development of anti-cancer drugs for CRC. In this review, we will discuss interaction between the Wnt/β-catenin and RAS-ERK pathways in the colorectal tumorigenesis by providing the mechanism of RAS stabilization by aberrant activation of Wnt/β-catenin. We will also discuss our small molecular anti-cancer approach controlling CRC by induction of specific degradations of both β-catenin and RAS via targeting Wnt/β-catenin pathway especially for the KYA1797K, a small molecule specifically binding at the regulator of G-protein signaling (RGS)-domain of Axin.

R-Ras2 is required for germinal center formation to aid B cells during energetically demanding processes.

PubMed

Mendoza, Pilar; Martínez-Martín, Nuria; Bovolenta, Elena R; Reyes-Garau, Diana; Hernansanz-Agustín, Pablo; Delgado, Pilar; Diaz-Muñoz, Manuel D; Oeste, Clara L; Fernández-Pisonero, Isabel; Castellano, Ester; Martínez-Ruiz, Antonio; Alonso-Lopez, Diego; Santos, Eugenio; Bustelo, Xosé R; Kurosaki, Tomohiro; Alarcón, Balbino

2018-05-29

Upon antigen recognition within peripheral lymphoid organs, B cells interact with T cells and other immune cells to transiently form morphological structures called germinal centers (GCs), which are required for B cell clonal expansion, immunoglobulin class switching, and affinity maturation. This process, known as the GC response, is an energetically demanding process that requires the metabolic reprogramming of B cells. We showed that the Ras-related guanosine triphosphate hydrolase (GTPase) R-Ras2 (also known as TC21) plays an essential, nonredundant, and B cell-intrinsic role in the GC response. Both the conversion of B cells into GC B cells and their expansion were impaired in mice lacking R-Ras2, but not in those lacking a highly related R-Ras subfamily member or both the classic H-Ras and N-Ras GTPases. In the absence of R-Ras2, activated B cells did not exhibit increased oxidative phosphorylation or aerobic glycolysis. We showed that R-Ras2 was an effector of both the B cell receptor (BCR) and CD40 and that, in its absence, B cells exhibited impaired activation of the PI3K-Akt-mTORC1 pathway, reduced mitochondrial DNA replication, and decreased expression of genes involved in glucose metabolism. Because most human B cell lymphomas originate from GC B cells or B cells that have undergone the GC response, our data suggest that R-Ras2 may also regulate metabolism in B cell malignancies. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Effects of mutant human Ki-ras{sup G12C} gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dance-Barnes, Stephanie T.; Kock, Nancy D.; Floyd, Heather S.

2008-08-15

Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras{sup G12C} allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 {mu}g/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras{sup G12C} allele in the lung, and resulted in themore » development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 {mu}g/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 {mu}g/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 {mu}g/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras{sup G12C} expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models.« less

Identification of cancer initiating cells in K-Ras driven lung adenocarcinoma.

PubMed

Mainardi, Sara; Mijimolle, Nieves; Francoz, Sarah; Vicente-Dueñas, Carolina; Sánchez-García, Isidro; Barbacid, Mariano

2014-01-07

Ubiquitous expression of a resident K-Ras(G12V) oncogene in adult mice revealed that most tissues are resistant to K-Ras oncogenic signals. Indeed, K-Ras(G12V) expression only induced overt tumors in lungs. To identify these transformation-permissive cells, we induced K-Ras(G12V) expression in a very limited number of adult lung cells (0.2%) and monitored their fate by X-Gal staining, a surrogate marker coexpressed with the K-Ras(G12V) oncoprotein. Four weeks later, 30% of these cells had proliferated to form small clusters. However, only SPC(+) alveolar type II (ATII) cells were able to form hyperplastic lesions, some of which progressed to adenomas and adenocarcinomas. In contrast, induction of K-Ras(G12V) expression in lung cells by intratracheal infection with adenoviral-Cre particles generated hyperplasias in all regions except the proximal airways. Bronchiolar and bronchioalveolar duct junction hyperplasias were primarily made of CC10(+) Clara cells. Some of them progressed to form benign adenomas. However, only alveolar hyperplasias, exclusively made up of SPC(+) ATII cells, progressed to yield malignant adenocarcinomas. Adenoviral infection induced inflammatory infiltrates primarily made of T and B cells. This inflammatory response was essential for the development of K-Ras(G12V)-driven bronchiolar hyperplasias and adenomas, but not for the generation of SPC(+) ATII lesions. Finally, activation of K-Ras(G12V) during embryonic development under the control of a Sca1 promoter yielded CC10(+), but not SPC(+), hyperplasias, and adenomas. These results, taken together, illustrate that different types of lung cells can generate benign lesions in response to K-Ras oncogenic signals. However, in adult mice, only SPC(+) ATII cells were able to yield malignant adenocarcinomas.

Loss of protein phosphatase 6 in mouse keratinocytes enhances K-rasG12D -driven tumor promotion.

PubMed

Kurosawa, Koreyuki; Inoue, Yui; Kakugawa, Yoichiro; Yamashita, Yoji; Kanazawa, Kosuke; Kishimoto, Kazuhiro; Nomura, Miyuki; Momoi, Yuki; Sato, Ikuro; Chiba, Natsuko; Suzuki, Mai; Ogoh, Honami; Yamada, Hidekazu; Miura, Koh; Watanabe, Toshio; Tanuma, Nobuhiro; Tachi, Masahiro; Shima, Hiroshi

2018-05-14

Here, we address the function of protein phosphatase 6 (PP6) loss on K-ras-initiated tumorigenesis in keratinocytes. To do so, we developed tamoxifen-inducible double mutant (K-ras G12D -expressing and Ppp6c-deficient) mice in which K-ras G12D expression is driven by the cytokeratin 14 (K14) promoter. Doubly-mutant mice showed early onset tumor formation in lip, nipples, external genitalia, anus and palms, and had to be sacrificed by three weeks after induction by tamoxifen, while comparably-treated K-ras G12D -expressing mice did not. HE-staining of lip tumors before euthanasia revealed that all were papillomas, some containing focal squamous cell carcinoma. Immunohistochemical analysis of lip of doubly-mutant versus K-ras G12D mice revealed that cell proliferation and cell size increased approximately two-fold relative to K-ras G12D -expressing mutants, and epidermal thickness of lip tissue greatly increased relative to that seen in K-ras G12D only mice. Moreover, AKT phosphorylation increased in K-ras G12D -expressing/Ppp6c-deficient cells, as did phosphorylation of the downstream effectors 4EBP1, S6, and GSK3, suggesting that protein synthesis and survival signals are enhanced in lip tissues of doubly-mutant mice. Finally, increased numbers of K14-positive cells were present in the suprabasal layer of doubly-mutant mice, indicating abnormal keratinocyte differentiation, and γH2AX-positive cells accumulated, indicating perturbed DNA repair. Taken together, Ppp6c deficiency enhances K-ras G12D -dependent tumor promotion. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord.

PubMed

Ohata, Shinya; Uga, Hideko; Okamoto, Hitoshi; Katada, Toshiaki

2018-06-27

Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord. Copyright © 2018 Elsevier Inc. All rights reserved.

RAS/ERK modulates TGFbeta-regulated PTEN expression in human pancreatic adenocarcinoma cells.

PubMed

Chow, Jimmy Y C; Quach, Khai T; Cabrera, Betty L; Cabral, Jennifer A; Beck, Stayce E; Carethers, John M

2007-11-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-beta might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFbeta and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFbeta surface receptors. Cells were treated with TGFbeta1 and separated into cytosolic/nuclear fractions for western blotting with phospho-SMAD2, SMAD 2, 4 phospho-ATP-dependent tyrosine kinases (Akt), Akt and PTEN antibodies. PTEN mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. The MEK1 inhibitor, PD98059, was used to block the downstream action of oncogenic K-RAS/ERK, as was a dominant-negative (DN) K-RAS construct. TGFbeta increased phospho-SMAD2 in both cytosolic and nuclear fractions. PD98059 treatment further increased phospho-SMAD2 in the nucleus of both pancreatic cell lines, and DN-K-RAS further improved SMAD translocation in K-RAS mutant CAPAN cells. TGFbeta treatment significantly suppressed PTEN protein levels concomitant with activation of Akt by 48 h through transcriptional reduction of PTEN mRNA that was evident by 6 h. TGFbeta-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFbeta. TGFbeta-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFbeta-induced transcriptional down-regulation of the tumor suppressor PTEN in a SMAD4-independent manner and could constitute a signaling switch mechanism from growth suppression to growth promotion in pancreatic cancers.

Primary Murine CD4+ T Cells Fail to Acquire the Ability to Produce Effector Cytokines When Active Ras Is Present during Th1/Th2 Differentiation

PubMed Central

Janardhan, Sujit V.; Marks, Reinhard; Gajewski, Thomas F.

2014-01-01

Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4+ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4+ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4+ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4+ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo. PMID:25397617

Sequential inductions of the ZEB1 transcription factor caused by mutation of Rb and then Ras proteins are required for tumor initiation and progression.

PubMed

Liu, Yongqing; Sánchez-Tilló, Ester; Lu, Xiaoqin; Huang, Li; Clem, Brian; Telang, Sucheta; Jenson, Alfred B; Cuatrecasas, Miriam; Chesney, Jason; Postigo, Antonio; Dean, Douglas C

2013-04-19

Rb1 restricts cell cycle progression, and it imposes cell contact inhibition to suppress tumor outgrowth. It also triggers oncogene-induced senescence to block Ras mutation. Loss of the Rb1 pathway, which is a hallmark of cancer cells, then provides a permissive environment for Ras mutation, and Ras is sufficient for invasive tumor formation in Rb1 family mutant mouse embryo fibroblasts (MEFs). These results demonstrate that sequential mutation of the Rb1 and Ras pathways comprises a tumor initiation axis. Both Rb1 and Ras regulate expression of the transcription factor ZEB1, thereby linking tumor initiation to the subsequent invasion and metastasis, which is induced by ZEB1. ZEB1 acts in a negative feedback loop to block expression of miR-200, which is thought to facilitate tumor invasion and metastasis. However, ZEB1 also represses cyclin-dependent kinase (cdk) inhibitors to control the cell cycle; its mutation in MEFs leads to induction of these inhibitors and premature senescence. Here, we provide evidence for two sequential inductions of ZEB1 during Ras transformation of MEFs. Rb1 constitutively represses cdk inhibitors, and induction of ZEB1 when the Rb1 pathway is lost is required to maintain this repression, allowing for the classic immortalization and loss of cell contact inhibition seen when the Rb1 pathway is lost. In vivo, we show that this induction of ZEB1 is required for Ras-initiated tumor formation. ZEB1 is then further induced by Ras, beyond the level seen with Rb1 mutation, and this Ras superinduction is required to reach a threshold of ZEB1 sufficient for repression of miR-200 and tumor invasion.

RAS Insight

Cancer.gov

David Heimbrook, now CEO of the Frederick National Laboratory for Cancer Research, played a major role in a large pharma as it tried to develop an anti-RAS drug. Lessons from that failure inform the RAS Initiative today.

Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid.

PubMed Central

Kinsella, B T; Erdman, R A; Maltese, W A

1991-01-01

ras proteins undergo posttranslational modification by a 15-carbon farnesyl isoprenoid at a cysteine within a defined COOH-terminal amino acid motif; i.e., Cys-Ali-Ali-Ser/Met (where Ali represents an aliphatic residue). In other low molecular mass GTP-binding proteins, cysteines are modified by 20-carbon geranylgeranyl groups within a Cys-Ali-Ali-Leu motif. We changed the terminal Ser-189 of Ha-ras p21 to Leu-189 by site-directed mutagenesis and found that the protein was modified by [3H]geranylgeranyl instead of [3H]farnesyl in an in vitro assay. Gel-permeation chromatography of [3H]mevalonate-labeled hydrocarbons released from immunoprecipitated ras proteins overexpressed in COS cells indicated that Ha-ras p21(Leu-189) was also a substrate for 20-carbon isoprenyl modification in vivo. Additional steps in Ha-ras p21 processing, normally initiated by farnesylation, appear to be supported by geranylgeranylation, based on metabolic labeling of Ha-ras p21(Leu-189) with [3H]palmitate and its subcellular localization in a particulate fraction from COS cells. These observations indicate that the amino acid occupying the terminal position (Xaa) in the Cys-Ali-Ali-Xaa motif constitutes a key structural feature by which Ha-ras p21 and other proteins with ras-like COOH-terminal isoprenylation sites are distinguished as substrates for farnesyl- or geranylgeranyltransferases. Images PMID:1924354

Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kinsella, B.T.; Erdman, R.A.; Maltese, W.A.

ras proteins undergo posttranslational modification by a 15-carbon farnesyl isoprenoid at a cysteine within a defined COOH-terminal amino acid motif; i.e., Cys-Ali-Ali-Ser/Met (where Ali represents an aliphatic residue). In other low molecular mass GTP-binding proteins, cysteines are modified by 20-carbon geranylgeranyl groups within a Cys-Ali-Ali-Leu motif. The authors changed the terminal Ser-189 of Ha-ras p21 to Leu-189 by site-directed mutagenesis and found that the protein was modified by ({sup 3}H)geranylgeranyl instead of ({sup 3}H)farnesyl in an in vitro assay. Gel-permeation chromatography of ({sup 3}H)mevalonate-labeled hydrocarbons released from immunoprecipitated ras proteins overexpressed in COS cells indicated that Ha-ras p21 (Leu-189) wasmore » also a substrate for 20-carbon isoprenyl modification in vivo. Additional steps in Ha-ras p21 processing, normally initiated by farnesylation, appear to be supported by geranylgeranylation, based on metabolic labeling of Ha-ras p21 (Leu-189) with ({sup 3}H) palmitate and its subcellular localization in a particulate fraction from COS cells. These observations indicate that the amino acid occupying the terminal position (Xaa) in the Cys-Ali-Ali-Xaa motif constitutes a key structural feature by which Ha-ras p21 and other proteins with ras-like COOH-terminal isoprenylation sites are distinguished as substrates for farnesyl- or geranylgeranyltransferases.« less

The Ras effector RASSF2 is a novel tumor-suppressor gene in human colorectal cancer.

PubMed

Akino, Kimishige; Toyota, Minoru; Suzuki, Hiromu; Mita, Hiroaki; Sasaki, Yasushi; Ohe-Toyota, Mutsumi; Issa, Jean-Pierre J; Hinoda, Yuji; Imai, Kohzoh; Tokino, Takashi

2005-07-01

Activation of Ras signaling is a hallmark of colorectal cancer (CRC), but the roles of negative regulators of Ras are not fully understood. Our aim was to address that question by surveying genetic and epigenetic alterations of Ras-Ras effector genes in CRC cells. The expression and methylation status of 6 RASSF family genes were examined using RT-PCR and bisulfite PCR in CRC cell lines and in primary CRCs and colorectal adenomas. Colony formation assays and flow cytometry were used to assess the tumor suppressor activities of RASSF1 and RASSF2. Immunofluorescence microscopy was used to determine the effect of altered RASSF2 expression on cell morphology. Mutations of K- ras , BRAF, and p53 were identified using single-strand conformation analysis and direct sequencing. Aberrant methylation and histone deacetylation of RASSF2 was associated with the gene's silencing in CRC. The activities of RASSF2, which were distinct from those of RASSF1, included induction of morphologic changes and apoptosis; moreover, its ability to prevent cell transformation suggests that RASSF2 acts as a tumor suppressor in CRC. Primary CRCs that showed K- ras /BRAF mutations also frequently showed RASSF2 methylation, and inactivation of RASSF2 enhanced K- ras -induced oncogenic transformation. RASSF2 methylation was also frequently identified in colorectal adenomas. RASSF2 is a novel tumor suppressor gene that regulates Ras signaling and plays a pivotal role in the early stages of colorectal tumorigenesis.

Studying the Immunomodulatory Effects of Small Molecule Ras Inhibitors in Animal Models of Rheumatoid Arthritis

DTIC Science & Technology

2016-10-01

2016 4. TITLE AND SUBTITLE Studying the Immunomodulatory Effects of Small Molecule Ras-Inhibitors in Animal Models of Rheumatoid Arthritis 5a...TERMS Ras GTPases; Rheumatoid Arthritis (RA); Farnesylthiosalicylic acid (FTS); T helper cells, disease-modifying antirheumatic drugs (DMARDs...anergy and to restore IL-2 production. Importantly, T cells from patients with Rheumatoid Arthritis (RA) display augmented activation of the Ras

The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

PubMed

Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D

1993-05-06

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

Local bone marrow renin-angiotensin system in primitive, definitive and neoplastic haematopoiesis.

PubMed

Haznedaroglu, Ibrahim C; Beyazit, Yavuz

2013-03-01

The locally active ligand peptides, mediators, receptors and signalling pathways of the haematopoietic BM (bone marrow) autocrine/paracrine RAS (renin-angiotensin system) affect the essential steps of definitive blood cell production. Haematopoiesis, erythropoiesis, myelopoiesis, formation of monocytic and lymphocytic lineages, thrombopoiesis and other stromal cellular elements are regulated by the local BM RAS. The local BM RAS is present and active even in primitive embryonic haematopoiesis. ACE (angiotensin-converting enzyme) is expressed on the surface of the first endothelial and haematopoietic cells, forming the marrow cavity in the embryo. ACE marks early haematopoietic precursor cells and long-term blood-forming CD34(+) BM cells. The local autocrine tissue BM RAS may also be active in neoplastic haematopoiesis. Critical RAS mediators such as renin, ACE, AngII (angiotensin II) and angiotensinogen have been identified in leukaemic blast cells. The local tissue RAS influences tumour growth and metastases in an autocrine and paracrine fashion via the modulation of numerous carcinogenic events, such as angiogenesis, apoptosis, cellular proliferation, immune responses, cell signalling and extracellular matrix formation. The aim of the present review is to outline the known functions of the local BM RAS within the context of primitive, definitive and neoplastic haematopoiesis. Targeting the actions of local RAS molecules could represent a valuable therapeutic option for the management of neoplastic disorders.

Clinical utility of RAS mutations in thyroid cancer: a blurred picture now emerging clearer.

PubMed

Xing, Mingzhao

2016-01-27

RAS mutations play an important role in thyroid tumorigenesis. Considerable effort has been made in the last decade to apply RAS mutations as molecular markers to the clinical management of thyroid nodules and thyroid cancer. Yet, for the low diagnostic sensitivities and specificities of RAS mutations, when used alone, and for their uncertain role in the clinical outcomes of thyroid cancer, it has been unclear how to appropriately use them to assist the management of thyroid nodules and thyroid cancer. Studies from recent years, now added from the Alexander group, have shed light on this issue, making a blurred clinical picture now emerge clearer-RAS mutations, when combined with other genetic markers, have high diagnostic negative predictive values for thyroid cancer; cytologically benign thyroid nodules, including those positive for RAS mutations, have long-term clinical stability when non-surgically managed; and differentiated thyroid cancers harboring RAS mutations alone have an excellent prognosis. This progress in understanding RAS mutations in thyroid cancer is showing a major impact on molecular-based practice in the management of thyroid cancer.Please see related research articles: http://dx.doi.org/10.1186/s12916-016-0554-1 and http://dx.doi.org/10.1186/s12916-015-0419-z.

Opposing tissue-specific roles of angiotensin in the pathogenesis of obesity, and implications for obesity-related hypertension

PubMed Central

Littlejohn, Nicole K.

2015-01-01

Metabolic disease, specifically obesity, has now become the greatest challenge to improving cardiovascular health. The renin-angiotensin system (RAS) exists as both a circulating hormone system and as a local paracrine signaling mechanism within various tissues including the brain, kidney, and adipose, and this system is strongly implicated in cardiovascular health and disease. Growing evidence also implicates the RAS in the control of energy balance, supporting the concept that the RAS may be mechanistically involved in the pathogenesis of obesity and obesity hypertension. Here, we review the involvement of the RAS in the entire spectrum of whole organism energy balance mechanisms, including behaviors (food ingestion and spontaneous physical activity) and biological processes (digestive efficiency and both aerobic and nonaerobic resting metabolic rates). We hypothesize that opposing, tissue-specific effects of the RAS to modulate these various components of energy balance can explain the apparently paradoxical results reported by energy-balance studies that involve stimulating, versus disrupting, the RAS. We propose a model in which such opposing and tissue-specific effects of the RAS can explain the failure of simple, global RAS blockade to result in weight loss in humans, and hypothesize that obesity-mediated uncoupling of endogenous metabolic rate control mechanisms can explain the phenomenon of obesity-related hypertension. PMID:26491099

Assertive Communication in Condom Negotiation: Insights From Late Adolescent Couples' Subjective Ratings of Self and Partner.

PubMed

Schmid, Amy; Leonard, Noelle R; Ritchie, Amanda S; Gwadz, Marya V

2015-07-01

Assertive communication has been associated with higher levels of condom use among youth using self-report survey methodology. The purpose of this study was to examine the subjective ratings of assertiveness among young, romantically involved couples in the context of a condom negotiation task. Using an innovative video-recall procedure, 32 couples (64 youth) engaged in a videotaped condom negotiation task and then rated self and partners' level of assertiveness. Both individual ratings of assertiveness and couple-level assertiveness were assessed using dyadic hierarchical linear modeling. Individuals' assertiveness was positively associated with condom use. Unexpectedly, the overall level of assertiveness in couples showed a curvilinear association with condom use. Very high and very low assertiveness was associated with lower condom use, whereas moderate levels of assertiveness were associated with higher condom use. Moderate levels of assertiveness during condom negotiation may facilitate condom use in young couples. Increasing condom use among romantic partners may require developing interventions that strengthen youths' ability to engage in assertive communication strategies that balance emotional intimacy with self-advocacy. Copyright © 2015 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.

Assertiveness training for undergraduate midwifery students.

PubMed

Warland, Jane; McKellar, Lois; Diaz, Monica

2014-11-01

Assertiveness can be defined as an interpersonal behaviour that promotes the fact all people in a relationship are equally important. All health professionals including midwives must work with and care for people. At times this will include facilitating interactions that require skilful negotiation and assertiveness. Yet embedding assertiveness education into undergraduate midwifery curricula has not been widely adopted. This paper explores one method of delivering assertiveness training in an undergraduate midwifery course and provides comment on the effectiveness of this strategy in developing assertiveness skills in a cohort of undergraduate midwifery students. We used an assertiveness survey which was administered immediately before and 3-4 months after an assertiveness training workshop. All students (n = 55) attending the training day were invited to participate. Of these 41 (77% response) chose to participate in the pre intervention survey and 32 participated (9 students lost to follow-up) in the follow up survey. There was an overall improvement in self-perceived assertiveness scores following the assertiveness training workshop. These findings provide encouraging evidence that educational institutions that offer specific and targeted assertiveness education will be rewarded with more assertive graduates. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effects of a single-session assertiveness music therapy role playing protocol for psychiatric inpatients.

PubMed

Silverman, Michael J

2011-01-01

The purpose of this study was to implement and measure the effectiveness of a single-session assertiveness music therapy role playing protocol for psychiatric inpatients. Participants (N=133) were randomly assigned by group to one of three conditions: (a) Assertiveness Music Therapy, (b) No Music Assertiveness, or (c) Music No Assertiveness. Participants in both assertiveness conditions role played a number of different commonly occurring scenarios at an inpatient psychiatric facility and in the community. There were no significant between-group differences in posttest quality of life, locus of control, or other subscales. However, participants in both assertiveness conditions tended to have slightly higher internal locus of control and overall quality of life scores than participants in the music no assertiveness condition. Additionally, the assertiveness music therapy condition had higher attendance rates than the other conditions. A higher percentage of participants from both the assertiveness music therapy and music no assertiveness conditions indicated they thought their session was the most helpful/therapeutic group therapy session in which they had participated; this was not the case for the assertiveness no music condition. Future research is warranted to measure the effects of protocols that can help psychiatric patients generalize skills learned in treatment.

RAS - Screens & Assays

Cancer.gov

A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

RAS - Screens & Assays - Drug Discovery

Cancer.gov

The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

Cancer.gov

The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

[Relationship of perception conflict and assertiveness in nurses].

PubMed

Stojčić, Živko; Perković, Lada; Stašević, Ina; Stojčić, Nevena; Ropac, Darko

2014-06-01

At their workplace, nurses are exposed to a number of conflict situations. On dealing with such situations, a significant role is played by assertiveness skills. Assertiveness is the necessity of efficient communication between nurses and patients. Thus, development of these skills can enhance patient confidence in the nursing profession. The aim of the study was to determine whether there are differences in assertiveness with respect to age and sex, and whether there is and what is the connection between assertiveness, potential sources of conflict at work, conflicts due to the behavior of associates, resolving conflicts and self-assessment in resolving conflicts. The survey included 87 hospital nurses. The questionnaire included assessment of assertiveness. On processing the results, we calculated the indicators of descriptive statistics, carried out the variance analysis and t-test, and calculated Pearson's correlation coefficients. It was found that the majority of subjects expressed a medium level of assertiveness, i.e. they could be considered as relatively assertive persons. There were significant differences in assertiveness according to age of the subjects and length of service, where the oldest age group was significantly less assertive. More assertive subjects frequently observed behaviors that may be a source of conflict and problems in the organization of work. At the same time, they often had conflicts because of such behavior, which indicated that more assertive subjects were bolder and more secure. More assertive subjects believed that they were more successful in resolving conflicts than non-assertive subjects.

Evaluation of assertiveness training for psychiatric patients.

PubMed

Lin, Yen-Ru; Wu, Mei-Hsuen; Yang, Cheng-I; Chen, Tsai-Hwei; Hsu, Chen-Chuan; Chang, Yue-Cune; Tzeng, Wen-Chii; Chou, Yuan-Hwa; Chou, Kuei-Ru

2008-11-01

To investigate the effectiveness of assertiveness training programmes on psychiatric patients' assertiveness, self-esteem and social anxiety. Assertiveness training programmes are designed to improve an individual's assertive beliefs and behaviours, which can help the individual change how they view themselves and establish self-confidence and social anxiety. It is useful for patients with depression, depressive phase of bipolar disorder, anxiety disorder or adjustment disorder. Experimental. There were 68 subjects (28, experimental group; 40, diagnosis-matched comparison group). Subjects in experimental groups participated in experimenter-designed assertiveness training twice a week (two hours each) for four weeks. The comparison groups participated the usual activities. Data were collected in the two groups at the same time: before, after and one month after training programme. Efficacy was measured by assertiveness, self-esteem and social anxiety inventories. A generalised estimating equation was used for analysis. After training, subjects had a significant increase in assertiveness immediately after the assertiveness training programme and one-month follow-up. There was a significant decrease in social anxiety after training, but the improvement was not significant after one month. Self-esteem did not increase significantly after training. With our sample of patients with mixed diagnoses, assertiveness seemed to be improved after assertiveness training. Patients would benefit more from the assertiveness training programme for the change in how they view themselves, improve their assertiveness, properly express their individual moods and thoughts and further establish self-confidence. The assertiveness training protocol could be provided as a reference guide to clinical nurses.

Decentralized State Estimation and Remedial Control Action for Minimum Wind Curtailment Using Distributed Computing Platform

DOE PAGES

Liu, Ren; Srivastava, Anurag K.; Bakken, David E.; ...

2017-08-17

Intermittency of wind energy poses a great challenge for power system operation and control. Wind curtailment might be necessary at the certain operating condition to keep the line flow within the limit. Remedial Action Scheme (RAS) offers quick control action mechanism to keep reliability and security of the power system operation with high wind energy integration. In this paper, a new RAS is developed to maximize the wind energy integration without compromising the security and reliability of the power system based on specific utility requirements. A new Distributed Linear State Estimation (DLSE) is also developed to provide the fast andmore » accurate input data for the proposed RAS. A distributed computational architecture is designed to guarantee the robustness of the cyber system to support RAS and DLSE implementation. The proposed RAS and DLSE is validated using the modified IEEE-118 Bus system. Simulation results demonstrate the satisfactory performance of the DLSE and the effectiveness of RAS. Real-time cyber-physical testbed has been utilized to validate the cyber-resiliency of the developed RAS against computational node failure.« less

Clinical study of 200 patients with recurrent aphthous stomatitis.

PubMed

Rodríguez-Archilla, Alberto; Raissouni, Tarik

2018-01-01

Recurrent aphthous stomatitis (RAS) affects approximately 20% of the general population. Its etiology is still unknown. To analyze this entity's clinical features. Data such as age, gender, family history of RAS, age at first episode onset, prodromal symptoms, number, size, morphology and localization of lesions, RAS clinical form, annual rate of recurrence, predisposing factors, symptoms and time for symptoms and lesions disappearance were assessed in 200 patients with RAS. Patients had RAS minor forms. Main clinical characteristics were family history of RAS (89%), first episode at ≥ 10 years of age (69%), prodromal symptoms (66%), one lesion per episode (63%), < 0.5 cm lesions (64%), rounded morphology (55%), localization at the tongue (27%), 3 recurrent episodes per year (36%), stress as predisposing factor (34%), symptom disappearance in 2 days (54%) and healing of lesions in 8 days (40%). Even when RAS is a common disorder of the oral mucosa, there is no curative treatment available. Therapeutic measures seek to reduce the pain and size of lesions, accelerate the time of recovery and decrease the rate of relapses. Copyright: © 2018 SecretarÍa de Salud.

The intracellular angiotensin system buffers deleterious effects of the extracellular paracrine system

PubMed Central

Villar-Cheda, Begoña; Costa-Besada, Maria A; Valenzuela, Rita; Perez-Costas, Emma; Melendez-Ferro, Miguel; Labandeira-Garcia, Jose L

2017-01-01

The ‘classical’ renin–angiotensin system (RAS) is a circulating system that controls blood pressure. Local/paracrine RAS, identified in a variety of tissues, including the brain, is involved in different functions and diseases, and RAS blockers are commonly used in clinical practice. A third type of RAS (intracellular/intracrine RAS) has been observed in some types of cells, including neurons. However, its role is still unknown. The present results indicate that in brain cells the intracellular RAS counteracts the intracellular superoxide/H2O2 and oxidative stress induced by the extracellular/paracrine angiotensin II acting on plasma membrane receptors. Activation of nuclear receptors by intracellular or internalized angiotensin triggers a number of mechanisms that protect the cell, such as an increase in the levels of protective angiotensin type 2 receptors, intracellular angiotensin, PGC-1α and IGF-1/SIRT1. Interestingly, this protective mechanism is altered in isolated nuclei from brains of aged animals. The present results indicate that at least in the brain, AT1 receptor blockers acting only on the extracellular or paracrine RAS may offer better protection of cells. PMID:28880266

Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Shenyuan; Long, Brian N.; Boris, Gabriel H.

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras–GTP complex, the switch I region undergoes amore » significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.« less

Targeted Sos1 deletion reveals its critical role in early T-cell development

PubMed Central

Kortum, Robert L.; Sommers, Connie L.; Alexander, Clayton P.; Pinski, John M.; Li, Wenmei; Grinberg, Alex; Lee, Jan; Love, Paul E.; Samelson, Lawrence E.

2011-01-01

Activation of the small G protein Ras is required for thymocyte differentiation. In thymocytes, Ras is activated by the Ras guanine exchange factors (RasGEFs) Sos1, Sos2, and RasGRP1. We report the development of a floxed allele of sos1 to assess the role of Sos1 during thymocyte development. Sos1 was required for pre–T-cell receptor (pre-TCR)– but not TCR-stimulated developmental signals. Sos1 deletion led to a partial block at the DN-to-DP transition. Sos1-deficient thymocytes showed reduced pre-TCR–stimulated proliferation, differentiation, and ERK phosphorylation. In contrast, TCR-stimulated positive selection, and negative selection under strong stimulatory conditions, remained intact in Sos1-deficient mice. Comparison of RasGEF expression at different developmental stages showed that relative to Sos2 and RasGRP1, Sos1 is most abundant in DN thymocytes, but least abundant in DP thymocytes. These data reveal that Sos1 is uniquely positioned to affect signal transduction early in thymocyte development. PMID:21746917

Single-molecule force measurement via optical tweezers reveals different kinetic features of two BRaf mutants responsible for cardio-facial-cutaneous (CFC) syndrome

PubMed Central

Wen, Cheng; Ye, Anpei

2013-01-01

BRaf (B- Rapid Accelerated Fibrosarcoma) protein is an important serine/threonine-protein kinase. Two domains on BRaf can independently bind its upstream kinase, Ras (Rat Sarcoma) protein. These are the Ras binding domain (RBD) and cysteine-rich-domain (CRD). Herein we use customized optical tweezers to compare the Ras binding process in two pathological mutants of BRaf responsible for CFC syndrome, abbreviated BRaf (A246P) and BRaf (Q257R). The two mutants differ in their kinetics of Ras-binding, though both bind Ras with similar increased overall affinity. BRaf (A246P) exhibits a slightly higher Ras/CRD unbinding force and a significantly higher Ras/RBD unbinding force versus the wild type. The contrary phenomenon is observed in the Q257R mutation. Simulations of the unstressed-off rate, koff(0), yield results in accordance with the changes revealed by the mean unbinding force. Our approach can be applied to rapidly assess other mutated proteins to deduce the effects of mutation on their kinetics compared to wild type proteins and to each other. PMID:24409384

Decentralized State Estimation and Remedial Control Action for Minimum Wind Curtailment Using Distributed Computing Platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ren; Srivastava, Anurag K.; Bakken, David E.

Intermittency of wind energy poses a great challenge for power system operation and control. Wind curtailment might be necessary at the certain operating condition to keep the line flow within the limit. Remedial Action Scheme (RAS) offers quick control action mechanism to keep reliability and security of the power system operation with high wind energy integration. In this paper, a new RAS is developed to maximize the wind energy integration without compromising the security and reliability of the power system based on specific utility requirements. A new Distributed Linear State Estimation (DLSE) is also developed to provide the fast andmore » accurate input data for the proposed RAS. A distributed computational architecture is designed to guarantee the robustness of the cyber system to support RAS and DLSE implementation. The proposed RAS and DLSE is validated using the modified IEEE-118 Bus system. Simulation results demonstrate the satisfactory performance of the DLSE and the effectiveness of RAS. Real-time cyber-physical testbed has been utilized to validate the cyber-resiliency of the developed RAS against computational node failure.« less

¹H, ¹³C and ¹⁵N resonance assignment for the human K-Ras at physiological pH.

PubMed

Vo, Uybach; Embrey, Kevin J; Breeze, Alexander L; Golovanov, Alexander P

2013-10-01

K-Ras, a member of the Ras family of small GTPases, is involved in cell growth, proliferation, differentiation and apoptosis and is frequently mutated in cancer. The activity of Ras is mediated by the inter-conversion between GTP- and GDP- bound states. This conversion is regulated by binding of effector proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Previously, NMR signals from these effector-binding regions of Ras often remained unassigned and largely unobservable due to conformational exchange and polysterism inherent to this protein. In this paper, we report the complete backbone and C(β), as well as partial H(α), H(β) and C(γ), NMR assignment for human K-Ras (residues 1-166) in the GDP-bound form at a physiological pH of 7.4. These data thereby make possible detailed monitoring of the functional cycle of Ras and its interactions with nucleotides and effector proteins through the observation of fingerprint signals from all the functionally important regions of the protein.

Group versus Individual Assertion Training.

ERIC Educational Resources Information Center

Linehan, Marsha M.; And Others

1979-01-01

Compared effectiveness of group assertion therapy with individual assertion therapy. Results indicated no significant differences between group v individual assertion training. Pre-, post-, and follow-up measures demonstrated that both treatments were effective in increasing assertive behavior skills and in reducing hostility and anger. Treatment…

The RAS Problem: Turning Off a Broken Switch

Cancer.gov

The RAS gene is commonly mutated in cancer and researchers are working to better understand how to develop drugs that can target the RAS protein, which for many years has been considered to be “undruggable.”

Role of Notch Signaling in Human Breast Cancer Pathogenesis

DTIC Science & Technology

2006-11-01

transform HMLE cells. Similarly, overexpression of ErbB2, a receptor tyrosine kinase upstream of Ras normally found overexpressed in many breast cancers ...Assess Notch-Ras cooperation in breast cancers in vivo: Since the major observation in this project has been the cooperation of Notch and Ras in HMLE ...metastasis. The in vitro cooperation between Notch and Ras in HMLE cells is mimicked in naturally arising breast cancers in vivo. Further dissection of the

Structure of the c-Ki-ras gene in a rat fibrosarcoma induced by 1,8-dinitropyrene.

PubMed Central

Tahira, T; Hayashi, K; Ochiai, M; Tsuchida, N; Nagao, M; Sugimura, T

1986-01-01

Restriction enzyme maps were made of the region around exons 1 and 2 of activated c-Ki-ras of a fibrosarcoma (1,8-DNP2) induced in a rat by 1,8-dinitropyrene. Nucleotide sequence analysis revealed that activated c-Ki-ras shows a G----T transversion in codon 12 and consequently encodes cysteine instead of glycine in normal rat c-Ki-ras. PMID:3023884

Studying the Immunomodulatory Effects of Small Molecule Ras-Inhibitors in Animal Models of Rheumatoid Arthritis

DTIC Science & Technology

2015-10-01

Models of Rheumatoid Arthritis PRINCIPAL INVESTIGATOR: Yoel Kloog RECIPIENT: Tel Aviv University TEL AVIV 69978 Israel REPORT DATE: October...TITLE AND SUBTITLE Studying the Immunomodulatory Effects of Small Molecule Ras- Inhibitors in Animal Models of Rheumatoid Arthritis 5a. CONTRACT NUMBER... Rheumatoid Arthritis (RA) display augmented activation of the Ras/Raf/MEK/ERK1/2 signaling pathway, and accordingly overexpression of active K-RAS in

Mitigation of Remedial Action Schemes by Decentralized Robust Governor Control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elizondo, Marcelo A.; Marinovici, Laurentiu D.; Lian, Jianming

This paper presents transient stability improvement by a new distributed hierarchical control architecture (DHC). The integration of remedial action schemes (RAS) to the distributed hierarchical control architecture is studied. RAS in power systems are designed to maintain stability and avoid undesired system conditions by rapidly switching equipment and/or changing operating points according to predetermined rules. The acceleration trend relay currently in use in the US western interconnection is an example of RAS that trips generators to maintain transient stability. The link between RAS and DHC is through fast acting robust turbine/governor control that can also improve transient stability. In thismore » paper, the influence of the decentralized robust turbine/governor control on the design of RAS is studied. Benefits of combining these two schemes are increasing power transfer capability and mitigation of RAS generator tripping actions; the later benefit is shown through simulations.« less

Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins.

PubMed Central

Goodman, L E; Judd, S R; Farnsworth, C C; Powers, S; Gelb, M H; Glomset, J A; Tamanoi, F

1990-01-01

Ras proteins are post-translationally modified by farnesylation. In the present investigation, we identified an activity in crude soluble extracts of yeast cells that catalyzes the transfer of a farnesyl moiety from farnesyl pyrophosphate to yeast RAS2 protein. RAS2 proteins having a C-terminal Cys-Ali-Ali-Xaa sequence (where Ali is an aliphatic amino acid and Xaa is the unspecified C-terminal amino acid) served as substrates for this reaction, whereas RAS2 proteins with an altered or deleted Cys-Ali-Ali-Xaa sequence did not. A yeast mutant, dpr1/ram1, originally isolated as a Ras-processing mutant was shown to be defective in farnesyltransferase activity. In addition, another mutant, ram2, also was defective in the transferase activity. These results demonstrate that at least two genes, DPR1/RAM1 and RAM2, are required for the farnesyltransferase activity in yeast. Images PMID:2124698

Yeast Ras regulates the complex that catalyzes the first step in GPI-anchor biosynthesis at the ER.

PubMed

Sobering, Andrew K; Watanabe, Reika; Romeo, Martin J; Yan, Benjamin C; Specht, Charles A; Orlean, Peter; Riezman, Howard; Levin, David E

2004-05-28

The yeast ERI1 gene encodes a small ER-localized protein that associates in vivo with GTP bound Ras2 in an effector loop-dependent manner. We showed previously that loss of Eri1 function results in hyperactive Ras phenotypes. Here, we demonstrate that Eri1 is a component of the GPI-GlcNAc transferase (GPI-GnT) complex in the ER, which catalyzes transfer of GlcNAc from UDP-GlcNAc to an acceptor phosphatidylinositol, the first step in the production of GPI-anchors for cell surface proteins. We also show that GTP bound Ras2 associates with the GPI-GnT complex in vivo and inhibits its activity, indicating that yeast Ras uses the ER as a signaling platform from which to negatively regulate the GPI-GnT. We propose that diminished GPI-anchor protein production contributes to hyperactive Ras phenotypes.

EGFR immunoexpression, RAS immunoexpression and their effects on survival in lung adenocarcinoma cases.

PubMed

Gundogdu, Ahmet Gokhan; Onder, Sevgen; Firat, Pinar; Dogan, Riza

2014-06-01

The impacts of epidermal growth factor receptor (EGFR) immunoexpression and RAS immunoexpression on the survival and prognosis of lung adenocarcinoma patients are debated in the literature. Twenty-six patients, who underwent pulmonary resections between 2002 and 2007 in our clinic, and whose pathologic examinations yielded adenocarcinoma, were included in the study. EGFR and RAS expression levels were examined by immunohistochemical methods. The results were compared with the survival, stage of the disease, nodal involvement, lymphovascular invasion, and pleural invasion. Nonparametric bivariate analyses were used for statistical analyses. A significant link between EGFR immunoexpression and survival has been identified while RAS immunoexpression and survival have been proven to be irrelevant. Neither EGFR, nor RAS has displayed a significant link with the stage of the disease, nodal involvement, lymphovascular invasion, or pleural invasion. Positive EGFR immunoexpression affects survival negatively, while RAS immunoexpression has no effect on survival in lung adenocarcinoma patients.

Multivalent small molecule pan-RAS inhibitors

PubMed Central

Welsch, Matthew E.; Kaplan, Anna; Chambers, Jennifer M.; Stokes, Michael E.; Bos, Pieter H.; Zask, Arie; Zhang, Yan; Sanchez-Martin, Marta; Badgley, Michael A.; Huang, Christine S.; Tran, Timothy H.; Akkiraju, Hemanth; Brown, Lewis M.; Nandakumar, Renu; Cremers, Serge; Yang, Wan S.; Tong, Liang; Olive, Kenneth P.; Ferrando, Adolfo; Stockwell, Brent R.

2017-01-01

SUMMARY Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, have potential use as chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers, and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins. PMID:28235199

Activation of K-ras by codon 13 mutations in C57BL/6 X C3H F1 mouse tumors induced by exposure to 1,3-butadiene.

PubMed

Goodrow, T; Reynolds, S; Maronpot, R; Anderson, M

1990-08-01

1,3-Butadiene has been detected in urban air, gasoline vapors, and cigarette smoke. It has been estimated that 65,000 workers are exposed to this chemical in occupational settings in the United States. Lymphomas, lung, and liver tumors were induced in female and male C57BL/6 X C3H F1 (hereafter called B6C3F1) mice by inhalation of 6.25 to 625 ppm 1,3-butadiene for 1 to 2 years. The objective of this study was to examine these tumors for the presence of activated protooncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA isolated from 7 of 9 lung tumors and 7 of 12 liver tumors induced morphological transformation of NIH 3T3 cells. Southern blot analysis indicated that the transformation induced by 6 lung and 3 liver tumor DNA samples was due to transfer of a K-ras oncogene. Four of the 7 liver tumors that were positive upon transfection contained an activated H-ras gene. The identity of the transforming gene in one of the lung tumors has not been determined but was not a member of the ras family or a met or raf gene. Eleven 1,3-butadiene-induced lymphomas were examined for transforming genes using the nude mouse tumorigenicity assay. Activated K-ras genes were detected in 2 of the 11 lymphomas assayed. DNA sequencing of polymerase chain reaction-amplified ras gene exons revealed that 9 of 11 of the activating K-ras mutations were G to C transversions in codon 13. One liver tumor contained an activated K-ras gene with mutations in both codons 60 and 61. The activating mutation in one of the K-ras genes from a lymphoma was not identified but DNA sequence analysis of amplified regions in proximity to codons 12, 13, and 61 demonstrated that the mutation was not located in or near these codons. Activation of K-ras genes by codon 13 mutations has not been found in any lung or liver tumors or lymphomas from untreated B6C3F1 mice. Thus, the K-ras activation found in 1,3-butadiene-induced B6C3F1 mouse tumors probably occurred as a result of genotoxic effects of this chemical. The oncogenes most frequently detected in human pulmonary adenocarcinomas are K-ras genes. Activated K-ras genes have also been found in some human lymphomas. This suggest that activation of K-ras may be important in the induction of human pulmonary adenocarcinomas and lymphomas.(ABSTRACT TRUNCATED AT 400 WORDS)

Inter-group and intra-group assertiveness: adolescents' social skills following cultural transition.

PubMed

Korem, Anat; Horenczyk, Gabriel; Tatar, Moshe

2012-08-01

The goals of this study were to examine intra-group and inter-group assertiveness among adolescents, and to compare these two domains of assertiveness between cultural groups in Israel. Measures of intra-group and inter-group assertiveness were developed, and questionnaires were administrated to 441 immigrants from the Former Soviet Union (FSU), 242 immigrants from Ethiopia and 333 non-immigrants. Compared to non-immigrants, FSU and Ethiopian immigrants' inter-group assertiveness was lower. Girls reported higher levels of inter-group assertiveness than boys. Each of the immigrant groups rates itself as equally assertive as the non-immigrant group and more assertive than the other immigrant group. Also, a difference between inter-group and intra-group assertiveness was found among the FSU immigrants. It is argued that adolescents' assertiveness following cultural transition is associated with socio-cultural context, and the implications of this conclusion are discussed. Copyright © 2011 The Foundation for Professionals in Services for Adolescents. Published by Elsevier Ltd. All rights reserved.

A Raf-competitive K-Ras binder can fail to functionally antagonize signaling.

PubMed

Kauke, Monique J; Tisdale, Alison W; Kelly, Ryan L; Braun, Christian J; Hemann, Michael T; Wittrup, K Dane

2018-05-02

Mutated in approximately 30% of human cancers, Ras GTPases are the most common drivers of oncogenesis and render tumors unresponsive to many standard therapies. Despite decades of research, no drugs directly targeting Ras are currently available. We have previously characterized a small protein antagonist of K-Ras, R11.1.6, and demonstrated its direct competition with Raf for Ras binding. Here we evaluate the effects of R11.1.6 on Ras signaling and cellular proliferation in a panel of human cancer cell lines. Through lentiviral transduction, we generated cell lines that constitutively or through induction with doxycycline express R11.1.6 or a control protein YW1 and show specific binding by R11.1.6 to endogenous Ras through microscopy and co-immunoprecipitation experiments. Genetically-encoded intracellular expression of this high-affinity Ras antagonist, however, fails to measurably disrupt signaling through either the MAPK or PI3K pathway. Consistently, cellular proliferation was unaffected as well. To understand this lack of signaling inhibition, we quantified the number of molecules of R11.1.6 expressed by the inducible cell lines and developed a simple mathematical model describing the competitive binding of Ras by R11.1.6 and Raf. This model supports a potential mechanism for the lack of biological effects that we observed, suggesting stoichiometric and thermodynamic barriers that should be overcome in pharmacological efforts to directly compete with downstream effector proteins localized to membranes at very high effective concentrations. Copyright ©2018, American Association for Cancer Research.

Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution

PubMed Central

Coyle, Scott M; Lim, Wendell A

2016-01-01

The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras’s ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease. DOI: http://dx.doi.org/10.7554/eLife.12435.001 PMID:26765565

Renoprotective effects of hepatocyte growth factor in the stenotic kidney

PubMed Central

Stewart, Nicholas

2013-01-01

Renal microvascular (MV) damage and loss contribute to the progression of renal injury in renal artery stenosis (RAS). Hepatocyte growth factor (HGF) is a powerful angiogenic and antifibrotic cytokine that we showed to be decreased in the stenotic kidney. We hypothesized that renal HGF therapy will improve renal function mainly by protecting the renal microcirculation. Unilateral RAS was induced in 15 pigs. Six weeks later, single-kidney RBF and GFR were quantified in vivo using multidetector computed tomography (CT). Then, intrarenal rh-HGF or vehicle was randomly administered into the stenotic kidney (RAS, n = 8; RAS+HGF, n = 7). Pigs were observed for 4 additional weeks before CT studies were repeated. Renal MV density was quantified by 3D micro-CT ex vivo and histology, and expression of angiogenic and inflammatory factors, apoptosis, and fibrosis was determined. HGF therapy improved RBF and GFR compared with vehicle-treated pigs. This was accompanied by improved renal expression of angiogenic cytokines (VEGF, p-Akt) and tissue-healing promoters (SDF-1, CXCR4, MMP-9), reduced MV remodeling, apoptosis, and fibrosis, and attenuated renal inflammation. However, HGF therapy did not improve renal MV density, which was similarly reduced in RAS and RAS+HGF compared with controls. Using a clinically relevant animal model of RAS, we showed novel therapeutic effects of a targeted renal intervention. Our results show distinct actions on the existing renal microcirculation and promising renoprotective effects of HGF therapy in RAS. Furthermore, these effects imply plasticity of the stenotic kidney to recuperate its function and underscore the importance of MV integrity in the progression of renal injury in RAS. PMID:23269649

Upregulation of c-mesenchymal epithelial transition expression and RAS mutations are associated with late lung metastasis and poor prognosis in colorectal carcinoma.

PubMed

Liu, Jianhua; Zeng, Weiqiang; Huang, Chengzhi; Wang, Junjiang; Xu, Lishu; Ma, Dong

2018-05-01

The present study aimed to investigate whether c-mesenchymal epithelial transition factor (C-MET) overexpression combined with RAS (including KRAS, NRAS and HRAS ) or BRAF mutations were associated with late distant metastases and the prognosis of patients with colorectal cancer (CRC). A total of 374 patients with stage III CRC were classified into 4 groups based on RAS/BRAF and C-MET status for comprehensive analysis. Mutations in RAS / BRAF were determined using Sanger sequencing and C-MET expression was examined using immunohistochemistry. The associations between RAS/BRAF mutations in combination with C-MET overexpression and clinicopathological variables including survival were evaluated. In addition, their predictive value for late distant metastases were statistically analyzed via logistic regression and receiver operating characteristic analysis. Among 374 patients, mutations in KRAS, NRAS, HRAS, BRAF and C-MET overexpression were observed in 43.9, 2.4, 0.3, 5.9 and 71.9% of cases, respectively. Considering RAS/BRAF mutations and C-MET overexpression, vascular invasion (P=0.001), high carcino-embryonic antigen level (P=0.031) and late distant metastases (P<0.001) were more likely to occur in patients of group 4. Furthermore, survival analyses revealed RAS/BRAF mutations may have a more powerful impact on survival than C-MET overexpression, although they were both predictive factors for adverse prognosis. Further logistic regression suggested that RAS/BRAF mutations and C-MET overexpression may predict late distant metastases. In conclusion, RAS/BRAF mutations and C-MET overexpression may serve as predictive indicators for metastatic behavior and poor prognosis of CRC.

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

PubMed Central

Jun, Jesse E.; Yang, Ming; Chen, Hang; Chakraborty, Arup K.

2013-01-01

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation. PMID:23589333

UV exposure, genetic targets in melanocytic tumors and transgenic mouse models.

PubMed

de Gruijl, Frank R; van Kranen, Henk J; van Schanke, Arne

2005-01-01

The genetic changes and corruption of kinase activity in melanomas appear to revolve around a central axis: mitogenic signaling along the RAS pathway down to transcription regulation by pRB. Epidemiological studies point to the importance of ultraviolet (UV) radiation in the etiology of melanoma, but where and how UV radiation is targeted to contribute to the oncogenic signaling remains obscure. Animal models of melanoma genesis could serve to clarify this issue, but many of these models are not responsive to UV exposure. Most interesting advances have been made by using transgenic mice that carry genetic defects that are known to be relevant to human melanoma: specifically, dysfunction in the tumor suppressive action of p16INK4a or a receptor tyrosine kinase/RAS pathway, that is constitutively activated in melanocytes. The latter types of mice appear to be most responsive to (neonatal) UV exposure. Whether this is due to a general increase in target cells by melanocytosis and a paucity or complete lack of pigment, or a possible UV-induced response of the promoter-enhancer of the transgene or a genuinely independent and additional genetic alteration caused by UV exposure needs to be established. Importantly, the full effect of UV radiation needs to be ascertained in mice with different pigmentation by varying the wavelengths, UV-B versus UV-A1, and the exposure schedules, i.e. neonatal versus adult and chronic versus intermittent overexposure. Intermittent UV-B overexposure deserves special attention because it most strongly evokes proliferative responses in melanocytes.

Optimizing depuration of salmon in RAS

USDA-ARS?s Scientific Manuscript database

Fish cultured within water recirculating aquaculture systems (RAS) can acquire "earthy" or "musty" off-flavors due to bioaccumulation of the compounds geosmin and 2-methylisoborneol (MIB), respectively, which are produced by certain bacterial species present in RAS biosolids and microbial biofilms. ...

Incidence and predictors of radial artery spasm during transradial coronary angiography and intervention.

PubMed

Jia, De-An; Zhou, Yu-Jie; Shi, Dong-Mei; Liu, Yu-Yang; Wang, Jian-Long; Liu, Xiao-Li; Wang, Zhi-Jian; Yang, Shi-Wei; Ge, Hai-Long; Hu, Bin; Yan, Zhen-Xian; Chen, Yi; Gao, Fei

2010-04-05

Radial artery spasm (RAS) is the most common complication in transradial coronary angiography and intervention. In this study, we designed to investigate the incidence of RAS during transradial procedures in Chinese, find out the independent predictors through multiple regression, and analyze the clinical effect of RAS during follow-up. Patients arranged to receive transradial coronary angiography and intervention were consecutively enrolled. The incidence of RAS was recorded. Univariate analysis was performed to find out the influence factors of RAS, and logistic regression analysis was performed to find out the independent predictors of RAS. The patients were asked to return 1 month later for the assessment of the radial access. The incidence of RAS was 7.8% (112/1427) in all the patients received transradial procedure. Univariate analysis indicates that young (P = 0.038), female (P = 0.026), small diameter of radial artery (P < 0.001), diabetes (P = 0.026), smoking (P = 0.019), moderate or severe pain during radial artery cannulation (P < 0.001), unsuccessful access at first attempt (P = 0.002), big sheath (P = 0.004), number of catheters (> 3) (P = 0.048), rapid baseline heart rate (P = 0.032) and long operation time (P = 0.021) were associated with RAS. Logistic regression showed that female (OR = 1.745, 95%CI: 1.148 - 3.846, P = 0.024), small radial artery diameter (OR = 4.028, 95%CI: 1.264 - 12.196, P = 0.008), diabetes (OR = 2.148, 95%CI: 1.579 - 7.458, P = 0.019) and unsuccessful access at first attempt (OR = 1.468, 95%CI: 1.212 - 2.591, P = 0.032) were independent predictors of RAS. Follow-up at (28 +/- 7) days after the procedure showed that, compared with non-spasm patients, the RAS patients had higher portion of pain (11.8% vs. 6.2%, P = 0.043). The occurrences of hematoma (7.3% vs. 5.6%, P = 0.518) and radial artery occlusion (3.6% vs. 2.6%, P = 0.534) were similar. The incidence of RAS during transradial coronary procedure was 7.8%. Logistic regression analysis showed that female, small radial artery diameter, diabetes and unsuccessful access at first attempt were the independent predictors of RAS.

Angiotensins in Alzheimer's disease - friend or foe?

PubMed

Kehoe, Patrick G; Miners, Scott; Love, Seth

2009-12-01

The renin-angiotensin system (RAS) is an important regulator of blood pressure. Observational and experimental studies suggest that alterations in blood pressure and components of the brain RAS contribute to the development and progression of Alzheimer's disease (AD), resulting in changes that can lead or contribute to cognitive decline. The complexity of the RAS and diversity of its interactions with neurological processes have recently become apparent but large gaps in our understanding still remain. Modulation of activity of components of the brain RAS offers substantial opportunities for the treatment and prevention of dementia, including AD. This paper reviews molecular, genetic, experimental and clinical data as well as the therapeutic opportunities that relate to the involvement of the RAS in AD.

Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation

PubMed Central

Šolman, Maja; Ligabue, Alessio; Blaževitš, Olga; Jaiswal, Alok; Zhou, Yong; Liang, Hong; Lectez, Benoit; Kopra, Kari; Guzmán, Camilo; Härmä, Harri; Hancock, John F; Aittokallio, Tero; Abankwa, Daniel

2015-01-01

Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms. Mutants are biochemically inconspicuous, while their clustering into nanoscale signaling complexes on the plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation. Our results describe an unprecedented mechanism of signaling protein activation in cancer. DOI: http://dx.doi.org/10.7554/eLife.08905.001 PMID:26274561

The Bisphenol A analogue Bisphenol S binds to K-Ras4B--implications for 'BPA-free' plastics.

PubMed

Schöpel, Miriam; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

2016-02-01

K-Ras4B is a small GTPase that belongs to the Ras superfamily of guanine nucleotide-binding proteins. GTPases function as molecular switches in cells and are key players in intracellular signalling. Ras has been identified as an oncogene and is mutated in more than 20% of human cancers. Here, we report that Bisphenol S binds into a binding pocket of K-Ras4B previously identified for various low molecular weight compounds. Our results advocate for more comprehensive safety studies on the toxicity of Bisphenol S, as it is frequently used for Bisphenol A-free food containers. © 2016 Federation of European Biochemical Societies.

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib

PubMed Central

Booth, Laurence; Roberts, Jane L.; Poklepovic, Andrew; Kirkwood, John; Avogadri-Connors, Francesca; Cutler Jr, Richard E.; Lalani, Alshad S.; Dent, Paul

2018-01-01

ABSTRACT The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins. PMID:29219657

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib.

PubMed

Booth, Laurence; Roberts, Jane L; Poklepovic, Andrew; Kirkwood, John; Sander, Cindy; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Dent, Paul

2018-02-01

The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated ras oncogene and SV40 T-antigen.

PubMed

Su, L N; Little, J B

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. Cells transfected with EJ ras alone showed no morphological alterations nor significant changes in radiosensitivity. Cell clones expressing ras and/or SV40 T-antigen showed a reduced requirement for serum supplements, an increase in aneuploidy and chromosomal aberrations, and enhanced growth in soft agar as an early cellular response to SV40 T-antigen expression. The sequential order of transfection with SV40 T-antigen and ras influenced radio-sensitivity but not the induction of morphological changes. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself.

H-Ras Modulates N-Methyl-d-aspartate Receptor Function via Inhibition of Src Tyrosine Kinase Activity*

PubMed Central

Thornton, Claire; Yaka, Rami; Dinh, Son; Ron, Dorit

2005-01-01

Tyrosine phosphorylation of the NR2A and NR2B subunits of the N-methyl-d-aspartate (NMDA) receptor by Src protein-tyrosine kinases modulates receptor channel activity and is necessary for the induction of long term potentiation (LTP). Deletion of H-Ras increases both NR2 tyrosine phosphorylation and NMDA receptor-mediated hippocampal LTP. Here we investigated whether H-Ras regulates phosphorylation and function of the NMDA receptor via Src family protein-tyrosine kinases. We identified Src as a novel H-Ras binding partner. H-Ras bound to Src but not Fyn both in vitro and in brain via the Src kinase domain. Cotransfection of H-Ras and Src inhibited Src activity and decreased NR2A tyrosine phosphorylation. Treatment of rat brain slices with Tat-H-Ras depleted NR2A from the synaptic membrane, decreased endogenous Src activity and NR2A phosphorylation, and decreased the magnitude of hip-pocampal LTP. No change was observed for NR2B. We suggest that H-Ras negatively regulates Src phosphorylation of NR2A and retention of NR2A into the synaptic membrane leading to inhibition of NMDA receptor function. This mechanism is specific for Src and NR2A and has implications for studies in which regulation of NMDA receptor-mediated LTP is important, such as synaptic plasticity, learning, and memory and addiction. PMID:12695509

Impacts of recurrent aphthous stomatitis on quality of life of 12- and 15-year-old Thai children.

PubMed

Krisdapong, Sudaduang; Sheiham, Aubrey; Tsakos, Georgios

2012-02-01

To assess the prevalence and characteristics of oral impacts attributed to recurrent aphthous stomatitis (RAS) in 12- and 15-year-olds Thais. A national oral health survey was conducted. Child-OIDP and OIDP indices were used to collect oral impacts in 1,100 12- and 871 15 year olds. RAS-related impacts were reported in 24.7% of 12 and 36.2% of 15 year olds. Girls were more likely than boys to report RAS-related impacts. Among all perceived causes of oral impacts, RAS ranked second for 12 and first for 15 year olds. Among 12 and 15 years olds, 79.8 and 86.8% respectively had impacts on eating, 81.0 and 84.4% on cleaning teeth and 51.7 and 60.3% on emotional stability. For individual children, impacts affected between 1-6 daily performances. Impacts were of 'little' and 'moderate' intensity for 12 and 15 year olds, respectively. RAS-related impacts occurred mostly in combination with impacts from other oral conditions. Combined with other oral conditions, the impacts were worse, in terms of score, intensity and extent, than when RAS occurred alone. RAS-related impacts were common in 12- and 15-year-old Thai children and mostly affected eating, cleaning teeth and emotional stability. RAS tended to occur with other conditions leading to more severe, more extensive impacts on quality of life.

Oncogenic Activation of Fibroblast Growth Factor Receptor-3 and RAS Genes as Non-Overlapping Mutual Exclusive Events in Urinary Bladder Cancer.

PubMed

Pandith, Arshad A; Hussain, Aashaq; Khan, Mosin S; Shah, Zafar A; Wani, M Saleem; Siddiqi, Mushtaq A

2016-01-01

Urinary bladder cancer is a common malignancy in the West and ranks as the 7th most common cancer in our region of Kashmir, India. FGFR3 mutations are frequent in superficial urothelial carcinoma (UC) differing from the RAS gene mutational pattern. The aim of this study was to analyze the frequency and association of FGFR3 and RAS gene mutations in UC cases. Paired tumor and adjacent normal tissue specimens of 65 consecutive UC patients were examined. DNA preparations were evaluated for the occurrence of FGFR3 and RAS gene mutations by PCR-SCCP and DNA sequencing. Somatic point mutations of FGFR3 were identified in 32.3% (21 of 65). The pattern and distribution were significantly associated with low grade/stage (<0.05). The overall mutations in exon 1 and 2 in all the forms of RAS genes aggregated to 21.5% and showed no association with any clinic-pathological parameters. In total, 53.8% (35 of 65) of the tumors studied had mutations in either a RAS or FGFR3 gene, but these were totally mutually exclusive in and none of the samples showed both the mutational events in mutually exclusive RAS and FGFR3. We conclude that RAS and FGFR3 mutations in UC are mutually exclusive and non-overlapping events which reflect activation of oncogenic pathways through different elements.

Noonan syndrome gain-of-function mutations in NRAS cause zebrafish gastrulation defects

PubMed Central

Runtuwene, Vincent; van Eekelen, Mark; Overvoorde, John; Rehmann, Holger; Yntema, Helger G.; Nillesen, Willy M.; van Haeringen, Arie; van der Burgt, Ineke; Burgering, Boudewijn; den Hertog, Jeroen

2011-01-01

SUMMARY Noonan syndrome is a relatively common developmental disorder that is characterized by reduced growth, wide-set eyes and congenital heart defects. Noonan syndrome is associated with dysregulation of the Ras–mitogen-activated-protein-kinase (MAPK) signaling pathway. Recently, two mutations in NRAS were reported to be associated with Noonan syndrome, T50I and G60E. Here, we report a mutation in NRAS, resulting in an I24N amino acid substitution, that we identified in an individual bearing typical Noonan syndrome features. The I24N mutation activates N-Ras, resulting in enhanced downstream signaling. Expression of N-Ras-I24N, N-Ras-G60E or the strongly activating mutant N-Ras-G12V, which we included as a positive control, results in developmental defects in zebrafish embryos, demonstrating that these activating N-Ras mutants are sufficient to induce developmental disorders. The defects in zebrafish embryos are reminiscent of symptoms in individuals with Noonan syndrome and phenocopy the defects that other Noonan-syndrome-associated genes induce in zebrafish embryos. MEK inhibition completely rescued the activated N-Ras-induced phenotypes, demonstrating that these defects are mediated exclusively by Ras-MAPK signaling. In conclusion, mutations in NRAS from individuals with Noonan syndrome activated N-Ras signaling and induced developmental defects in zebrafish embryos, indicating that activating mutations in NRAS cause Noonan syndrome. PMID:21263000

The TAT-RasGAP317-326 anti-cancer peptide can kill in a caspase-, apoptosis-, and necroptosis-independent manner

PubMed Central

Puyal, Julien; Margue, Christiane; Michel, Sébastien; Kreis, Stephanie; Kulms, Dagmar; Barras, David; Nahimana, Aimable; Widmann, Christian

2016-01-01

Tumor cell resistance to apoptosis, which is triggered by many anti-tumor therapies, remains a major clinical problem. Therefore, development of more efficient therapies is a priority to improve cancer prognosis. We have previously shown that a cell-permeable peptide derived from the p120 Ras GTPase-activating protein (RasGAP), called TAT-RasGAP317-326, bears anti-malignant activities in vitro and in vivo, such as inhibition of metastatic progression and tumor cell sensitization to cell death induced by various anti-cancer treatments. Recently, we discovered that this RasGAP-derived peptide possesses the ability to directly kill some cancer cells. TAT-RasGAP317-326 can cause cell death in a manner that can be either partially caspase-dependent or fully caspase-independent. Indeed, TAT-RasGAP317-326-induced toxicity was not or only partially prevented when apoptosis was inhibited. Moreover, blocking other forms of cell death, such as necroptosis, parthanatos, pyroptosis and autophagy did not hamper the killing activity of the peptide. The death induced by TAT-RasGAP317-326 can therefore proceed independently from these modes of death. Our finding has potentially interesting clinical relevance because activation of a death pathway that is distinct from apoptosis and necroptosis in tumor cells could lead to the generation of anti-cancer drugs that target pathways not yet considered for cancer treatment. PMID:27602963

RAS testing in metastatic colorectal cancer: advances in Europe.

PubMed

Van Krieken, J Han J M; Rouleau, Etienne; Ligtenberg, Marjolijn J L; Normanno, Nicola; Patterson, Scott D; Jung, Andreas

2016-04-01

Personalized medicine shows promise for maximizing efficacy and minimizing toxicity of anti-cancer treatment. KRAS exon 2 mutations are predictive of resistance to epidermal growth factor receptor-directed monoclonal antibodies in patients with metastatic colorectal cancer. Recent studies have shown that broader RAS testing (KRAS and NRAS) is needed to select patients for treatment. While Sanger sequencing is still used, approaches based on various methodologies are available. Few CE-approved kits, however, detect the full spectrum of RAS mutations. More recently, "next-generation" sequencing has been developed for research use, including parallel semiconductor sequencing and reversible termination. These techniques have high technical sensitivities for detecting mutations, although the ideal threshold is currently unknown. Finally, liquid biopsy has the potential to become an additional tool to assess tumor-derived DNA. For accurate and timely RAS testing, appropriate sampling and prompt delivery of material is critical. Processes to ensure efficient turnaround from sample request to RAS evaluation must be implemented so that patients receive the most appropriate treatment. Given the variety of methodologies, external quality assurance programs are important to ensure a high standard of RAS testing. Here, we review technical and practical aspects of RAS testing for pathologists working with metastatic colorectal cancer tumor samples. The extension of markers from KRAS to RAS testing is the new paradigm for biomarker testing in colorectal cancer.

Effect of Primary Tumor Location on Second- or Later-line Treatment Outcomes in Patients With RAS Wild-type Metastatic Colorectal Cancer and All Treatment Lines in Patients With RAS Mutations in Four Randomized Panitumumab Studies.

PubMed

Boeckx, Nele; Koukakis, Reija; Op de Beeck, Ken; Rolfo, Christian; Van Camp, Guy; Siena, Salvatore; Tabernero, Josep; Douillard, Jean-Yves; André, Thierry; Peeters, Marc

2018-03-08

The primary tumor location has a prognostic impact in metastatic colorectal cancer (mCRC). We report the results from retrospective analyses assessing the effect of tumor location on prognosis and efficacy of second- and later-line panitumumab treatment in patients with RAS wild-type (WT) mCRC and on prognosis in all lines of treatment in patients with RAS mutant (MT) mCRC. RAS WT data (n = 483) from 2 randomized phase III panitumumab trials (ClinicalTrials.gov identifiers, NCT00339183 and NCT00113763) were analyzed for treatment outcomes stratified by tumor location. The second analysis assessed the effect of tumor location in RAS MT patients (n = 1205) from 4 panitumumab studies (ClinicalTrials.gov identifiers, NCT00364013, NCT00819780, NCT00339183, and NCT00113763). Primary tumors located in the cecum to transverse colon were coded as right-sided; those located from the splenic flexure to the rectum were coded as left-sided. Of all patients, the tumor location was ascertained for 83% to 88%; 71% to 77% of patients had left-sided tumors. RAS WT patients with right-sided tumors did worse for all efficacy parameters compared with those with left-sided tumors. The patients with left-sided tumors had better outcomes with panitumumab than with the comparator treatment. Because of the low patient numbers, no conclusions could be drawn for right-sided mCRC. The prognostic effect of tumor location on survival was unclear for RAS MT patients. These retrospective analyses have confirmed that RAS WT right-sided mCRC is associated with a poor prognosis, regardless of the treatment. RAS WT patients with left-sided tumors benefitted from the addition of panitumumab in second or later treatment lines. Further research is warranted to determine the optimum management of right-sided mCRC and RAS MT tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity

PubMed Central

Hosogane, Masaki; Funayama, Ryo; Nishida, Yuichiro; Nagashima, Takeshi; Nakayama, Keiko

2013-01-01

Oncogenic signaling pathways regulate gene expression in part through epigenetic modification of chromatin including DNA methylation and histone modification. Trimethylation of histone H3 at lysine-27 (H3K27), which correlates with transcriptional repression, is regulated by an oncogenic form of the small GTPase Ras. Although accumulation of trimethylated H3K27 (H3K27me3) has been implicated in transcriptional regulation, it remains unclear whether Ras-induced changes in H3K27me3 are a trigger for or a consequence of changes in transcriptional activity. We have now examined the relation between H3K27 trimethylation and transcriptional regulation by Ras. Genome-wide analysis of H3K27me3 distribution and transcription at various times after expression of oncogenic Ras in mouse NIH 3T3 cells identified 115 genes for which H3K27me3 level at the gene body and transcription were both regulated by Ras. Similarly, 196 genes showed Ras-induced changes in transcription and H3K27me3 level in the region around the transcription start site. The Ras-induced changes in transcription occurred before those in H3K27me3 at the genome-wide level, a finding that was validated by analysis of individual genes. Depletion of H3K27me3 either before or after activation of Ras signaling did not affect the transcriptional regulation of these genes. Furthermore, given that H3K27me3 enrichment was dependent on Ras signaling, neither it nor transcriptional repression was maintained after inactivation of such signaling. Unexpectedly, we detected unannotated transcripts derived from intergenic regions at which the H3K27me3 level is regulated by Ras, with the changes in transcript abundance again preceding those in H3K27me3. Our results thus indicate that changes in H3K27me3 level in the gene body or in the region around the transcription start site are not a trigger for, but rather a consequence of, changes in transcriptional activity. PMID:24009517

The Reciprocal Relationship Between Sexual Victimization and Sexual Assertiveness

PubMed Central

Livingston, Jennifer A.; Testa, Maria; VanZile-Tamsen, Carol

2007-01-01

Low sexual assertiveness has been proposed as a possible mechanism through which sexual revictimization occurs, yet evidence for this has been mixed. In this study, prospective path analysis was used to examine the relationship between sexual refusal assertiveness and sexual victimization over time among a community sample of women. Results provide support for a reciprocal relationship, with historical victimization predicting low sexual assertiveness and low sexual assertiveness predicting subsequent victimization. The effect of recent sexual victimization on subsequent sexual assertiveness also was replicated prospectively. These findings suggest that strengthening sexual assertiveness may help reduce vulnerability to future victimization. PMID:17322273

The reciprocal relationship between sexual victimization and sexual assertiveness.

PubMed

Livingston, Jennifer A; Testa, Maria; VanZile-Tamsen, Carol

2007-03-01

Low sexual assertiveness has been proposed as a possible mechanism through which sexual revictimization occurs, yet evidence for this has been mixed. In this study, prospective path analysis was used to examine the relationship between sexual refusal assertiveness and sexual victimization over time among a community sample of women. Results provide support for a reciprocal relationship, with historical victimization predicting low sexual assertiveness and low sexual assertiveness predicting subsequent victimization. The effect of recent sexual victimization on subsequent sexual assertiveness also was replicated prospectively. These findings suggest that strengthening sexual assertiveness may help reduce vulnerability to future victimization.

RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3*

PubMed Central

Cailliau, Katia; Lescuyer, Arlette; Burnol, Anne-Françoise; Cuesta-Marbán, Álvaro; Widmann, Christian; Browaeys-Poly, Edith

2015-01-01

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling. PMID:26109071

Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

PubMed

Esteller, M; Toyota, M; Sanchez-Cespedes, M; Capella, G; Peinado, M A; Watkins, D N; Issa, J P; Sidransky, D; Baylin, S B; Herman, J G

2000-05-01

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

[Clinical relevance of the K-ras oncogene in colorectal cancer: experience in a Mexican population].

PubMed

Cabrera-Mendoza, F; Gainza-Lagunes, S; Castañeda-Andrade, I; Castro-Zárate, A

2014-01-01

Colorectal cancer is frequent in the developed countries, with a cancer-specific mortality rate of 33%. Different biomarkers are associated with overall survival and the prediction of monoclonal treatment effectiveness. The presence of mutations in the K-ras oncogene alters the response to target therapy with cetuximab and could be an independent prognostic factor. To analyze the difference in survival between patients with mutated K-ras and those with K-ras wild-type status. Thirty-one clinical records were retrospectively analyzed of patients presenting with colorectal cancer that underwent K-ras sequencing through real-time polymerase chain reaction within the time frame of 2009 to 2012 at the Hospital de Alta Especialidad de Veracruz of the Instituto para la Salud y Seguridad Social de los Trabajadores del Estado (HAEV-ISSSTE). Survival analysis for patients with and without K-ras mutation was performed using the Kaplan Meier method. Contrast of covariates was performed using logarithmic transformations. No statistically significant difference was found in relation to survival in the patients with mutated K-ras vs. those with K-ras wild-type (P=.416), nor were significant differences found when analyzing the covariants and survival in the patients with mutated K-ras: ECOG scale (P=.221); age (less than, equal to or greater than 65years, P=.441); clinical stage according to the AJCC (P=.057), and primary lesion site (P=.614). No relation was found between the K-ras oncogene mutation and reduced survival, in contrast to what has been established in the international medical literature. Further studies that include both a larger number of patients and those receiving monoclonal treatment, need to be conducted. There were only 5 patients in the present study that received cetuximab, resulting in a misleading analysis. Copyright © 2013 Asociación Mexicana de Gastroenterología. Published by Masson Doyma México S.A. All rights reserved.

Tg.rasH2 Mice and not CByB6F1 Mice Should Be Used for 28-Day Dose Range Finding Studies Prior to 26-Week Tg.rasH2 Carcinogenicity Studies.

PubMed

Paranjpe, Madhav G; Belich, Jessica; Vidmar, Tom J; Elbekai, Reem H; McKeon, Marie; Brown, Caren

Our recent retrospective analysis of data, collected from 29 Tg.rasH2 mouse carcinogenicity studies, determined how successful the strategy of choosing the high dose for the 26-week studies was based on the estimated maximum tolerated dose (EMTD) derived from earlier 28-day dose range finding (DRF) studies conducted in CByB6F1 mice. Our analysis demonstrated that the high doses applied at EMTD in the 26-week Tg.rasH2 studies failed to detect carcinogenic effects. To investigate why the dose selection process failed in the 26-week carcinogenicity studies, the initial body weights, terminal body weights, body weight gains, food consumption, and mortality from the first 4 weeks of 26-week studies with Tg.rasH2 mice were compared with 28-day DRF studies conducted with CByB6F1 mice. Both the 26-week and the earlier respective 28-day studies were conducted with the exact same vehicle, test article, and similar dose levels. The analysis of our results further emphasizes that the EMTD and subsequent lower doses, determined on the basis of the 28-day studies in CByB6F1 mice, may not be an accurate strategy for selecting appropriate dose levels for the 26-week carcinogenicity studies in Tg.rasH2 mice. Based on the analysis presented in this article, we propose that the Tg.rasH2 mice and not the CByB6F1 mice should be used in future DRF studies. The Tg.rasH2 mice demonstrate more toxicity than the CByB6F1 mice, possibly because of their smaller size compared to CByB6F1 mice. Also, the Tg.rasH2 males appear to be more sensitive than the female Tg.rasH2 mice.

RAS signaling and anti-RAS therapy: lessons learned from genetically engineered mouse models, human cancer cells, and patient-related studies.

PubMed

Fang, Bingliang

2016-01-01

Activating mutations of oncogenic RAS genes are frequently detected in human cancers. The studies in genetically engineered mouse models (GEMMs) reveal that Kras-activating mutations predispose mice to early onset tumors in the lung, pancreas, and gastrointestinal tract. Nevertheless, most of these tumors do not have metastatic phenotypes. Metastasis occurs when tumors acquire additional genetic changes in other cancer driver genes. Studies on clinical specimens also demonstrated that KRAS mutations are present in premalignant tissues and that most of KRAS mutant human cancers have co-mutations in other cancer driver genes, including TP53, STK11, CDKN2A, and KMT2C in lung cancer; APC, TP53, and PIK3CA in colon cancer; and TP53, CDKN2A, SMAD4, and MED12 in pancreatic cancer. Extensive efforts have been devoted to develop therapeutic agents that target enzymes involved in RAS posttranslational modifications, that inhibit downstream effectors of RAS signaling pathways, and that kill RAS mutant cancer cells through synthetic lethality. Recent clinical studies have revealed that sorafenib, a pan-RAF and VEGFR inhibitor, has impressive benefits for KRAS mutant lung cancer patients. Combination therapy of MEK inhibitors with either docetaxel, AKT inhibitors, or PI3K inhibitors also led to improved clinical responses in some KRAS mutant cancer patients. This review discusses knowledge gained from GEMMs, human cancer cells, and patient-related studies on RAS-mediated tumorigenesis and anti-RAS therapy. Emerging evidence demonstrates that RAS mutant cancers are heterogeneous because of the presence of different mutant alleles and/or co-mutations in other cancer driver genes. Effective subclassifications of RAS mutant cancers may be necessary to improve patients' outcomes through personalized precision medicine. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3.

PubMed

Cailliau, Katia; Lescuyer, Arlette; Burnol, Anne-Françoise; Cuesta-Marbán, Álvaro; Widmann, Christian; Browaeys-Poly, Edith

2015-08-07

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A 26-week carcinogenicity study of 2-amino-3-methylimidazo[4,5-f]quinoline in rasH2 mice.

PubMed

Okamura, Miwa; Moto, Mitsuyoshi; Muguruma, Masako; Ito, Tadashi; Jin, Meilan; Kashida, Yoko; Mitsumori, Kunitoshi

2006-01-01

To evaluate the carcinogenic susceptibility of rasH2 mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 7-week-old rasH2 mice and their wild-type littermates (non-Tg mice) of both the sexes were fed a diet containing 0 or 300 ppm IQ for 26 weeks. Microscopical examinations revealed that the proliferative lesions of the forestomach, including squamous cell hyperplasias, papillomas, and carcinomas, were frequently encountered in male and female rasH2 mice fed with IQ. In non-Tg mice, no significant differences in the incidence of forestomach lesions were observed between the 0 ppm and 300 ppm groups. Histopathological changes such as periportal hepatocellular hypertrophy and oval cell proliferation in the liver were more apparent in female rasH2 and non-Tg mice than in males, and the incidence of hepatocellular altered foci significantly increased in female rasH2 mice in the 300 ppm group as compared to that in the 0 ppm group. These results suggest that the carcinogenic potential of IQ can be detected in rasH2 mice by a 26-week, short-term carcinogenicity test.

Very low density lipoprotein receptor regulates dendritic spine formation in a RasGRF1/CaMKII dependent manner

PubMed Central

DiBattista, Amanda Marie; Dumanis, Sonya B.; Song, Jung Min; Bu, Guojun; Weeber, Edwin; Rebeck, G. William; Hoe, Hyang-Sook

2015-01-01

Very Low Density Lipoprotein Receptor (VLDLR) is an apolipoprotein E receptor involved in synaptic plasticity, learning, and memory. However, it is unknown how VLDLR can regulate synaptic and cognitive function. In the present study, we found that VLDLR is present at the synapse both pre- and post-synaptically. Overexpression of VLDLR significantly increases, while knockdown of VLDLR decreases, dendritic spine number in primary hippocampal cultures. Additionally, knockdown of VLDLR significantly decreases synaptophysin puncta number while differentially regulating cell surface and total levels of glutamate receptor subunits. To identify the mechanism by which VLDLR induces these synaptic effects, we investigated whether VLDLR affects dendritic spine formation through the Ras signaling pathway, which is involved in spinogenesis and neurodegeneration. Interestingly, we found that VLDLR interacts with RasGRF1, a Ras effector, and knockdown of RasGRF1 blocks the effect of VLDLR on spinogenesis. Moreover, we found that VLDLR did not rescue the deficits induced by the absence of Ras signaling proteins CaMKIIα or CaMKIIβ. Taken together, our results suggest that VLDLR requires RasGRF1/CaMKII to alter dendritic spine formation. PMID:25644714

Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

PubMed Central

Sasaki, Atsuo T.; Chun, Cheryl; Takeda, Kosuke; Firtel, Richard A.

2004-01-01

During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P3, the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P3 asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal. PMID:15534002

Human Mut T Homolog 1 (MTH1): a roadblock for the tumor-suppressive effects of oncogenic RAS-induced ROS.

PubMed

Rai, Priyamvada

2012-01-01

Oncogenic RAS-induced reactive oxygen species (ROS) trigger barriers to cell transformation and cancer progression through tumor-suppressive responses such as cellular senescence or cell death. We have recently shown that oncogenic RAS-induced DNA damage and attendant premature senescence can be prevented by overexpressing human MutT Homolog 1 (MTH1), the major mammalian detoxifier of the oxidized DNA precursor, 8-oxo-dGTP. Paradoxically, RAS-induced ROS are also able to participate in tumor progression via transformative processes such as mitogenic signaling, the epithelial-mesenchymal transition (EMT), anoikis inhibition, and PI3K/Akt-mediated survival signaling. Here we provide a preliminary insight into the influence of MTH1 levels on the EMT phenotype and Akt activation in RAS-transformed HMLE breast epithelial cells. Within this context, we will discuss the implications of MTH1 upregulation in oncogenic RAS-sustaining cells as a beneficial adaptive change that inhibits ROS-mediated cell senescence and participates in the maintenance of ROS-associated tumor-promoting mechanisms. Accordingly, targeting MTH1 in RAS-transformed tumor cells will not only induce proliferative defects but also potentially enhance therapeutic cytotoxicity by shifting cellular response away from pro-survival mechanisms.

Facebook Use between College Resident Advisors' and Their Residents: A Mixed Methods Approach.

PubMed

Kacvinsky, Lauren E; Moreno, Megan A

2014-01-01

Facebook use is nearly ubiquitous among college students. Studies have shown links between Facebook displays of depression or problem drinking and risk of these problems. This project aimed to determine whether Facebook could be used to help Resident Advisors (RAs) identify college students at risk for depression or problem drinking. Interviews were conducted with college freshmen to investigate whether they were Facebook "friends" with their RA. Focus groups were conducted with RAs to determine their views on Facebook friending their dormitory residents and using Facebook to help identify at-risk students. 72 freshmen were interviewed and 25 RAs participated in focus groups; both agreed it is common for RAs and residents to be Facebook friends. RAs commonly noted references to depression and problem drinking on residents' Facebook pages, which often led to in-person discussions with the resident. This study provides support that RAs use Facebook to identify issues that may impact their student residents. RAs emphasized benefits of in-person interactions in order to provide support and obtain additional details about the situation. Universities could consider whether providing RA education about Facebook interactions with residents merits encouragement within their existing RA training programs.

Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms.

PubMed

Tartaglia, Marco; Gelb, Bruce D

2010-12-01

RAS GTPases control a major signaling network implicated in several cellular functions, including cell fate determination, proliferation, survival, differentiation, migration, and senescence. Within this network, signal flow through the RAF-MEK-ERK pathway-the first identified mitogen-associated protein kinase (MAPK) cascade-mediates early and late developmental processes controlling morphology determination, organogenesis, synaptic plasticity, and growth. Signaling through the RAS-MAPK cascade is tightly controlled; and its enhanced activation represents a well-known event in oncogenesis. Unexpectedly, in the past few years, inherited dysregulation of this pathway has been recognized as the cause underlying a group of clinically related disorders sharing facial dysmorphism, cardiac defects, reduced postnatal growth, ectodermal anomalies, variable cognitive deficits, and susceptibility to certain malignancies as major features. These disorders are caused by heterozygosity for mutations in genes encoding RAS proteins, regulators of RAS function, modulators of RAS interaction with effectors, or downstream signal transducers. Here, we provide an overview of the phenotypic spectrum associated with germline mutations perturbing RAS-MAPK signaling, the unpredicted molecular mechanisms converging toward the dysregulation of this signaling cascade, and major genotype-phenotype correlations. © 2010 New York Academy of Sciences.

Realgar bioleaching solution suppress ras excessive activation by increasing ROS in Caenorhabditis elegans.

PubMed

Zhi, De Juan; Feng, Na; Liu, Dong Ling; Hou, Rong Li; Wang, Mei Zu; Ding, Xiao Xia; Li, Hong Yu

2014-03-01

Although realgar bioleaching solution (RBS) has been proved to be a potential candidate for cancer therapy, the mechanisms of RBS anticancer are still far from being completely understood. Dosed with RBS in C. elegans, the multivulva phenotype resulting from oncogenic ras gain-of-function was inhibited in a dose dependent manner. It could be abrogated by concurrent treatment C. elegans with RBS and the radical scavenger DMSO. However, RBS could not induce DAF-16 nuclear translocation in TJ356 or the increase of HSP 16.2 expression in CL2070, which both could be aroused visible GFP fluorescent variation to represent for oxidative stress generation. Treatment C. elegans with superoxide anion generator paraquat, similar results were also obtained. Our results indicated that RBS suppress excessive activated ras by increasing reactive oxygen species (ROS) in C. elegans. Secondly, ROS induced by RBS significantly accumulated on a higher level in C. elegans with a mutational ras than that with wild ras, thus leading to oxidative stress on ras gain-of-function background rather than on normal ras context. Our results firstly demonstrated that using C. elegans as a model organism for evaluating prooxidant drug candidates for cancer therapy.

Long-term follow-up of chronic pancreatitis patients with K-ras mutation in the pancreatic juice.

PubMed

Kamisawa, Terumi; Takuma, Kensuke; Tabata, Taku; Egawa, Naoto; Yamaguchi, Toshikazu

2011-01-01

Pancreatic cancer is known to occur during the course of chronic pancreatitis in some patients. This study aimed to identify a high risk group for developing pancreatic cancer associated with chronic pancreatitis, particularly the presence of K-ras mutations in the pancreatic juice. K-ras mutation was analyzed by enriched polymerase chain reaction-enzyme linked mini-sequence assay in endoscopically-collected pancreatic juice of 21 patients with chronic pancreatitis between 1995 and 2000. All of them were followed-up for 6.0 +/- 3.8 (mean +/- SD) years (range, 2.1-14.2 years). K-ras point mutation was observed in the pancreatic juice of 11 patients with chronic pancreatitis (2+, n=2; 1+, n=6; +/-, n=3). Of these, 2 chronic pancreatitis patients with 2+K-ras point mutation developed pancreatic cancer 4.5 and 10.8 years, respectively, after the examination. Two chronic pancreatitis patients with K-ras mutation developed pancreatic cancer 4.5 and 10.8 years later. Semiquantitative analysis of K-ras mutation in endoscopically-collected pancreatic juice appears to be a useful tool for identifying chronic pancreatitis patients at high risk for developing pancreatic cancer.

Renin-angiotensin system: an old player with novel functions in skeletal muscle.

PubMed

Cabello-Verrugio, Claudio; Morales, María Gabriela; Rivera, Juan Carlos; Cabrera, Daniel; Simon, Felipe

2015-05-01

Skeletal muscle is a tissue that shows the most plasticity in the body; it can change in response to physiological and pathological stimuli. Among the diseases that affect skeletal muscle are myopathy-associated fibrosis, insulin resistance, and muscle atrophy. A common factor in these pathologies is the participation of the renin-angiotensin system (RAS). This system can be functionally separated into the classical and nonclassical RAS axis. The main components of the classical RAS pathway are angiotensin-converting enzyme (ACE), angiotensin II (Ang-II), and Ang-II receptors (AT receptors), whereas the nonclassical axis is composed of ACE2, angiotensin 1-7 [Ang (1-7)], and the Mas receptor. Hyperactivity of the classical axis in skeletal muscle has been associated with insulin resistance, atrophy, and fibrosis. In contrast, current evidence supports the action of the nonclassical RAS as a counter-regulator axis of the classical RAS pathway in skeletal muscle. In this review, we describe the mechanisms involved in the pathological effects of the classical RAS, advances in the use of pharmacological molecules to inhibit this axis, and the beneficial effects of stimulation of the nonclassical RAS pathway on insulin resistance, atrophy, and fibrosis in skeletal muscle. © 2015 Wiley Periodicals, Inc.

Is there a role of food additives in recurrent aphthous stomatitis? A prospective study with patch testing.

PubMed

Gülseren, Duygu; Hapa, Asli; Ersoy-Evans, Sibel; Elçin, Gonca; Karaduman, Ayşen

2017-03-01

Recurrent aphthous stomatitis (RAS) is a common disease of the oral mucosa with an unknown etiology. This study aimed to determine if food additives play a role in the etiology of RAS as well as to determine if patch testing can be used to detect which allergens cause RAS. This prospective study included 24 patients with RAS and 22 healthy controls. All the participants underwent patch testing for 23 food additives. In total, 21 (87.5%) RAS patients and 3 (13.6%) controls had positive patch test reactions to ≥1 allergens; the difference in the patch test positivity rate between groups was significant (P < 0.05). The most common allergen that elicited positive patch test results in the patient group was cochineal red (n = 15 [62.5%]), followed by azorubine (n = 11 [45.8%]) and amaranth (n = 6 [25%]). The present findings show that food additives might play a role in the etiology of RAS and that patch testing could be a method for determining the etiology of RAS. © 2016 The International Society of Dermatology.

Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP.

PubMed

Matheny, Sharon A; Chen, Chiyuan; Kortum, Robert L; Razidlo, Gina L; Lewis, Robert E; White, Michael A

2004-01-15

The signal transduction cascade comprising Raf, mitogen-activated protein (MAP) kinase kinase (MEK) and MAP kinase is a Ras effector pathway that mediates diverse cellular responses to environmental cues and contributes to Ras-dependent oncogenic transformation. Here we report that the Ras effector protein Impedes Mitogenic signal Propagation (IMP) modulates sensitivity of the MAP kinase cascade to stimulus-dependent activation by limiting functional assembly of the core enzymatic components through the inactivation of KSR, a scaffold/adaptor protein that couples activated Raf to its substrate MEK. IMP is a Ras-responsive E3 ubiquitin ligase that, on activation of Ras, is modified by auto-polyubiquitination, which releases the inhibition of Raf-MEK complex formation. Thus, Ras activates the MAP kinase cascade through simultaneous dual effector interactions: induction of Raf kinase activity and derepression of Raf-MEK complex formation. IMP depletion results in increased stimulus-dependent MEK activation without alterations in the timing or duration of the response. These observations suggest that IMP functions as a threshold modulator, controlling sensitivity of the cascade to stimulus and providing a mechanism to allow adaptive behaviour of the cascade in chronic or complex signalling environments.

The frequencies of calcium oscillations are optimized for efficient calcium-mediated activation of Ras and the ERK/MAPK cascade.

PubMed

Kupzig, Sabine; Walker, Simon A; Cullen, Peter J

2005-05-24

Ras proteins are binary switches that, by cycling through inactive GDP- and active GTP-bound conformations, regulate multiple cellular signaling pathways, including those that control growth and differentiation. For some time, it has been known that receptor-mediated increases in the concentration of intracellular free calcium ([Ca(2+)](i)) can modulate Ras activation. Increases in [Ca(2+)](i) often occur as repetitive Ca(2+) spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca(2+) oscillations increase in frequency with the amplitude of receptor stimuli, a phenomenon critical for the induction of selective cellular functions. Here, we show that Ca(2+) oscillations are optimized for Ca(2+)-mediated activation of Ras and signaling through the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. We present additional evidence that Ca(2+) oscillations reduce the effective Ca(2+) threshold for the activation of Ras and that the oscillatory frequency is optimized for activation of Ras and the ERK/MAPK pathway. Our results describe a hitherto unrecognized link between complex Ca(2+) signals and the modulation of the Ras/ERK/MAPK signaling cascade.

Literature review : performance of RAP/RAS mixes and new direction.

DOT National Transportation Integrated Search

2014-04-01

In the last several years reclaimed asphalt pavement (RAP) and recycled asphalt shingles (RAS) have been : widely used in asphalt mixes in Texas. The use of RAP/RAS can significantly reduce the initial cost of : asphalt mixtures, conserve energy, and...

Mechanism of Ras Activation by TGFBeta

DTIC Science & Technology

2002-07-01

32P-labeled oligonucleotides to each reaction. The reactions were incubated at room temperature for 20 min. For supershift assays, 1 p\\ of antibodies ...formation of this TGFß3-inducible complex. In addition, as shown in Fig. 4A, left side, addition of either a pan-Fos or pan-Jun antibody completely blocked...addition to c-Jun 30770 A Ras/MAPK/Smads and TGFß1 Production RasNI7E3 -RasN17 +RiisN17 Antibodies TGFß I " - Jun Fits Ig(J

Assertive Training: Teaching Women Not to Discriminate Against Themselves.

ERIC Educational Resources Information Center

Butler, Pamela E.

The process of assertive training is described with emphasis placed on its applicability to problems in female assertiveness. Male and female members of four assertive training groups were compared on the Wolpe-Lazarus assertive inventory. Male members obtained a significantly lower score than female members i.e., men reported themselves as…

The Effects of Assertive Training on Performance in Highly Anxious Adolescents.

ERIC Educational Resources Information Center

Wehr, Sara H.; Kaufman, Melvin E.

1987-01-01

Investigated the effects of assertive training on measures of assertiveness, state anxiety, and mathematics performance in highly anxious ninth graders (N=96). Found that assertive training resulted in increased assertiveness and decreased state anxiety, with no significant effect on mathematics performance, and no significant effect due to sex.…

The test of truth: an experimental investigation of the norm of assertion.

PubMed

Turri, John

2013-11-01

Assertion is fundamental to our lives as social and cognitive beings. Philosophers have recently built an impressive case that the norm of assertion is factive. That is, you should make an assertion only if it is true. Thus far the case for a factive norm of assertion been based on observational data. This paper adds experimental evidence in favor of a factive norm from six studies. In these studies, an assertion's truth value dramatically affects whether people think it should be made. Whereas nearly everyone agreed that a true assertion supported by good evidence should be made, most people judged that a false assertion supported by good evidence should not be made. The studies also suggest that people are consciously aware of criteria that guide their evaluation of assertions. Evidence is also presented that some intuitive support for a non-factive norm of assertion comes from a surprising tendency people have to misdescribe cases of blameless rule-breaking as cases where no rule is broken. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of an assertiveness training program on nursing and medical students' assertiveness, self-esteem, and interpersonal communication satisfaction.

PubMed

Lin, Yen-Ru; Shiah, I-Shin; Chang, Yue-Cune; Lai, Tzu-Ju; Wang, Kwua-Yun; Chou, Kuei-Ru

2004-11-01

This study's objective was to evaluate the effect of an assertiveness training program on nursing and medical students' assertiveness, self-esteem, and interpersonal communication satisfaction. Using a longitudinal research design, 69 participants whose scores on the Assertive Scale were < or = 50% (i.e., low assertiveness) and who were willing to participate were included and assigned to an experimental group (33 subjects) or comparison group (36 participants; participants were matched with the experimental group by grade and sex). Participants in the experimental group received eight 2-h sessions of assertiveness training once a week. Data were collected before and after training and again one month after the end of the training using the Rotter's Internal versus External Control of Reinforcement Scale, Sex Role Inventory, Assertive Scale, Esteem Scale, and Interpersonal Communication Satisfaction Inventory. The generalized estimated equation (GEE) method was used for statistical analysis. The assertiveness and self-esteem of the experimental group were significantly improved in nursing and medical students after assertiveness training, although interpersonal communication satisfaction of the experimental group was not significantly improved after the training program.

Biotransformation of glyceryl trinitrate occurs concurrently with relaxation of rabbit aorta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brien, J.F.; McLaughlin, B.E.; Breedon, T.H.

1986-05-01

This study was conducted to test the hypothesis that biotransformation of glyceryl trinitrate (GTN) is involved in GTN-induced relaxation of vascular smooth muscle. Isolated rabbit aortic strips (RAS) were contracted submaximally with phenylephrine (PE) and then were incubated with 0.5 microM (/sup 14/C)GTN in a time course study. GTN-induced relaxation (inhibition of PE-induced tone) of RAS was monitored and tissue GTN and glyceryl-1,2- and 1,3-dinitrate (GDN) concentrations were measured by thin-layer chromatography and liquid scintillation spectrometry at 0.5, 1, 2 and 20 min after incubation. Biotransformation of GTN to GDN occurred during GTN-induced relaxation of RAS. The tissue GDN concentrationmore » was dependent on the time duration of incubation with GTN and was related to the magnitude of GTN-induced tissue relaxation. At the 20-min interval, the GDN concentration in the incubation medium indicated appreciable efflux of GDN metabolites from the RAS. In the biotransformation of GTN by RAS, there was about 4-fold preferential formation of 1,2-GDN compared with 1,3-GDN. RAS were made tolerant to GTN in vitro by incubation with 500 microM GTN for 1 hr. After washing, GTN-tolerant and nontolerant (incubation with vehicle for 1 hr) RAS were contracted submaximally with PE, and then were incubated with 0.5 microM (/sup 14/C)GTN for 2 min. GTN-induced relaxation of RAS and tissue GDN concentration were significantly less for GTN-tolerant tissue compared with nontolerant tissue. Tissue GTN concentration was similar for both GTN-tolerant and nontolerant RAS, which indicated that the tissue uptake of GTN was similar and that GTN biotransformation was diminished in tolerant tissue.(ABST« less

Changes in the renin angiotensin system during the development of colorectal cancer liver metastases.

PubMed

Neo, Jaclyn H; Ager, Eleanor I; Angus, Peter W; Zhu, Jin; Herath, Chandana B; Christophi, Christopher

2010-04-10

Blockade of the renin angiotensin system (RAS) via angiotensin I converting enzyme (ACE) inhibition reduces growth of colorectal cancer (CRC) liver metastases in a mouse model. In this work we defined the expression of the various components of the RAS in both tumor and liver during the progression of this disease. Immunohistochemistry and quantitative RT-PCR was used to examine RAS expression in a mouse CRC liver metastases model. CRC metastases and liver tissue was assessed separately at key stages of CRC liver metastases development in untreated (control) mice and in mice treated with the ACE inhibitor captopril (750 mg/kg/day). Non-tumor induced (sham) mice indicated the effect of tumors on normal liver RAS. The statistical significance of multiple comparisons was determined using one-way analysis of variance followed by Bonferroni adjustment with SAS/STAT software. Reduced volume of CRC liver metastases with captopril treatment was evident. Local RAS of CRC metastases differed from the surrounding liver, with lower angiotensin II type 1 receptor (AT1R) expression but increased ANG-(1-7) receptor (MasR) compared to the liver. The AT1R localised to cancer and stromal infiltrating cells, while other RAS receptors were detected in cancer cells only. Tumor induction led to an initial increase in AT1R and ACE expression while captopril treatment significantly increased ACE expression in the final stages of tumor growth. Conversely, captopril treatment decreased expression of AT1R and angiotensinogen. These results demonstrate significant changes in RAS expression in the tumor-bearing captopril treated liver and in CRC metastases. The data suggests the existence of a tumor-specific RAS that can be independently targeted by RAS blockade.

Changes in the renin angiotensin system during the development of colorectal cancer liver metastases

PubMed Central

2010-01-01

Background Blockade of the renin angiotensin system (RAS) via angiotensin I converting enzyme (ACE) inhibition reduces growth of colorectal cancer (CRC) liver metastases in a mouse model. In this work we defined the expression of the various components of the RAS in both tumor and liver during the progression of this disease. Methods Immunohistochemistry and quantitative RT-PCR was used to examine RAS expression in a mouse CRC liver metastases model. CRC metastases and liver tissue was assessed separately at key stages of CRC liver metastases development in untreated (control) mice and in mice treated with the ACE inhibitor captopril (750 mg/kg/day). Non-tumor induced (sham) mice indicated the effect of tumors on normal liver RAS. The statistical significance of multiple comparisons was determined using one-way analysis of variance followed by Bonferroni adjustment with SAS/STAT software. Results Reduced volume of CRC liver metastases with captopril treatment was evident. Local RAS of CRC metastases differed from the surrounding liver, with lower angiotensin II type 1 receptor (AT1R) expression but increased ANG-(1-7) receptor (MasR) compared to the liver. The AT1R localised to cancer and stromal infiltrating cells, while other RAS receptors were detected in cancer cells only. Tumor induction led to an initial increase in AT1R and ACE expression while captopril treatment significantly increased ACE expression in the final stages of tumor growth. Conversely, captopril treatment decreased expression of AT1R and angiotensinogen. Conclusions These results demonstrate significant changes in RAS expression in the tumor-bearing captopril treated liver and in CRC metastases. The data suggests the existence of a tumor-specific RAS that can be independently targeted by RAS blockade. PMID:20380732

RB mutation and RAS overexpression induce resistance to NK cell-mediated cytotoxicity in glioma cells.

PubMed

Orozco-Morales, Mario; Sánchez-García, Francisco Javier; Golán-Cancela, Irene; Hernández-Pedro, Norma; Costoya, Jose A; de la Cruz, Verónica Pérez; Moreno-Jiménez, Sergio; Sotelo, Julio; Pineda, Benjamín

2015-01-01

Several theories aim to explain the malignant transformation of cells, including the mutation of tumor suppressors and proto-oncogenes. Deletion of Rb (a tumor suppressor), overexpression of mutated Ras (a proto-oncogene), or both, are sufficient for in vitro gliomagenesis, and these genetic traits are associated with their proliferative capacity. An emerging hallmark of cancer is the ability of tumor cells to evade the immune system. Whether specific mutations are related with this, remains to be analyzed. To address this issue, three transformed glioma cell lines were obtained (Rb(-/-), Ras(V12), and Rb(-/-)/Ras(V12)) by in vitro retroviral transformation of astrocytes, as previously reported. In addition, Ras(V12) and Rb(-/-)/Ras(V12) transformed cells were injected into SCID mice and after tumor growth two stable glioma cell lines were derived. All these cells were characterized in terms of Rb and Ras gene expression, morphology, proliferative capacity, expression of MHC I, Rae1δ, and Rae1αβγδε, mult1, H60a, H60b, H60c, as ligands for NK cell receptors, and their susceptibility to NK cell-mediated cytotoxicity. Our results show that transformation of astrocytes (Rb loss, Ras overexpression, or both) induced phenotypical and functional changes associated with resistance to NK cell-mediated cytotoxicity. Moreover, the transfer of cell lines of transformed astrocytes into SCID mice increased resistance to NK cell-mediated cytotoxicity, thus suggesting that specific changes in a tumor suppressor (Rb) and a proto-oncogene (Ras) are enough to confer resistance to NK cell-mediated cytotoxicity in glioma cells and therefore provide some insight into the ability of tumor cells to evade immune responses.

Fibroblast growth factor 2 restrains Ras-driven proliferation of malignant cells by triggering RhoA-mediated senescence.

PubMed

Costa, Erico T; Forti, Fábio L; Matos, Tatiana G F; Dermargos, Alexandre; Nakano, Fábio; Salotti, Jacqueline; Rocha, Kátia M; Asprino, Paula F; Yoshihara, Celina K; Koga, Marianna M; Armelin, Hugo A

2008-08-01

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant Y1 adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated beta-galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Y1 adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either Y1 or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Ras-dependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RhoA-GTP. Surprisingly, attempts to select FGF2-resistant cells from the Y1 and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Ras-dependent malignant cells could rarely overcome.

The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells.

PubMed

Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival.

Impact of Nrf2 on tumour growth and drug sensitivity in oncogenic K-ras-transformed cells in vitro and in vivo.

PubMed

Shao, Jiajia; Glorieux, Christophe; Liao, Jianwei; Chen, Ping; Lu, Wenhua; Liang, Zhenhao; Wen, Shijun; Hu, Yumin; Huang, Peng

2018-06-01

K-ras is one of the most common oncogenes in human cancers, and its aberrant activation may lead to malignant transformation associated with oxidative stress and activation of the transcription factor Nrf2 that regulates multiple detoxification enzymes. The purpose of this research was to use gene editing technology to evaluate the role of Nrf2 in affecting tumour growth and drug sensitivity of K-ras G12V -transformed cells. We showed that induction of K-ras G12V caused a significant activation of Nrf2 associated with increased expression of its target genes NAD(P)H:quinone oxidoreductase 1 (NQO1) and haem oxygenase-1 (HO-1). Interestingly, knock-out of Nrf2 by CRISPR/Cas9 in K-ras G12V -expressing cells only impacted the expression of NQO1 but not HO-1. We also found that Nrf2 knock-out caused high reactive oxygen species (ROS) stress, suppression of cell proliferation, increased apoptosis in vitro, and a decrease of tumour growth in vivo. Furthermore, abrogation of Nrf2 significantly increased the sensitivity of K-ras G12V cells to multiple anticancer agents including phenethyl isothiocyanate (PEITC), doxorubicin, etoposide, and cisplatin. These results show that genetic abrogation of Nrf2 impairs the malignant phenotype of K-Ras G12V -transformed cells in vitro and in vivo, and demonstrates the critical role of Nrf2 in promoting cell survival and drug resistance in cells harbouring oncogenic K-ras. As such, inhibition of Nrf2 would be an attractive strategy to increase the therapeutic effect and overcome drug resistance in cancer with oncogenic K-ras activation.

The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells

PubMed Central

Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival. PMID:25616580

DRoP: a water analysis program identifies Ras-GTP-specific pathway of communication between membrane-interacting regions and the active site.

PubMed

Kearney, Bradley M; Johnson, Christian W; Roberts, Daniel M; Swartz, Paul; Mattos, Carla

2014-02-06

Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras. Copyright © 2013 Elsevier Ltd. All rights reserved.

Loss of NF1 in cutaneous melanoma is associated with RAS activation and MEK dependence.

PubMed

Nissan, Moriah H; Pratilas, Christine A; Jones, Alexis M; Ramirez, Ricardo; Won, Helen; Liu, Cailian; Tiwari, Shakuntala; Kong, Li; Hanrahan, Aphrothiti J; Yao, Zhan; Merghoub, Taha; Ribas, Antoni; Chapman, Paul B; Yaeger, Rona; Taylor, Barry S; Schultz, Nikolaus; Berger, Michael F; Rosen, Neal; Solit, David B

2014-04-15

Melanoma is a disease characterized by lesions that activate ERK. Although 70% of cutaneous melanomas harbor activating mutations in the BRAF and NRAS genes, the alterations that drive tumor progression in the remaining 30% are largely undefined. Vemurafenib, a selective inhibitor of RAF kinases, has clinical utility restricted to BRAF-mutant tumors. MEK inhibitors, which have shown clinical activity in NRAS-mutant melanoma, may be effective in other ERK pathway-dependent settings. Here, we investigated a panel of melanoma cell lines wild type for BRAF and NRAS to determine the genetic alteration driving their transformation and their dependence on ERK signaling in order to elucidate a candidate set for MEK inhibitor treatment. A cohort of the BRAF/RAS wild type cell lines with high levels of RAS-GTP had loss of NF1, a RAS GTPase activating protein. In these cell lines, the MEK inhibitor PD0325901 inhibited ERK phosphorylation, but also relieved feedback inhibition of RAS, resulting in induction of pMEK and a rapid rebound in ERK signaling. In contrast, the MEK inhibitor trametinib impaired the adaptive response of cells to ERK inhibition, leading to sustained suppression of ERK signaling and significant antitumor effects. Notably, alterations in NF1 frequently co-occurred with RAS and BRAF alterations in melanoma. In the setting of BRAF(V600E), NF1 loss abrogated negative feedback on RAS activation, resulting in elevated activation of RAS-GTP and resistance to RAF, but not MEK, inhibitors. We conclude that loss of NF1 is common in cutaneous melanoma and is associated with RAS activation, MEK-dependence, and resistance to RAF inhibition. ©2014 AACR.

Efficacy and safety of nab-paclitaxel in patients with previously treated metastatic colorectal cancer: a phase II COLO-001 trial.

PubMed

Ducreux, Michel; Bennouna, Jaafar; Adenis, Antoine; Conroy, Thierry; Lièvre, Astrid; Portales, Fabienne; Jeanes, Julie; Li, Li; Romano, Alfredo

2017-01-01

This single-arm, phase II trial evaluated nab-paclitaxel monotherapy in pretreated patients with metastatic colorectal cancer (mCRC). Patients with mCRC (RAS wild-type and RAS mutant cohorts) received nab-paclitaxel 125 mg/m 2 days 1, 8, and 15 (28-day cycle). The primary endpoint was investigator-assessed progression-free survival (PFS) rate at week 8; secondary endpoints included overall survival, overall response rate, and safety. Stage 1 planned enrollment was 15 patients per cohort per Simon 2-stage design. Stage 2 enrollment was to continue unless ≤8 of the first 15 patients per cohort achieved PFS at 8 weeks. Stage 1 enrolled 41 patients (RAS wild type: n = 18; RAS mutant: n = 23). In both RAS cohorts, 3 of 15 patients initially enrolled were progression-free at week 8 (20%; 95% CI 4.0-48.0). Median PFS was 8.1 weeks (95% CI 7.7-8.6) and 7.9 weeks (95% CI 7.6-8.0) for RAS wild-type and RAS mutant cohorts, respectively. There were no complete or partial responses. The overall disease control rate was 16% (95% CI 6.0-32.0), and rates were similar in the RAS wild-type and RAS mutant cohorts (18 and 15%, respectively). No new safety signals were reported; the most common grade ≥3 adverse events included neutropenia, asthenia, and peripheral neuropathy. This study did not progress to stage 2 per the preplanned statistical stopping rule. In patients with heavily pretreated mCRC, nab-paclitaxel did not demonstrate promising antitumor activity; further assessment of nab-paclitaxel monotherapy in this population of patients is not supported. NCT02103062.

Transcriptional and translational control of ornithine decarboxylase during Ras transformation.

PubMed Central

Shantz, Lisa M

2004-01-01

ODC (ornithine decarboxylase) activity is induced following ras activation. However, the Ras effector pathways responsible are unknown. These experiments used NIH-3T3 cells expressing partial-loss-of-function Ras mutants to activate selectively pathways downstream of Ras and examined the contribution of each pathway to ODC induction. Overexpression of Ras12V, a constitutively active mutant, resulted in ODC activities up to 20-fold higher than controls. Stable transfections of Ras partial-loss-of-function mutants and constitutively active forms of MEK (MAPK kinase) and Akt indicated that activation of more than one Ras effector pathway is necessary for the complete induction of ODC activity. The increase in ODC activity in Ras12V-transformed cells is not owing to a substantial change in ODC protein half-life, which increased by <2-fold. Northern-blot analysis and reporter assays suggested that the mechanism of ODC induction involves both a modest increase in the transcription of ODC mRNA and a much more considerable increase in the translation of mRNA into protein. ODC transcription was controlled through a pathway dependent on Raf/MEK/ERK (where ERK stands for extracellular-signal-regulated kinase) activation, whereas activation of the phosphoinositide 3-kinase and the Raf/MEK/ERK pathways were necessary for translational regulation of ODC. The increase in ODC synthesis was accompanied by changes in phosphorylation of eukaryotic initiation factor 4E and its binding protein 4E-BP1. Results show that the phosphoinositide 3-kinase pathway regulates phosphorylation of both proteins, whereas the Raf/MEK/ERK pathway affects only the eukaryotic initiation factor 4E phosphorylation. PMID:14519103

Restrictive allograft syndrome (RAS): a novel form of chronic lung allograft dysfunction.

PubMed

Sato, Masaaki; Waddell, Thomas K; Wagnetz, Ute; Roberts, Heidi C; Hwang, David M; Haroon, Ayesha; Wagnetz, Dirk; Chaparro, Cecilia; Singer, Lianne G; Hutcheon, Michael A; Keshavjee, Shaf

2011-07-01

Bronchiolitis obliterans syndrome (BOS) with small-airway pathology and obstructive pulmonary physiology may not be the only form of chronic lung allograft dysfunction (CLAD) after lung transplantation. Characteristics of a form of CLAD consisting of restrictive functional changes involving peripheral lung pathology were investigated. Patients who received bilateral lung transplantation from 1996 to 2009 were retrospectively analyzed. Baseline pulmonary function was taken as the time of peak forced expiratory volume in 1 second (FEV(1)). CLAD was defined as irreversible decline in FEV(1) < 80% baseline. The most accurate threshold to predict irreversible decline in total lung capacity and thus restrictive functional change was at 90% baseline. Restrictive allograft syndrome (RAS) was defined as CLAD meeting this threshold. BOS was defined as CLAD without RAS. To estimate the effect on survival, Cox proportional hazards models and Kaplan-Meier analyses were used. Among 468 patients, CLAD developed in 156; of those, 47 (30%) showed the RAS phenotype. Compared with the 109 BOS patients, RAS patients showed significant computed tomography findings of interstitial lung disease (p < 0.0001). Prevalence of RAS was approximately 25% to 35% of all CLAD over time. Patient survival of RAS was significantly worse than BOS after CLAD onset (median survival, 541 vs 1,421 days; p = 0.0003). The RAS phenotype was the most significant risk factor of death among other variables after CLAD onset (hazard ratio, 1.60; confidential interval, 1.23-2.07). RAS is a novel form of CLAD that exhibits characteristics of peripheral lung fibrosis and significantly affects survival of lung transplant patients. Copyright © 2011 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

Structure of the Dominant Negative S17N Mutant of Ras

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nassar, N.; Singh, K; Garcia-Diaz, M

2010-01-01

The use of the dominant negative mutant of Ras has been crucial in elucidating the cellular signaling of Ras in response to the activation of various membrane-bound receptors. Although several point mutants of Ras exhibit a dominant negative effect, the asparagine to serine mutation at position 17 (S17N) remains the most popular and the most effective at inhibiting the activation of endogenous Ras. It is now widely accepted that the dominant negative effect is due to the ability of the mutant to sequester upstream activators and its inability to activate downstream effectors. Here, we present the crystal structure of RasS17Nmore » in the GDP-bound form. In the three molecules that populate the asymmetric unit, the Mg{sup 2+} ion that normally coordinates the {beta}-phosphate is absent because of steric hindrance from the Asn17 side chain. Instead, a Ca{sup 2+} ion is coordinating the {alpha}-phosphate. Also absent from one molecule is electron density for Phe28, a conserved residue that normally stabilizes the nucleotide's guanine base. Except for Phe28, the nucleotide makes conserved interactions with Ras. Combined, the inability of Phe28 to stabilize the guanine base and the absence of a Mg{sup 2+} ion to neutralize the negative charges on the phosphates explain the weaker affinity of GDP for Ras. Our data suggest that the absence of the Mg{sup 2+} should also dramatically affect GTP binding to Ras and the proper positioning of Thr35 necessary for the activation of switch 1 and the binding to downstream effectors, a prerequisite for the triggering of signaling pathways.« less

K-ras Mutations as the Earliest Driving Force in a Subset of Colorectal Carcinomas

PubMed Central

MARGETIS, NIKOLAOS; KOULOUKOUSSA, MYRSINI; PAVLOU, KYRIAKI; VRAKAS, SPYRIDON; MARIOLIS-SAPSAKOS, THEODOROS

2017-01-01

K-ras oncogene is a key factor in colorectal cancer. Based on published and our data we propose that K-ras could be the oncogene responsible for the inactivation of the tumor-suppressor gene APC, currently considered as the initial step in colorectal tumorigenesis. K-ras fulfills the criteria of the oncogene-induced DNA damage model, as it can provoke well- established causes for inactivating tumor-suppressors, i.e. DNA double-strand breaks (causing allele deletion) and ROS production (responsible for point mutation). The model we propose is a variation of the currently existing model and hypothesizes that, in a subgroup of colorectal carcinomas, K-ras mutation may precede APC inactivation, representing the earliest driving force and, probably, an early biomarker of colorectal carcinogenesis. This observation is clinically useful, since it may modify the preventive colorectal cancer strategy, restricting numerically patients undergoing colonoscopies to those bearing K-ras mutation in their colorectum, either in benign polyps or the normal accompanying mucosa. PMID:28652417

HTLV-1 Tax protein cooperates with Ras in protecting cells from apoptosis.

PubMed

Vajente, Nicola; Trevisan, Roberta; Saggioro, Daniela

2009-02-01

Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) plays a critical role in HTLV-I-correlated diseases through its ability to deregulate the expression of a vast array of cellular genes. We have previously shown that Tax counteracts apoptosis induced by stimuli triggering mitochondria apoptotic pathway, most likely by activating CREB-mediated transcription and affecting the phosphorylation levels of CREB at Ser-133. Here, we report data that indicate the oncoprotein Ras as a possible mediator of Tax-induced apoptosis protection and suggest a possible role of Tax in Ras activation. In addition, using inhibitors of down stream effectors of Ras, we found that ERK signaling is the most relevant for Tax-mediated apoptosis protection. As a whole, our findings provide intriguing evidence of a possible link between Ras signaling and Tax capability to counteract apoptosis and to enhance P-CREB levels, and implicates a potential role for Ras in HTLV-1-induced diseases.

EGFR immunoexpression, RAS immunoexpression and their effects on survival in lung adenocarcinoma cases

PubMed Central

Onder, Sevgen; Firat, Pinar; Dogan, Riza

2014-01-01

Background The impacts of epidermal growth factor receptor (EGFR) immunoexpression and RAS immunoexpression on the survival and prognosis of lung adenocarcinoma patients are debated in the literature. Methods Twenty-six patients, who underwent pulmonary resections between 2002 and 2007 in our clinic, and whose pathologic examinations yielded adenocarcinoma, were included in the study. EGFR and RAS expression levels were examined by immunohistochemical methods. The results were compared with the survival, stage of the disease, nodal involvement, lymphovascular invasion, and pleural invasion. Nonparametric bivariate analyses were used for statistical analyses. Results A significant link between EGFR immunoexpression and survival has been identified while RAS immunoexpression and survival have been proven to be irrelevant. Neither EGFR, nor RAS has displayed a significant link with the stage of the disease, nodal involvement, lymphovascular invasion, or pleural invasion. Conclusions Positive EGFR immunoexpression affects survival negatively, while RAS immunoexpression has no effect on survival in lung adenocarcinoma patients. PMID:24977003

Positive feedback can lead to dynamic nanometer-scale clustering on cell membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wehrens, Martijn; Rein ten Wolde, Pieter; Mugler, Andrew, E-mail: amugler@purdue.edu

2014-11-28

Clustering of molecules on biological membranes is a widely observed phenomenon. A key example is the clustering of the oncoprotein Ras, which is known to be important for signal transduction in mammalian cells. Yet, the mechanism by which Ras clusters form and are maintained remains unclear. Recently, it has been discovered that activated Ras promotes further Ras activation. Here we show using particle-based simulation that this positive feedback is sufficient to produce persistent clusters of active Ras molecules at the nanometer scale via a dynamic nucleation mechanism. Furthermore, we find that our cluster statistics are consistent with experimental observations ofmore » the Ras system. Interestingly, we show that our model does not support a Turing regime of macroscopic reaction-diffusion patterning, and therefore that the clustering we observe is a purely stochastic effect, arising from the coupling of positive feedback with the discrete nature of individual molecules. These results underscore the importance of stochastic and dynamic properties of reaction diffusion systems for biological behavior.« less

Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging.

PubMed

Yasuda, Ryohei; Harvey, Christopher D; Zhong, Haining; Sobczyk, Aleksander; van Aelst, Linda; Svoboda, Karel

2006-02-01

To understand the biochemical signals regulated by neural activity, it is necessary to measure protein-protein interactions and enzymatic activity in neuronal microcompartments such as axons, dendrites and their spines. We combined two-photon excitation laser scanning with fluorescence lifetime imaging to measure fluorescence resonance energy transfer at high resolutions in brain slices. We also developed sensitive fluorescent protein-based sensors for the activation of the small GTPase protein Ras with slow (FRas) and fast (FRas-F) kinetics. Using FRas-F, we found in CA1 hippocampal neurons that trains of back-propagating action potentials rapidly and reversibly activated Ras in dendrites and spines. The relationship between firing rate and Ras activation was highly nonlinear (Hill coefficient approximately 5). This steep dependence was caused by a highly cooperative interaction between calcium ions (Ca(2+)) and Ras activators. The Ras pathway therefore functions as a supersensitive threshold detector for neural activity and Ca(2+) concentration.

[Farnesyl transferase inhibitors (anti-Ras). A new class of anticancer agents].

PubMed

Levy, R

Ras genes are frequently activated in human tumours. The role of their product, the P21 proteins, in the transduction of the mitogenic signal makes them attractive targets for an anti-neoplastic therapy. The p21 ras proteins are linked to the plasma membrane and transformed into an active form for signal transmission. Their effect is to mediate the effects of growth factors. Two drug families, the Benzodiazepine peptidomimetics and the CAAX tetrapeptides which inhibit the farnesylation of P21-Ras proteins abolish the transforming properties of mutated P21. These promising drugs could rapidly have clinical applications. They have been shown to be highly active at precise concentrations on ras-transformed cells but at the same concentrations are not toxic for untransformed cells. They do not effect other similar enzyme systems within the cell, underlining their selective capacity. Theoretically anti-ras therapy could only suspend cell transformation although it might be possible that if given long enough, a lethal threshold could be reached.

Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro.

PubMed

Parrell, Daniel; Zhang, Yang; Olenic, Sandra; Kroos, Lee

2017-10-01

RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli , we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli , including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His 6 -MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His 6 -MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL. IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are typically inferred from genetic studies in the native bacterium. Evidence for direct effects has come sometimes from coexpression of the enzyme and potential substrate in a heterologous host and rarely from biochemical reconstitution of cleavage in vitro We applied these two approaches to the B. subtilis enzyme RasP and its proposed substrates RsiW and FtsL. We discovered potential pitfalls and solutions in heterologous coexpression experiments in E. coli , providing evidence that both substrates are cleaved by RasP in vivo but, surprisingly, that only RsiW was cleaved in vitro , suggesting that FtsL has an additional requirement. Copyright © 2017 American Society for Microbiology.

Defeat mutant KRAS with synthetic lethality

PubMed Central

Pang, Xiufeng; Liu, Mingyao

2017-01-01

ABSTRACT Ras proteins are considered as the founding members of a large superfamily of small GTPases that control fundamental cellular functions. Mutationally activated RAS genes were discovered in human cancer cells more than 3 decades ago, but intensive efforts on Ras structure, biochemistry, function and signaling continue even now. Because mutant Ras proteins are inherently difficult to inhibit and have yet been therapeutically conquered, it was designated as “the Everest of oncogenes” in the cancer genome landscape, further promoting a “renaissance” in RAS research. Different paths to directly or indirectly targeting mutant Ras signaling are currently under investigation in the hope of finding an efficacious regimen. Inhibitors directly binding to KRASG12C to block its downstream signaling have been revealed, supporting the notion of Ras' druggability. An alternative indirect approach by targeting synthetic lethal interactors of mutant RAS is underway. We recently employed a synthetic lethal drug screen plus a combinatorial strategy using a panel of clinical agents and discovered that KRAS-mutant cancers were fragile to the combined inhibition of polo-like kinase 1 (Plk1) and RhoA/Rho kinase (ROCK). The combined regimen of BI-2536 (a Plk1 inhibitor) and fasudil (a ROCK inhibitor) promoted a significant inhibition of patient-derived lung cancer xenografts and prolonged the survival of LSL-KRASG12D mice. In this commentary, we will summarize the state-of-the art for the direction of synthetic lethality, and also speculate on the future development of this approach. PMID:27463838

K-ras mutations and HLA-DR expression in large bowel adenomas.

PubMed Central

Norheim Andersen, S.; Breivik, J.; Løvig, T.; Meling, G. I.; Gaudernack, G.; Clausen, O. P.; Schjölberg, A.; Fausa, O.; Langmark, F.; Lund, E.; Rognum, T. O.

1996-01-01

A total of 72 sporadic colorectal adenomas in 56 patients were studied for the presence of point mutations in codons 12 and 13 of the K-ras gene and for HLA-DR antigen expression related to clinicopathological variables. Forty K-ras mutations in 39 adenomas were found (54%): 31 (77%) in codon 12 and nine (23%) in codon 13. There was a strong relationship between the incidence of K-ras mutations and adenoma type, degree of dysplasia and sex. The highest frequency of K-ras mutations was seen in large adenomas of the villous type with high-grade dysplasia. Fourteen out of 15 adenomas obtained from 14 women above 65 years of age carried mutations. HLA-DR positivity was found in 38% of the adenomas, large tumours and those with high-grade dysplasia having the strongest staining. Coexpression of K-ras mutations and HLA-DR was found significantly more frequently in large and highly dysplastic adenomas, although two-way analysis of variance showing size and grade of dysplasia to be the most important variable. None of the adenomas with low-grade dysplasia showed both K-ras mutation and HLA-DR positivity (P = 0.004). K-ras mutation is recognised as an early event in colorectal carcinogenesis. The mutation might give rise to peptides that may be presented on the tumour cell surface by class II molecules, and thereby induce immune responses against neoplastic cells. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8679466

Autosomal dominant deficiency of the interleukin-17F in recurrent aphthous stomatitis: Possible novel mutation in a new entity.

PubMed

Zare Bidoki, Alireza; Massoud, Ahmad; Najafi, Shamsolmoulouk; Mohammadzadeh, Mahsa; Rezaei, Nima

2018-05-15

Recurrent Aphthous Stomatitis (RAS) is a common oral inflammatory disease with unknown pathogenesis. Although the immune system alterations could be involved in predisposition of individuals to oral candidiasis, precise etiologies of RAS have not been understood yet. A recent study showed that autosomal dominant IL17F deficiency could cause chronic mucocutaneous candidiasis. Considering the inflammatory nature of interleukin (IL)-17F and RAS, this study was performed to check any disease-associated mutation in a number of patients with RAS. Sixty-two Iranian individuals with RAS were investigated in this study. After DNA extraction using a phenol-chloroform method from the whole blood, amplification was accomplished by polymerase chain reaction and the products were sequenced using a 3730 ABI sequencer. The results of sequencing revealed a missense, heterozygous mutation of IL17F, converting a threonine to proline in a patient with RAS (T79P). The Poly-phen software suggested a damaging probability predicting this substitution to have a harmful effect on IL-17F protein function. This mutation was checked in fifty healthy individuals, and was not detected in any of them. This is the first study showing that a mutation in IL-17F is associated with susceptibility to RAS. However, functional studies and further studies on more patients with RAS are required to confirm such association. Copyright © 2018 Elsevier B.V. All rights reserved.

Hyperactivation of Ha-ras oncogene, but not Ink4a/Arf deficiency, triggers bladder tumorigenesis

PubMed Central

Mo, Lan; Zheng, Xiaoyong; Huang, Hong-Ying; Shapiro, Ellen; Lepor, Herbert; Cordon-Cardo, Carlos; Sun, Tung-Tien; Wu, Xue-Ru

2007-01-01

Although ras is a potent mitogenic oncogene, its tumorigenicity depends on cellular context and cooperative events. Here we show that low-level expression of a constitutively active Ha-ras in mouse urothelium induces simple urothelial hyperplasia that is resistant to progression to full-fledged bladder tumors even in the absence of Ink4a/Arf. In stark contrast, doubling of the gene dosage of the activated Ha-ras triggered early-onset, rapidly growing, and 100% penetrant tumors throughout the urinary tract. Tumor initiation required superseding a rate-limiting step between simple and nodular hyperplasia, the latter of which is marked by the emergence of mesenchymal components and the coactivation of AKT and STAT pathways as well as PTEN inactivation. These results indicate that overactivation of Ha-ras is both necessary and sufficient to induce bladder tumors along a low-grade, noninvasive papillary pathway, and they shed light on the recent findings that ras activation, via point mutation, overexpression, or intensified signaling from FGF receptor 3, occurs in 70%–90% of these tumors in humans. Our results highlight the critical importance of the dosage/strength of Ha-ras activation in dictating its tumorigenicity — a mechanism of oncogene activation not fully appreciated to date. Finally, our results have clinical implications, as inhibiting ras and/or its downstream effectors, such as AKT and STAT3/5, could provide alternative means to treat low-grade, superficial papillary bladder tumors, the most common tumor in the urinary system. PMID:17256055

Real-time PCR-based method for the rapid detection of extended RAS mutations using bridged nucleic acids in colorectal cancer.

PubMed

Iida, Takao; Mizuno, Yukie; Kaizaki, Yasuharu

2017-10-27

Mutations in RAS and BRAF are predictors of the efficacy of anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer (mCRC). Therefore, simple, rapid, cost-effective methods to detect these mutations in the clinical setting are greatly needed. In the present study, we evaluated BNA Real-time PCR Mutation Detection Kit Extended RAS (BNA Real-time PCR), a real-time PCR method that uses bridged nucleic acid clamping technology to rapidly detect mutations in RAS exons 2-4 and BRAF exon 15. Genomic DNA was extracted from 54 formalin-fixed paraffin-embedded (FFPE) tissue samples obtained from mCRC patients. Among the 54 FFPE samples, BNA Real-time PCR detected 21 RAS mutations (38.9%) and 5 BRAF mutations (9.3%), and the reference assay (KRAS Mutation Detection Kit and MEBGEN™ RASKET KIT) detected 22 RAS mutations (40.7%). The concordance rate of detected RAS mutations between the BNA Real-time PCR assay and the reference assays was 98.2% (53/54). The BNA Real-time PCR assay proved to be a more simple, rapid, and cost-effective method for detecting KRAS and RAS mutations compared with existing assays. These findings suggest that BNA Real-time PCR is a valuable tool for predicting the efficacy of early anti-EGFR therapy in mCRC patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Decreased diacylglycerol metabolism enhances ERK activation and augments CD8+ T cell functional responses.

PubMed

Riese, Matthew J; Grewal, Jashanpreet; Das, Jayajit; Zou, Tao; Patil, Vineet; Chakraborty, Arup K; Koretzky, Gary A

2011-02-18

Modulation of T cell receptor signal transduction in CD8(+) T cells represents a novel strategy toward enhancing the immune response to tumor. Recently, levels of guanine exchange factors, RasGRP and SOS, within T cells have been shown to represent a key determinant in the regulation of the analog to the digital activation threshold of Ras. One important for regulating activation levels of RasGRP is diacylglycerol (DAG), and its levels are influenced by diacylglycerol kinase-ζ (DGKζ), which metabolizes DAG into phosphatidic acid, terminating DAG-mediated Ras signaling. We sought to determine whether DGKζ-deficient CD8(+) T cells demonstrated enhanced in vitro responses in a manner predicted by the current model of Ras activation and to evaluate whether targeting this threshold confers enhanced CD8(+) T cell responsiveness to tumor. We observed that DGKζ-deficient CD8(+) T cells conform to most predictions of the current model of how RasGRP levels influence Ras activation. But our results differ in that the EC(50) value of stimulation is not altered for any T cell receptor stimulus, a finding that suggests a further degree of complexity to how DGKζ deficiency affects signals important for Ras and ERK activation. Additionally, we found that DGKζ-deficient CD8(+) T cells demonstrate enhanced responsiveness in a subcutaneous lymphoma model, implicating the analog to a digital conversion threshold as a novel target for potential therapeutic manipulation.

What breaks a leader: the curvilinear relation between assertiveness and leadership.

PubMed

Ames, Daniel R; Flynn, Francis J

2007-02-01

The authors propose that individual differences in assertiveness play a critical role in perceptions about leaders. In contrast to prior work that focused on linear effects, the authors argue that individuals seen either as markedly low in assertiveness or as high in assertiveness are generally appraised as less effective leaders. Moreover, the authors claim that observers' perceptions of leaders as having too much or too little assertiveness are widespread. The authors linked the curvilinear effects of assertiveness to underlying tradeoffs between social outcomes (a high level of assertiveness worsens relationships) and instrumental outcomes (a low level of assertiveness limits goal achievement). In 3 studies, the authors used qualitative and quantitative approaches and found support for their account. The results suggest that assertiveness (and other constructs with nonlinear effects) might have been overlooked in research that has been focused on identifying what makes a leader rather than on identifying what breaks a leader. ((c) 2007 APA, all rights reserved).

32 CFR 842.118 - Assertable claims.

Code of Federal Regulations, 2011 CFR

2011-07-01

... it merits assertion. Claims for $150 or less need not be asserted; they should be asserted only if... party offers payment and demands a release from the United States before paying damages to the injured...

32 CFR 842.118 - Assertable claims.

Code of Federal Regulations, 2010 CFR

2010-07-01

... it merits assertion. Claims for $150 or less need not be asserted; they should be asserted only if... party offers payment and demands a release from the United States before paying damages to the injured...

Trial and evaluation of assertion training involving nursing students.

PubMed

Nishina, Yuko; Tanigaki, Shizuko

2013-09-01

The concept of assertion and conceptual/practical methods of assertion (assertiveness) training were originally developed in the United States and Europe. These principles were embraced and adapted in Japan in 1970's. However, only a few studies relating to assertion (assertiveness) have been undertaken thus far in Japan, especially so in the domain of nursing students in comparison with other countries. The purpose of this study was to design and implement assertion training with nursing students and to clarify its effects. The participants were all volunteers, invited from a class of 3rd year nursing students. Ten students (intervention group) participated in the assertion training comprised of five sessions in February 2006. Fifty-six students (control group) were participated only in the questionnaire. Both groups were asked to complete the same questionnaire twice, before and after the assertion training. The questionnaire measured levels of assertiveness, social skills, self-esteem, social support and satisfaction with university life. The results and variances, both before and after assertion training, between the intervention group and the control group were analyzed. The effectiveness of the assertion training was determined by changes in pre and post training questionnaire scores. The scores for social skills in the control group had a tendency to decline while the scores for social skills in the intervention group remained constant. Although there were no statistically significant results in the intervention group, the present study highlights areas appropriate for further study.

Trial and Evaluation of Assertion Training Involving Nursing Students

PubMed Central

Nishina, Yuko; Tanigaki, Shizuko

2013-01-01

Background The concept of assertion and conceptual/practical methods of assertion (assertiveness) training were originally developed in the United States and Europe. These principles were embraced and adapted in Japan in 1970’s. However, only a few studies relating to assertion (assertiveness) have been undertaken thus far in Japan, especially so in the domain of nursing students in comparison with other countries. The purpose of this study was to design and implement assertion training with nursing students and to clarify its effects. Methods The participants were all volunteers, invited from a class of 3rd year nursing students. Ten students (intervention group) participated in the assertion training comprised of five sessions in February 2006. Fifty-six students (control group) were participated only in the questionnaire. Both groups were asked to complete the same questionnaire twice, before and after the assertion training. The questionnaire measured levels of assertiveness, social skills, self-esteem, social support and satisfaction with university life. The results and variances, both before and after assertion training, between the intervention group and the control group were analyzed. The effectiveness of the assertion training was determined by changes in pre and post training questionnaire scores. Results The scores for social skills in the control group had a tendency to decline while the scores for social skills in the intervention group remained constant. Conclusion Although there were no statistically significant results in the intervention group, the present study highlights areas appropriate for further study. PMID:24174705

Dimensions of assertiveness: factor analysis of five assertion inventories.

PubMed

Henderson, M; Furnham, A

1983-09-01

Five self report assertiveness inventories were factor analyzed. In each case two major factors emerged, accounting for approximately one-quarter to a third of the variance. The findings emphasize the multidimensional nature of current measures of assertiveness, and suggest the construction of a more systematic and psychometrically evaluated scale that would yield subscale scores assessing the separate dimensions of assertiveness.

Irish nursing students' changing levels of assertiveness during their pre-registration programme.

PubMed

Begley, Cecily M; Glacken, Michèle

2004-10-01

Stress and bullying have been found to be common problems in a number of studies of Irish nursing and midwifery. Victims of bullying need high levels of assertiveness to enable them to withstand the stress of victimization. It was deemed important to measure nursing students' level of assertiveness prior to, and near completion of, their pre-registration education programme. Aim. To ascertain nursing students' perceived levels of assertiveness prior to, and nearing the completion of, their three-year pre-registration programme. Ethical approval was given. The students commencing general nurse education programmes in two schools in Southern Ireland agreed to take part (n=72). A questionnaire adapted from a number of assertiveness scales, and tested for validity and reliability in this population, was used to collect data. In general, students' reported assertiveness levels rose as they approached completion of their three-year education programme. The resource constrained health service of the 21st century requires nurses who are assertive to meet the needs of its users. Nursing students' assertiveness skills could be augmented through concentrated efforts from nurse educationalists and clinicians to reduce the communication theory practice gap in nurse education today. To address the multi-dimensional nature of assertiveness, strategies to increase assertiveness should operate at the individual, interface and organisational level. The students in this study reported an increase in levels of assertiveness as they approached completion of their three-year education programme. To function as effective, safe practitioners registered nurses need to be assertive, therefore education in assertiveness should be an integral part of their preparation. The precise composition and mode of delivery of this education requires exploration and evaluation.

Autophagy in Ras-induced malignant transformation: fatal or vital?

PubMed

Mariño, Guillermo; Martins, Isabelle; Kroemer, Guido

2011-04-08

In this issue of Molecular Cell, Elgendy et al. suggest that Ras-induced autophagy may kill tumor cells on the verge of oncogenic transformation, providing a contrast to recent reports indicating that autophagy is required for optimal growth of Ras-driven cancers. Copyright © 2011 Elsevier Inc. All rights reserved.

Northwestern’s Kelleher Laboratory Develops Top-Down KRAS Isoform Assay to Detect Protein Mutations and Modifications | Office of Cancer Clinical Proteomics Research

Cancer.gov

Mutations in the RAS genes — KRAS, HRAS, and NRAS — have been identified in approximately 30% of all human cancers. While RAS gene family members encode proteins that are pivotal for cytoplasmic cell signaling, RAS oncogenes

Upregulating Apoptotic Signaling in Neurofibromatosis

DTIC Science & Technology

2009-09-01

Schwannoma) cells that are NF1 deficient cells were used. Rat pheochromocytoma PC12 cells were selected as a control, in which Ras signaling is normal. The...Down and Detection kit. The baseline level of GTP bound Ras was detected in PC12 cells (rat pheochromocytoma ) in which Ras signaling is intact. In

Thermodynamics between RAP/RAS and virgin aggregates during asphalt concrete production : a literature review.

DOT National Transportation Integrated Search

2015-09-01

In hot-mix asphalt (HMA) plants, virgin aggregates are heated and dried separately before being mixed with : RAP/RAS and virgin asphalt binder. RAP/RAS materials are not heated or dried directly by a burner to avoid : burning of aged binder coating o...

Control of Innate and Adaptive Lymphocytes by the RAR-Retinoic Acid Axis.

PubMed

Kim, Chang H

2018-02-01

Lymphocytes, such as T cells, B cells, and innate lymphoid cells (ILCs), play central roles in regulating immune responses. Retinoic acids (RAs) are vitamin A metabolites, produced and metabolized by certain tissue cells and myeloid cells in a tissue-specific manner. It has been established that RAs induce gut-homing receptors on T cells, B cells, and ILCs. A mounting body of evidence indicates that RAs exert far-reaching effects on functional differentiation and fate of these lymphocytes. For example, RAs promote effector T cell maintenance, generation of induced gut-homing regulatory and effector T cell subsets, antibody production by B cells, and functional maturation of ILCs. Key functions of RAs in regulating major groups of innate and adaptive lymphocytes are highlighted in this article.

Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils.

PubMed

Philips, M R; Pillinger, M H; Staud, R; Volker, C; Rosenfeld, M G; Weissmann, G; Stock, J B

1993-02-12

In human neutrophils, as in other cell types, Ras-related guanosine triphosphate-binding proteins are directed toward their regulatory targets in membranes by a series of posttranslational modifications that include methyl esterification of a carboxyl-terminal prenylcysteine residue. In intact cells and in a reconstituted in vitro system, the amount of carboxyl methylation of Ras-related proteins increased in response to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP). Activation of Ras-related proteins by guanosine-5'-O-(3-thiotriphosphate) had a similar effect and induced translocation of p22rac2 from cytosol to plasma membrane. Inhibitors of prenylcysteine carboxyl methylation effectively blocked neutrophil responses to FMLP. These findings suggest a direct link between receptor-mediated signal transduction and the carboxyl methylation of Ras-related proteins.

Oxidative stress and myeloperoxidase levels in saliva of patients with recurrent aphthous stomatitis.

PubMed

Cağlayan, F; Miloglu, O; Altun, O; Erel, O; Yilmaz, A B

2008-11-01

Recurrent aphthous stomatitis (RAS) is the most common oral ulcerative condition affecting 5-25% of the general population. The aim of this study was to evaluate the oxidative stress parameters in saliva of patients with RAS and to investigate the relationship among these parameters in either group. The study involved 50 patients with RAS of whom 24 were male and 26 were female, and 25 healthy controls of whom 13 were male and 12 were female. There was no statistically significant difference in the salivary total antioxidant capacity, total oxidant status, oxidative stress index levels, and myeloperoxidase activity between patients with RAS and those in the control group. The results show that reactive oxygen species may not play a role in the etiology of RAS.

A little something from physics for medicine (Scientific session of the Physical Sciences Division of the Russian Academy of Sciences, 23 April 2014)

NASA Astrophysics Data System (ADS)

2014-12-01

A scientific session of the Physical Sciences Division of the Russian Academy of Sciences (RAS), entitled "A little something from physics for medicine", was held on 23 April 2014 at the conference hall of the Lebedev Physical Institute, RAS. The agenda posted on the website of the Physical Sciences Division, RAS, http://www.gpad.ac.ru, included the following reports: (1) Rumyantsev S A (D Rogachev Federal Research and Clinical Center of Pediatric Hematology, Oncology, and Immunology, Moscow) "Translational medicine as a basis of progress in hematology/oncology"; (2) Akulinichev S V (Institute for Nuclear Research, RAS, Moscow) "Promising nuclear medicine research at the INR, RAS"; (3) Nikitin P P (Prokhorov General Physics Institute, RAS, Moscow) "Biosensorics: new possibilities provided by marker-free optical methods and magnetic nanoparticles for medical diagnostics"; (4) Alimpiev S S, Nikiforov S M, Grechnikov A A (Prokhorov General Physics Institute, RAS, Moscow) "New approaches in laser mass-spectrometry of organic objects". The publication of the article based on the oral report No. 2 is presented below. • Promising nuclear medicine research in the Institute for Nuclear Research, Russian Academy of Sciences, V V Akulinichev Physics-Uspekhi, 2014, Volume 57, Number 12, Pages 1239-1243

Effect of blood estrogen and progesterone on severity of minor RAS

NASA Astrophysics Data System (ADS)

Utami, S.; Rahardjo, T. W. B.; Baziad, A.; Alwadris, T. T.; Auerkari, E. I.

2018-05-01

Recurrent Aphthous Stomatitis (RAS) is a chronic inflammatory disease that attacks oral mucosa. Estrogen stimulates proliferation and buccal mucosa cornification and generally acts as an immunoinhibitor that can increase antibody synthesis through humoral response, while progesterone has immunosuppressive potential. This study aimed to examine the levels of blood estrogen and progesterone in females with Minor RAS of varying severity. Subjects were 42 women, 17–40 years old, with Minor RAS but without hormonal therapy or hysterectomy. They were investigated to see whether the severity of pain was related to condition of onset, recurrence and site or amount of lesions. Blood was taken on the 21st – 22nd day of the menstrual cycle to test both hormone levels, using Microparticle Enzyme Immunoassay for the estrogen level, and Competitive Radioimmunoassay for the progesterone level. There was no significant relation between estrogen (p=0.530) or progesterone (p=0.717) level and the severity of Minor RAS. There is a tendency of normal estrogen level on both mild (62.5%) and severe (37.5%) Minor RAS. However, the progesterone level tends to be low both in mild (61.3%) and severe (38.7%) Minor RAS. It is assumed that the decrease of progesterone level also decreases the anti-inflammation function.

Inhibition of RAS activation due to a homozygous ezrin variant in patients with profound intellectual disability.

PubMed

Riecken, Lars Björn; Tawamie, Hasan; Dornblut, Carsten; Buchert, Rebecca; Ismayel, Amina; Schulz, Alexander; Schumacher, Johannes; Sticht, Heinrich; Pohl, Katja J; Cui, Yan; Reis, André; Morrison, Helen; Abou Jamra, Rami

2015-02-01

Gain-of-function alterations in several components and modulators of the Ras-MAPK pathway lead to dysregulation of the pathway and cause a broad spectrum of autosomal dominant developmental disorders, collectively known as RASopathies. These findings demonstrate the importance of tight multilevel Ras regulation to safeguard signaling output and prevent aberrant activity. We have recently identified ezrin as a novel regulatory element required for Ras activation. Homozygosity mapping and exome sequencing have now revealed the first presumably disease-causing variant in the coding gene EZR in two siblings with a profound intellectual disability. Localization and membrane targeting of the altered ezrin protein appeared normal but molecular modeling suggested protein interaction surfaces to be disturbed. Functional analysis revealed that the altered ezrin protein is no longer able to bind Ras and facilitate its activation. Furthermore, expression of the altered ezrin protein in different cell lines resulted in abnormal cellular processes, including reduced proliferation and neuritogenesis, thus revealing a possible mechanism for its phenotype in humans. To our knowledge, this is the first report of an autosomal recessively inherited loss-of-function mutation causing reduced Ras activity and thus extends and complements the pathogenicity spectrum of known Ras-MAPK pathway disturbances. © 2014 WILEY PERIODICALS, INC.

High Background Incidence of Spontaneous Subcapsular Adrenal Gland Hyperplasia of Tg.rasH2 Mice Used in 26-week Carcinogenicity Studies.

PubMed

Boyle, Molly H; Paranjpe, Madhav G; Creasy, Dianne M

2018-06-01

The Tg.rasH2 model was accepted by regulatory agencies worldwide for 26-week carcinogenicity assays as an alternative to the standard 2-year assays in conventional mice in 2003. Several references documenting spontaneous nonneoplastic findings and incidences of spontaneous tumors in the Tg.rasH2 mice have been published. The purpose of this publication is to add adrenal gland subcapsular hyperplasia to the database pertaining to spontaneous lesions noted in Tg.rasH2 mice, review physiology related to this finding, and discuss its significance. The incidence of spontaneous subcapsular adrenal gland hyperplasia was determined in control Tg.rasH2 mice from nine 26-week carcinogenicity studies carried out within the last 5 years at two contract research organizations. Incidence of this finding ranged from 56% to 79% in males and 88% to 100% in females, with an incidence average of 62% in males and 93% in females. Adrenal gland subcapsular hyperplasia is a common finding in male and female Tg.rasH2 mice that did not progress to neoplasia in Tg.rasH2 mice. In general, it tends to be more frequent and severe in females in comparison to males.

Combination use of medicines from two classes of renin-angiotensin system blocking agents: risk of hyperkalemia, hypotension, and impaired renal function.

PubMed

Esteras, Raquel; Perez-Gomez, Maria Vanessa; Rodriguez-Osorio, Laura; Ortiz, Alberto; Fernandez-Fernandez, Beatriz

2015-08-01

European and United States regulatory agencies recently issued warnings against the use of dual renin-angiotensin system (RAS) blockade therapy through the combined use of angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs) or aliskiren in any patient, based on absence of benefit for most patients and increased risk of hyperkalemia, hypotension, and renal failure. Special emphasis was made not to use these combinations in patients with diabetic nephropathy. The door was left open to therapy individualization, especially for patients with heart failure, when the combined use of an ARB and ACEI is considered absolutely essential, although renal function, electrolytes and blood pressure should be closely monitored. Mineralocorticoid receptor antagonists were not affected by this warning despite increased risk of hyperkalemia. We now critically review the risks associated with dual RAS blockade and answer the following questions: What safety issues are associated with dual RAS blockade? Can the safety record of dual RAS blockade be improved? Is it worth trying to improve the safety record of dual RAS blockade based on the potential benefits of the combination? Is dual RAS blockade dead? What is the role of mineralocorticoid antagonists in combination with other RAS blocking agents: RAAS blockade?

Prx I Suppresses K-ras-Driven Lung Tumorigenesis by Opposing Redox-Sensitive ERK/Cyclin D1 Pathway

PubMed Central

Park, Young-Ho; Kim, Sun-Uk; Lee, Bo-Kyoung; Kim, Hyun-Sun; Song, In-Sung; Shin, Hye-Jun; Han, Ying-Hao; Chang, Kyu-Tae; Kim, Jin-Man; Lee, Dong-Seok; Kim, Yeul-Hong; Choi, Chang-Min; Kim, Bo-Yeon

2013-01-01

Abstract Aims: Coupled responses of mutated K-ras and oxidative stress are often an important etiological factor in non–small-cell lung cancer (NSCLC). However, relatively few studies have examined the control mechanism of oxidative stress in oncogenic K-ras-driven NSCLC progression. Here, we studied whether the redox signaling pathway governed by peroxiredoxin I (Prx I) is involved in K-rasG12D-mediated lung adenocarcinogenesis. Results: Using human-lung adenocarcinoma tissues and lung-specific K-rasG12D-transgenic mice, we found that Prx I was significantly up-regulated in the tumor regions via activation of nuclear erythroid 2-related factor 2 (Nrf2) transcription. Interestingly, the increased reactive oxygen species (ROS) by null mutation of Prx I greatly promoted K-rasG12D-driven lung tumorigenesis in number and size, which appeared to require the activation of the ROS-dependent extracellular signal-regulated kinase (ERK)/cyclin D1 pathway. Innovation: Taken together, these results suggest that Prx I functions as an Nrf2-dependently inducible tumor suppressant in K-ras-driven lung adenocarcinogenesis by opposing ROS/ERK/cyclin D1 pathway activation. Conclusion: These findings provide a better understanding of oxidative stress-mediated lung tumorigenesis. Antioxid. Redox Signal. 19, 482–496. PMID:23186333

Ras oncogenes in oral cancer: the past 20 years.

PubMed

Murugan, Avaniyapuram Kannan; Munirajan, Arasambattu Kannan; Tsuchida, Nobuo

2012-05-01

Oral squamous cell carcinoma (OSCC) of head and neck is associated with high morbidity and mortality in both Western and Asian countries. Several risk factors for the development of oral cancer are very well established, including tobacco chewing, betel quid, smoking, alcohol drinking and human papilloma virus (HPV) infection. Apart from these risk factors, many genetic factors such as oncogenes, tumor suppressor genes and regulatory genes are identified to involve in oral carcinogenesis with these risk factors dependent and independent manner. Ras is one of the most frequently genetically deregulated oncogene in oral cancer. In this review, we analyze the past 22years of literature on genetic alterations such as mutations and amplifications of the isoforms of the ras oncogene in oral cancer. Further, we addressed the isoform-specific role of the ras in oral carcinogenesis. We also discussed how targeting the Akt and MEK, downstream effectors of the PI3K/Akt and MAPK pathways, respectively, would probably pave the possible molecular therapeutic target for the ras driven tumorigenesis in oral cancer. Analysis of these ras isoforms may critically enlighten specific role of a particular ras isoform in oral carcinogenesis, enhance prognosis and pave the way for isoform-specific molecular targeted therapy in OSCC. Copyright © 2011 Elsevier Ltd. All rights reserved.

Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules.

PubMed

Wasylyk, Christine; Zheng, Hong; Castell, Christelle; Debussche, Laurent; Multon, Marie-Christine; Wasylyk, Bohdan

2008-03-01

Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

Rhythmic auditory stimulation improves gait more than NDT/Bobath training in near-ambulatory patients early poststroke: a single-blind, randomized trial.

PubMed

Thaut, M H; Leins, A K; Rice, R R; Argstatter, H; Kenyon, G P; McIntosh, G C; Bolay, H V; Fetter, M

2007-01-01

The effectiveness of 2 different types of gait training in stroke rehabilitation, rhythmic auditory stimulation (RAS) versus neurodevelopmental therapy (NDT)/Bobath- based training, was compared in 2 groups of hemiparetic stroke patients over a 3-week period of daily training (RAS group, n = 43; NDT/Bobath group =35). Mean entry date into the study was 21.3 days poststroke for the RAS group and 22.3 days for the control group. Patients entered the study as soon as they were able to complete 5 stride cycles with handheld assistance. Patients were closely equated by age, gender, and lesion site. Motor function in both groups was pre-assessed by the Barthel Index and the Fugl-Meyer Scales. Pre- to posttest measures showed a significant improvement in the RAS group for velocity (P = .006), stride length (P = .0001), cadence (P = .0001) and symmetry (P = .0049) over the NDT/Bobath group. Effect sizes for RAS over NDT/Bobath training were 13.1 m/min for velocity, 0.18 m for stride length, and 19 steps/min for cadence. The data show that after 3 weeks of gait training, RAS is an effective therapeutic method to enhance gait training in hemiparetic stroke rehabilitation. Gains were significantly higher for RAS compared to NDT/Bobath training.

Cancer stem cell drugs target K-ras signaling in a stemness context

PubMed Central

Najumudeen, A K; Jaiswal, A; Lectez, B; Oetken-Lindholm, C; Guzmán, C; Siljamäki, E; Posada, I M D; Lacey, E; Aittokallio, T; Abankwa, D

2016-01-01

Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC. PMID:26973241

Brain Renin-Angiotensin System and Microglial Polarization: Implications for Aging and Neurodegeneration

PubMed Central

Labandeira-Garcia, Jose L.; Rodríguez-Perez, Ana I.; Garrido-Gil, Pablo; Rodriguez-Pallares, Jannette; Lanciego, Jose L.; Guerra, Maria J.

2017-01-01

Microglia can transform into proinflammatory/classically activated (M1) or anti-inflammatory/alternatively activated (M2) phenotypes following environmental signals related to physiological conditions or brain lesions. An adequate transition from the M1 (proinflammatory) to M2 (immunoregulatory) phenotype is necessary to counteract brain damage. Several factors involved in microglial polarization have already been identified. However, the effects of the brain renin-angiotensin system (RAS) on microglial polarization are less known. It is well known that there is a “classical” circulating RAS; however, a second RAS (local or tissue RAS) has been observed in many tissues, including brain. The locally formed angiotensin is involved in local pathological changes of these tissues and modulates immune cells, which are equipped with all the components of the RAS. There are also recent data showing that brain RAS plays a major role in microglial polarization. Level of microglial NADPH-oxidase (Nox) activation is a major regulator of the shift between M1/proinflammatory and M2/immunoregulatory microglial phenotypes so that Nox activation promotes the proinflammatory and inhibits the immunoregulatory phenotype. Angiotensin II (Ang II), via its type 1 receptor (AT1), is a major activator of the NADPH-oxidase complex, leading to pro-oxidative and pro-inflammatory effects. However, these effects are counteracted by a RAS opposite arm constituted by Angiotensin II/AT2 receptor signaling and Angiotensin 1–7/Mas receptor (MasR) signaling. In addition, activation of prorenin-renin receptors may contribute to activation of the proinflammatory phenotype. Aged brains showed upregulation of AT1 and downregulation of AT2 receptor expression, which may contribute to a pro-oxidative pro-inflammatory state and the increase in neuron vulnerability. Several recent studies have shown interactions between the brain RAS and different factors involved in microglial polarization, such as estrogens, Rho kinase (ROCK), insulin-like growth factor-1 (IGF-1), tumor necrosis factor α (TNF)-α, iron, peroxisome proliferator-activated receptor gamma, and toll-like receptors (TLRs). Metabolic reprogramming has recently been involved in the regulation of the neuroinflammatory response. Interestingly, we have recently observed a mitochondrial RAS, which is altered in aged brains. In conclusion, dysregulation of brain RAS plays a major role in aging-related changes and neurodegeneration by exacerbation of oxidative stress (OS) and neuroinflammation, which may be attenuated by pharmacological manipulation of RAS components. PMID:28515690

K-ras mutation promotes ionizing radiation-induced invasion and migration of lung cancer in part via the Cathepsin L/CUX1 pathway.

PubMed

Wang, Long; Zhao, Yifan; Xiong, Yajie; Wang, Wenjuan; Fei, Yao; Tan, Caihong; Liang, Zhongqin

2018-01-15

K-ras mutation is involved in cancer progression including invasion and migration, but the underlying mechanism is not yet clear. Cathepsin L is a lysosomal cysteine protease and has recently been associated with invasion and migration in human cancers when it is overexpressed. Our recent studies have shown that ionizing radiation (IR) enhanced expression of cathepsin L and increased invasion and migration of tumor cells, but the molecular mechanism is still unclear. In the present study, the effects of K-ras mutation and IR induced invasion and migration of lung cancer as well as the underlying mechanisms were investigated both in vitro and in vivo. Firstly, the levels of cathepsin L and epithelial mesenchymal transition (EMT) marker proteins remarkably changed in A549 (K-ras mutant) after irradiation compared with H1299 (K-ras wild), thereby promoting invasion and migration. Additionally, cathepsin L and its downstream transcription factor CUX1/p110 were increased after irradiation in A549 transfected with CUX1/p200, and the proteolytic processing of CUX1 by cathepsin L was remarkably increased after co-transfection of CUX1/p200 and cathepsin L-lentivirus in H1299. In addition, delivery of a mutant K-ras (V12) into HEK 293 cells stimulated EMT after irradiation due to the accumulation of cathepsin L. Moreover, mutated K-ras was associated with IR-induced cathepsin L and EMT in BALB/c nude mice. Finally, the level of cathepsin L expression was higher in samples carrying a K-ras mutation than in wild-type K-ras samples and the mesenchymal markers were upregulated in the samples of mutant K-ras, whereas the epithelial marker E-cadherin was downregulated in non-small cell lung cancers tissues. In conclusion, the findings demonstrated that mutated K-ras promotes cathepsin L expression and plays a pivotal role in EMT of human lung cancer. The regulatory effect of IR-induced cathepsin L on lung cancer invasion and migration was partially attributed to the Cathepsin L /CUX1-mediated EMT signaling pathway. This study will provide cathepsin L as a potential target for tumor therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Mutations that abolish the ability of Ha-Ras to associate with Raf-1.

PubMed

Shirouzu, M; Koide, H; Fujita-Yoshigaki, J; Oshio, H; Toyama, Y; Yamasaki, K; Fuhrman, S A; Villafranca, E; Kaziro, Y; Yokoyama, S

1994-08-01

Recent studies have revealed that Ras can associate physically with Raf. In the present study, we tested 34 mutants of Ha-Ras carrying substitution(s) in the region of residues 23-71 for their ability to associate with Raf-1. Mouse Ba/F3 cell lysates were incubated with each mutant Ras protein, in either the guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)- or the guanosine 5'-[beta-thio]diphosphate (GDP beta S)-bound form, and the anti-Ras antibody Y13-238. The immunoprecipitates were analysed for the presence of Raf-1 by Western blotting with an anti-Raf-1 antibody. Six mutants of Ras, E31K, P34G, T35S, D38N, D57A and A59T, failed to bind Raf-1. Mutations N26G, V29A, S39A, Y40W, R41A, V44A, V45E, L56A and T58A partially reduced the ability to bind Raf-1. All the other mutants could associate with Raf-1 with nearly the same efficiency as that of wild-type Ras. Thus, the Raf-I-binding ability of Ras appears to be affected by mutations in the N-terminal region, and in particular, by those in and neighboring the effector region (residues 32-40) and in the region (residues 56-59) flanking the N-terminal of Switch II. The abilities to bind Raf-1 and to induce neurite outgrowth of pheochromocytoma (PC) 12 cells correlate to each other for 22 Ras mutants. However, mutation A59T, which does not reduce the neurite-inducing or transforming activities, abolishes the ability to bind Raf-1. In contrast, mutations Y32F, K42A and L53A, which impair the neurite-inducing activity of Ras, have no effect on the Ras.Raf-1 association. Partially reduced Raf-1-binding ability was observed for mutants V29A, S39A, Y40W, R41A, V44A, L56A and T58A, which exhibit full neurite-inducing activity, and also for mutant V45E, which has no activity of neurite induction.

Aspects of Predisposition to Assertiveness, Resistance to Assertiveness and Insight into Assertiveness Based Upon Race and Sex.

DTIC Science & Technology

1977-12-15

reflecting assertive behavior are presented to the subjects and (b) the subjects respond by using a scale that permits him or her to indicate his...responses of sub— - jects are not assessed in terms of how assertive ~j~y perceive them to be. For example, an item might ask a subject to scale how...possible actions. This time we want you to tell us how strong or assertive each action is, in your opinion. Each action can be rated on a scale from 1 to 5

[Self-esteem: a comparison study between eating disorders and social phobia].

PubMed

Eiber, R; Vera, L; Mirabel-Sarron, C; Guelfi, J-D

2003-01-01

Eating disorder patients evidenced very often a low self-esteem. Self-esteem in eating disorder patients is excessively based on body dissatisfaction. In eating disorders there seems to be a link between body image dissatisfaction and social anxiety. We hypothesised: self-esteem would be as low in eating disorder patients as in social phobia patients; self-esteem would be lower in eating disorder patients with social phobia than in patients with social phobia alone; self-esteem would be lower in eating disorder patients with depressive cognitions than in social phobia patients with depressive cognitions; self-esteem could have different characteristics in the two disorders; self-esteem would be as low in anorexia as in bulimia; 103 eating disorder patients (33 restrictive anorectics, 34 anorectics-bulimics, 36 bulimics) and 26 social phobia patients diagnosed according to DSM IV and ICD-10 criteria have been investigated by the Self-Esteem Inventory of Coopersmith, the Assertiveness Schedule of Rathus, the Fear Survey Schedule of Wolpe (FSS III) and the Beck Depression Inventory (BDI). Patients were free of medication and presented no episode of major depression according to DSM IV criteria. Evaluations took place before any psychotherapy. Self-esteem in eating disorder patients is reduced at the same level as in social phobia patients; 86.1% of the total sample and 84.5% of the eating disorder patients have a very low self-esteem (score 33 in the SEI). Eating disorder patients have significantly higher scores in the Social (p=0.016) and Professional (p=0.0225) sub-scales of the SEI than social phobia patients. Eating disorder patients show higher scores on the Assertiveness Schedule of Rathus (p=0.0013) than social phobia patients. Eating disorder patients disclose higher scores on the BDI (p=0.0003) but eating disorder patients with depressive cognitions do not differ from social phobia patients with depressive cognitions in the level of self-esteem. The FSS III scores are significantly lower in eating disorder patients (p<0.0001). There is a difference in the nature of the deficit of self-esteem between the two patient populations. Self-esteem is not influenced by the Body Mass Index (BMI) and is identically reduced in all groups of eating disorder patients. Whereas eating disorder patients have the same complaints compared to social phobia, they differ significantly from social phobia patients in their characteristics of social phobia and self-esteem.

The SWAN Scientific Discourse Ontology

PubMed Central

Ciccarese, Paolo; Wu, Elizabeth; Kinoshita, June; Wong, Gwendolyn T.; Ocana, Marco; Ruttenberg, Alan

2015-01-01

SWAN (Semantic Web Application in Neuromedicine) is a project to construct a semantically-organized, community-curated, distributed knowledge base of Theory, Evidence, and Discussion in biomedicine. Unlike Wikipedia and similar approaches, SWAN’s ontology is designed to represent and foreground both harmonizing and contradictory assertions within the total community discourse. Releases of the software, content and ontology will be initially by and for the Alzheimer Disease (AD) research community, with the obvious potential for extension into other disease research areas. The Alzheimer Research Forum, a 4,000-member web community for AD researchers, will host SWAN’s initial public release, currently scheduled for late 2007. This paper presents the current version of SWAN’s ontology of scientific discourse and presents our current thinking about its evolution including extensions and alignment with related communities, projects and ontologies. PMID:18583197

The effects of assertiveness training in patients with schizophrenia: a randomized, single-blind, controlled study.

PubMed

Lee, Tso-Ying; Chang, Shih-Chin; Chu, Hsin; Yang, Chyn-Yng; Ou, Keng-Liang; Chung, Min-Huey; Chou, Kuei-Ru

2013-11-01

In this study, we investigated the effects of group assertiveness training on assertiveness, social anxiety and satisfaction with interpersonal communication among patients with chronic schizophrenia. Only limited studies highlighted the effectiveness of group assertiveness training among inpatients with schizophrenia. Given the lack of group assertiveness training among patients with schizophrenia, further development of programmes focusing on facilitating assertiveness, self-confidence and social skills among inpatients with chronic schizophrenia is needed. This study used a prospective, randomized, single-blinded, parallel-group design. This study employed a prospective, randomized, parallel-group design. Seventy-four patients were randomly assigned to experimental group receiving 12 sessions of assertiveness training, or a supportive control group. Data collection took place for the period of June 2009-July 2010. Among patients with chronic schizophrenia, assertiveness, levels of social anxiety and satisfaction with interpersonal communication significantly improved immediately after the intervention and at the 3-month follow-up in the intervention group. The results of a generalized estimating equation (GEE) indicated that: (1) assertiveness significantly improved from pre- to postintervention and was maintained until the follow-up; (2) anxiety regarding social interactions significantly decreased after assertiveness training; and (3) satisfaction with interpersonal communication slightly improved after the 12-session intervention and at the 3-month follow-up. Assertivenss training is a non-invasive and inexpensive therapy that appears to improve assertiveness, social anxiety and interpersonal communication among inpatients with chronic schizophrenia. These findings may provide a reference guide to clinical nurses for developing assertiveness-training protocols. © 2013 Blackwell Publishing Ltd.

Scientific session of the General meeting of the Physical Sciences Division of the Russian Academy of Sciences (7 December 2015)

NASA Astrophysics Data System (ADS)

2016-05-01

A scientific session of the General meeting of the Physical Sciences Division of the Russian Academy of Sciences (RAS) was held in the conference hall of the Lebedev Physical Institute, RAS on 7 December 2015. The papers collected in this issue were written based on talks given at the session (the program of the session is available on the RAS Physical Sciences Division website http://www.gpad.ac.ru). (1) Loshchenov V B (Prokhorov General Physics Institute, RAS, Moscow) "Pharmacodynamics of a nanophotosensitizer under irradiation by an electromagnetic field: from THz to Cherenkov radiation"; (2) Zhuikov B L (Institute for Nuclear Research, RAS, Moscow) "Successes and problems in the development of medical radioisotope production in Russia"; (3) Tikhonov Yu A (Budker Institute of Nuclear Physics, SB RAS, Novosibirsk) "Applying nuclear physics methods in healthcare"; (4) Turchin I V (Institute of Applied Physics, RAS, Nizhny Novgorod) "Methods of biomedical optical imaging: from subcellular structures to tissues and organs"; (5) Breus T K, Petrukovich A A (Space Research Institute, RAS, Moscow), Binhi V N (Prokhorov General Physics Institute, RAS, Moscow; Lomonosov Moscow State University, Moscow) "Magnetic factor in solar-terrestrial relations and its impact on the human body: physical problems and prospects for research"; (6) Makarov D I (Special Astrophysical Observatory, RAS, Nizhnii Arkhyz, Zelenchukskii region, Karachai-Cherkessian Republic) "Studying the Local University". Papers based on oral reports 2, 4, and 5 are presented below. • Successes and problems in the development of medical radioisotope production in Russia, B L Zhuikov Physics-Uspekhi, 2016, Volume 59, Number 5, Pages 481-486 • Methods of biomedical optical imaging: from subcellular structures to tissues and organs, I V Turchin Physics-Uspekhi, 2016, Volume 59, Number 5, Pages 487-501 • Magnetic factor in solar-terrestrial relations and its impact on the human body: physical problems and prospects for research, T K Breus, V N Binhi, A A Petrukovich Physics-Uspekhi, 2016, Volume 59, Number 5, Pages 502-510

Combined use of drugs inhibiting the renin-angiotensin system: prescribing patterns and risk of acute kidney injury in German nursing home residents.

PubMed

Dörks, Michael; Herget-Rosenthal, Stefan; Hoffmann, Falk; Jobski, Kathrin

2018-01-01

In 2012, the European Medicines Agency reviewed the safety of dual renin-angiotensin system (RAS) blockade because of potentially increased risks for inter alia acute kidney injury (AKI). Since residents of nursing homes are particularly vulnerable to adverse drug outcomes, the aims of our study were to describe RAS-inhibiting drug use in German nursing home residents and examine the risk of AKI associated with dual RAS blockade. Based on claims data, a nested case-control study within a cohort of RAS-inhibiting drug users was conducted. Using conditional logistic regression, confounder-adjusted odds ratios (aORs) and 95% confidence intervals (CI) were obtained for the risk of AKI associated with dual RAS blockade. Subgroup analyses were performed in patients with diabetes or chronic kidney disease and both comorbidities. Of all 127,227 nursing home residents, the study cohort included 64,567 (50.7%) who were treated with at least one RAS-inhibiting drug. More than three quarters of the study population were female (77.1%). Mean age was 86.0 ± 6.8 years. Most residents were treated with angiotensin-converting enzyme inhibitors (77.8%), followed by angiotensin II receptor blockers (21.6%) and aliskiren (0.2%). Annual prevalence of dual RAS blockade declined from 9.6 (95% CI 7.8-11.8) in 2010 to 4.7 (95% CI 4.0-5.4) per 1,000 users in 2014. In the overall cohort, AKI was not significantly associated with dual RAS blockade (aOR 1.99; 0.77-5.17). However, significantly increased aORs were observed when considering patients with diabetes (3.47; 1.27-9.47), chronic kidney disease (4.74; 1.24-18.13) or both (11.17; 2.65-47.15). Prescribing of drugs inhibiting the RAS is common in German nursing homes. Though the prevalence of dual RAS blockade declined, our study showed an increased risk of AKI in patients with diabetes and/or chronic kidney disease. Therefore, cautious use is warranted in these vulnerable patients.

Final Analysis of Outcomes and RAS/BRAF Status in a Randomized Phase 3 Study of Panitumumab and Best Supportive Care in Chemorefractory Wild Type KRAS Metastatic Colorectal Cancer.

PubMed

Kim, Tae Won; Elme, Anneli; Park, Joon Oh; Udrea, Anghel Adrian; Kim, Sun Young; Ahn, Joong Bae; Valencia, Ricardo Villalobos; Krishnan, Srinivasan; Manojlovic, Nebojsa; Guan, Xuesong; Lofton-Day, Catherine; Jung, A Scott; Vrdoljak, Eduard

2018-03-21

Tumor rat sarcoma gene (RAS) status is a negative predictive biomarker for anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer (mCRC). We analyzed outcomes according to RAS and v-Raf murine sarcoma viral oncogene homolog B (BRAF) mutational status, and evaluated early tumor shrinkage (ETS) and depth of response (DpR) for patients with wild type RAS. Patients with confirmed metastatic colon or rectum adenocarcinoma, wild type Kristen rat sarcoma gene tumor exon 2 status, clinical/radiologic disease progression or toxicity during irinotecan or oxaliplatin treatment, and no previous anti-EGFR therapy were randomized 1:1 to receive best supportive care (BSC) with or without panitumumab (6.0 mg/kg, intravenously, on day 1 of each 14-day cycle) in this open-label, multicenter, phase III study (20100007). RAS and BRAF mutation status were determined using Sanger sequencing. ETS was evaluated as maximum percentage change from baseline to week 8; DpR was calculated as the percentage change for tumor shrinkage at nadir versus baseline. Overall, 270 patients had RAS wild type mCRC (panitumumab with BSC, n = 142; BSC, n = 128). For patients with wild type RAS tumors, median overall survival (OS; hazard ratio [HR], 0.72; P = .015) and progression-free survival (PFS; HR, 0.45; P < .0001) were improved with panitumumab with BSC versus BSC. Similar improvements were seen for patients with wild type RAS, and wild type BRAF tumors (OS: HR, 0.75; P = .04; PFS: HR, 0.45; P < .0001). Median DpR was 16.9% for the evaluable panitumumab with BSC wild type RAS population. Overall, 69.5% experienced any type of tumor shrinkage at week 8; 38.2% experienced ≥ 20% shrinkage. Similar improvements in OS and PFS were seen with stratification according to ETS. This analysis showed that panitumumab improved outcomes in wild type RAS mCRC and indicated that ETS and DpR could be used as additional efficacy markers. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The ability of GAP1IP4BP to function as a Rap1 GTPase-activating protein (GAP) requires its Ras GAP-related domain and an arginine finger rather than an asparagine thumb.

PubMed

Kupzig, Sabine; Bouyoucef-Cherchalli, Dalila; Yarwood, Sam; Sessions, Richard; Cullen, Peter J

2009-07-01

GAP1(IP4BP) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) that includes GAP1(m), CAPRI, and RASAL. Composed of a central Ras GAP-related domain (RasGRD), surrounded by amino-terminal C2 domains and a carboxy-terminal PH/Btk domain, these proteins, with the notable exception of GAP1(m), possess an unexpected arginine finger-dependent GAP activity on the Ras-related protein Rap1 (S. Kupzig, D. Deaconescu, D. Bouyoucef, S. A. Walker, Q. Liu, C. L. Polte, O. Daumke, T. Ishizaki, P. J. Lockyer, A. Wittinghofer, and P. J. Cullen, J. Biol. Chem. 281:9891-9900, 2006). Here, we have examined the mechanism through which GAP1(IP4BP) can function as a Rap1 GAP. We show that deletion of domains on either side of the RasGRD, while not affecting Ras GAP activity, do dramatically perturb Rap1 GAP activity. By utilizing GAP1(IP4BP)/GAP1(m) chimeras, we establish that although the C2 and PH/Btk domains are required to stabilize the RasGRD, it is this domain which contains the catalytic machinery required for Rap1 GAP activity. Finally, a key residue in Rap1-specific GAPs is a catalytic asparagine, the so-called asparagine thumb. By generating a molecular model describing the predicted Rap1-binding site in the RasGRD of GAP1(IP4BP), we show that mutagenesis of individual asparagine or glutamine residues that lie in close proximity to the predicted binding site has no detectable effect on the in vivo Rap1 GAP activity of GAP1(IP4BP). In contrast, we present evidence consistent with a model in which the RasGRD of GAP1(IP4BP) functions to stabilize the switch II region of Rap1, allowing stabilization of the transition state during GTP hydrolysis initiated by the arginine finger.

Pharmacological potential of exercise and RAS vasoactive peptides for prevention of diseases.

PubMed

Petriz, Bernardo de Assis; de Almeida, Jeeser Alves; Migliolo, Ludovico; Franco, Octavio Luiz

2013-09-01

The Renin-Angiotensin-System (RAS) molecular network has been widely studied, especially with attention to angiotensin II, the main effector peptide among RAS. The relation of Ang II to hypertension pathogenesis has led to research being extended to other molecules from the RAS, such as angiotensin III and IV, angiotensin (1-5), and angiotensin (1-9). Moreover, great pharmacologic advances have been made in hypertension treatment by inhibiting renin and angiotensin converting enzymes and blocking the bonding of angiotensin II to its receptor AT1. Thus, RAS molecular signaling and its effect on blood pressure as well as its relationship to renal function and cardiovascular disease are still being investigated. It is a great challenge to fully cover and understand all molecules from the RAS, especially those that interfere with or have vasoactive properties. Some of these targets respond to exercise, stimulating nitric oxide synthesis and endothelial vasodilation. The activation of these specific molecules via exercise is a systematic way of controlling high blood pressure without pharmacological treatment. Angiotensin (1-7) has been focused due to its vasodilation properties and its responses to exercise, improving vascular function. Thus, stimulation of the ACE2/Ang (1-7)/Mas axis has been gaining ground as a prospective clinical means to attenuate cardiovascular diseases such as hypertension by modulating RAS activity. This review focuses on the vasoactive peptides from the RAS, their responses to exercise and possible trends for pharmacological development. In several cases where exercise training is not achievable, cardiovascular drug therapy with vasodilator peptides may possibly be an option.

Circadian rhythm of blood pressure and the renin-angiotensin system in the kidney.

PubMed

Ohashi, Naro; Isobe, Shinsuke; Ishigaki, Sayaka; Yasuda, Hideo

2017-05-01

Activation of the intrarenal renin-angiotensin system (RAS) has a critical role in the pathophysiology of the circadian rhythm of blood pressure (BP) and renal injury, independent of circulating RAS. Although it is clear that the circulating RAS has a circadian rhythm, reports of a circadian rhythm in tissue-specific RAS are limited. Clinical studies evaluating intrarenal RAS activity by urinary angiotensinogen (AGT) levels have indicated that urinary AGT levels were equally low during both the daytime and nighttime in individuals without chronic kidney disease (CKD) and that urinary AGT levels were higher during the daytime than at nighttime in patients with CKD. Moreover, urinary AGT levels of the night-to-day (N/D) ratio of urinary AGT were positively correlated with the levels of N/D of urinary protein, albumin excretion and BP. In addition, animal studies have demonstrated that the expression of intrarenal RAS components, such as AGT, angiotensin II (AngII) and AngII type 1 receptor proteins, increased and peaked at the same time as BP and urinary protein excretion during the resting phase, and the amplitude of the oscillations of these proteins was augmented in a chronic progressive nephritis animal compared with a control. Thus, the circadian rhythm of intrarenal RAS activation may lead to renal damage and hypertension, which both are associated with diurnal variations in BP. It is possible that augmented glomerular permeability increases AGT excretion levels into the tubular lumen and that circadian fluctuation of glomerular permeability influences the circadian rhythm of the intrarenal RAS.

Comparative health technology assessment of robotic-assisted, direct manual laparoscopic and open surgery: a prospective study.

PubMed

Turchetti, Giuseppe; Pierotti, Francesca; Palla, Ilaria; Manetti, Stefania; Freschi, Cinzia; Ferrari, Vincenzo; Cuschieri, Alfred

2017-02-01

Despite many publications reporting on the increased hospital cost of robotic-assisted surgery (RAS) compared to direct manual laparoscopic surgery (DMLS) and open surgery (OS), the reported health economic studies lack details on clinical outcome, precluding valid health technology assessment (HTA). The present prospective study reports total cost analysis on 699 patients undergoing general surgical, gynecological and thoracic operations between 2011 and 2014 in the Italian Public Health Service, during which period eight major teaching hospitals treated the patients. The study compared total healthcare costs of RAS, DMLS and OS based on prospectively collected data on patient outcome in addition to healthcare costs incurred by the three approaches. The cost of RAS operations was significantly higher than that of OS and DMLS for both gynecological and thoracic operations (p < 0.001). The study showed no significant difference in total costs between OS and DMLS. Total costs of general surgery RAS were significantly higher than those of OS (p < 0.001), but not against DMLS general surgery. Indirect costs were significantly lower in RAS compared to both DMLS general surgery and OS gynecological surgery due to the shorter length of hospital stay of RAS approach (p < 0.001). Additionally, in all specialties compared to OS, patients treated by RAS experienced a quicker recovery and significantly less pain during the hospitalization and after discharge. The present HTA while confirming higher total healthcare costs for RAS operations identified significant clinical benefits which may justify the increased expenditure incurred by this approach.

Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth

PubMed Central

2012-01-01

Background Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX). Results We found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice. Conclusion Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents. PMID:22917272

Head-to-head comparison of H2-receptor antagonists and proton pump inhibitors in the treatment of erosive esophagitis: A meta-analysis

PubMed Central

Wang, Wei-Hong; Huang, Jia-Qing; Zheng, Ge-Fan; Xia, Harry Hua-Xiang; Wong, Wai-Man; Lam, Shiu-Kum; Wong, Benjamin Chun-Yu

2005-01-01

AIM: To systematically evaluate the efficacy of H2-receptor antagonists (H2RAs) and proton pump inhibitors in healing erosive esophagitis (EE). METHODS: A meta-analysis was performed. A literature search was conducted in PubMed, Medline, Embase, and Cochrane databases to include randomized controlled head-to-head comparative trials evaluating the efficacy of H2RAs or proton pump inhibitors in healing EE. Relative risk (RR) and 95% confidence interval (CI) were calculated under a random-effects model. RESULTS: RRs of cumulative healing rates for each comparison at 8 wk were: high dose vs standard dose H2RAs, 1.17 (95%CI, 1.02-1.33); standard dose proton pump inhibitors vs standard dose H2RAs, 1.59 (95%CI, 1.44-1.75); standard dose other proton pump inhibitors vs standard dose omeprazole, 1.06 (95%CI, 0.98-1.06). Proton pump inhibitors produced consistently greater healing rates than H2RAs of all doses across all grades of esophagitis, including patients refractory to H2RAs. Healing rates achieved with standard dose omeprazole were similar to those with other proton pump inhibitors in all grades of esophagitis. CONCLUSION: H2RAs are less effective for treating patients with erosive esophagitis, especially in those with severe forms of esophagitis. Standard dose proton pump inhibitors are significantly more effective than H2RAs in healing esophagitis of all grades. Proton pump inhibitors given at the recommended dose are equally effective for healing esophagitis. PMID:15996033

Restrictive allograft syndrome after lung transplantation: new radiological insights.

PubMed

Dubbeldam, Adriana; Barthels, Caroline; Coolen, Johan; Verschakelen, Johny A; Verleden, Stijn E; Vos, Robin; Verleden, Geert M; De Wever, Walter

2017-07-01

To describe the CT changes in patients with restrictive allograft syndrome (RAS) after lung transplantation, before and after clinical diagnosis. This retrospective study included 22 patients with clinical diagnosis of RAS. Diagnosis was based on a combination of forced expiratory volume (FEV1) decline (≥20 %) and total lung capacity (TLC) decline (≥10 %). All available CT scans after transplantation were analyzed for the appearance and evolution of lung abnormalities. In 14 patients, non-regressing nodules and reticulations predominantly affecting the upper lobes developed an average of 13.9 months prior to the diagnosis of RAS. Median graft survival after onset of non-regressing abnormalities was 33.5 months, with most patients in follow-up (9/14). In eight patients, a sudden appearance of diffuse consolidations mainly affecting both upper and lower lobes was seen an average of 2.8 months prior to the diagnosis of RAS. Median graft survival was 6.4 months after first onset of non-regressing abnormalities, with graft loss in most patients (6/8). RAS has been previously described as a homogenous group. However, our study shows two different groups of RAS-patients: one with slow progression and one with fast progression. The two groups show different onset and progression patterns of CT abnormalities. • RAS is the newest discovered form of chronic lung allograft dysfunction (CLAD). • RAS is not a homogenous group, as survival varies greatly between patients. • In this study, we see two different CT onset and progression patterns. • These two different CT patterns also correlate with a different survival rate.

MEK-1 Activates C-Raf Through a Ras-Independent Mechanism

PubMed Central

Leicht, Deborah T.; Balan, Vitaly; Zhu, Jun; Kaplun, Alexander; Bronisz, Agnieszka; Rana, Ajay; Tzivion, Guri

2013-01-01

C-Raf is a member of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK. Using 32P-metabolic labeling and 2D-phosphopeptide mapping experiments, we show that MEK increases C-Raf phosphorylation by up-to 10-fold. This increase was associated with C-Raf kinase activation, matching the activity seen with growth factor stimulation. Consequently, coexpression of wildtype C-Raf and MEK was sufficient for full and constitutive activation of ERK. Notably, the ability of MEK to activate C-Raf was completely Ras independent, since mutants impaired in Ras binding that are irresponsive to growth factors or Ras were fully activated by MEK. The ability of MEK to activate C-Raf was only partially dependent on MEK kinase activity but required MEK binding to C-Raf, suggesting that the binding results in a conformational change that increases C-Raf susceptibility to phosphorylation and activation or in the stabilization of the phosphorylated-active form. These findings propose a novel Ras-independent mechanism for activating C-Raf and the MAPK pathway without the need for mutations in the pathway. This mechanism could be of significance in pathological conditions or cancers overexpressing C-Raf and MEK or in conditions where C-Raf-MEK interaction is enhanced due to the downregulation of RKIP and MST2. PMID:23360980

The Significance of Ras Activity in Pancreatic Cancer Initiation

PubMed Central

Logsdon, Craig D.; Lu, Weiqin

2016-01-01

The genetic landscape of pancreatic cancer shows nearly ubiquitous mutations of K-RAS. However, oncogenic K-Rasmt alone is not sufficient to lead to pancreatic ductal adenocarcinoma (PDAC) in either human or in genetically modified adult mouse models. Many stimulants, such as high fat diet, CCK, LPS, PGE2 and others, have physiological effects at low concentrations that are mediated in part through modest increases in K-Ras activity. However, at high concentrations, they induce inflammation that, in the presence of oncogenic K-Ras expression, substantially accelerates PDAC formation. The mechanism involves increased activity of oncogenic K-Rasmt. Unlike what has been proposed in the standard paradigm for the role of Ras in oncogenesis, oncogenic K-Rasmt is now known to not be constitutively active. Rather, it can be activated by standard mechanisms similar to wild-type K-Ras, but its activity is sustained for a prolonged period. Furthermore, if the level of K-Ras activity exceeds a threshold at which it begins to generate its own activators, then a feed-forward loop is formed between K-Ras activity and inflammation and pathological processes including oncogenesis are initiated. Oncogenic K-Rasmt activation, a key event in PDAC initiation and development, is subject to complex regulatory mechanisms. Reagents which inhibit inflammation, such as the Cox2 inhibitor celecoxib, block the feed-forward loop and prevent induction of PDAC in models with endogenous oncogenic K-Rasmt. Increased understanding of the role of activating and inhibitory mechanisms on oncogenic K-Rasmt activity is of paramount importance for the development of preventive and therapeutic strategies to fight against this lethal disease. PMID:26929740

Suitability Measurement and Analysis for Fort Bliss Naval Air Facility OLS. Opportune Landing Site Program

DTIC Science & Technology

2008-09-01

bushes on the RAS. ERDC/CRREL TR-08-16 71 d. Delphinium virescens.4 e. Nolina microcarpa .5 f. Lygodesmia exigua.6 Figure 27 (cont’d...4 There are a few of these plants along the RAS. 5 There are a few nolinas on the RAS

Product (P1) from project 0-6738 : performance studies and future directions for mixes containing RAP and RAS.

DOT National Transportation Integrated Search

2013-01-01

In recent years both reclaimed asphalt pavement (RAP) and recycled asphalt shingles (RAS) have been widely used in asphalt mixes by the asphalt paving industry in Texas. The use of RAP and RAS can save tax payers money, and it is also good for the...

The Impact of Gatekeeper Training for Suicide Prevention on University Resident Assistants

ERIC Educational Resources Information Center

Taub, Deborah J.; Servaty-Seib, Heather L.; Miles, Nathan; Lee, Ji-Yeon; Wachter Morris, Carrie A.; Prieto-Welch, Susan L.; Werden, Donald

2013-01-01

Resident assistants (RAs) can serve as important suicide prevention gatekeepers. The purpose of the study was to determine if training improved RAs' crisis communications skills and suicide-related knowledge and to determine if the knowledge elements predicted crisis communications skills. New RAs showed significant improvement in all areas from…

Resident Assistants as Rule Enforcers versus Friends: An Exploratory Study of Role Conflict

ERIC Educational Resources Information Center

Everett, Diane D.; Loftus, Zachary V.

2011-01-01

Using data from both qualitative interviews and quantitative questionnaires from 32 undergraduate resident assistants (RAs) at a private, residential university, this study explored RAs' role conflict derived from their simultaneous positions as rule enforcers and friends, vis-a-vis their residents. Most RAs reported that they were friends with…

Comparison of the Effects of RAS vs. Kain-Fritsch Convective Schemes on Katrina Forecasts with GEOS-5

NASA Technical Reports Server (NTRS)

Miller, Timothy L.; Cohen, Charles; Paxton, Jessica; Robertson, F. R. (Pete)

2009-01-01

Global forecasts were made with the 0.25-degree latitude version of GEOS-5, with the RAS scheme and with the Kain-Fritsch scheme. Examination was made of the Katrina (2005) hurricane simulation. Replacement of the RAS convective scheme with the K-F scheme results in a much more vigorous Katrina, closer to reality. Still, the result is not as vigorous as reality. In terms of wind maximum, the gap was closed by 50%. The result seems to be due to the RAS scheme drying out the boundary layer, thus hampering the grid-scale secondary circulation and attending cyclone development. The RAS case never developed a full warm core, whereas the K-F case did. Not shown here: The K-F scheme also resulted in a more vigorous storm than when GEOS-5 is run with no convective parameterization. Also not shown: An experiment in which the RAS firing level was moved up by 3 model levels resulted in a stronger, warm-core storm, though not as strong as the K-F case. Effects on storm track were noticed, but not studied.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Jinqi; Cook, Aaron A.; Bergmeier, Wolfgang

The dynamic regulation of ERK1 and -2 (ERK1/2) is required for precise signal transduction controlling cell proliferation, differentiation, and survival. However, the underlying mechanisms regulating the activation of ERK1/2 are not completely understood. In this study, we show that phosphorylation of RasGRP2, a guanine nucleotide exchange factor (GEF), inhibits its ability to activate the small GTPase Rap1 that ultimately leads to decreased activation of ERK1/2 in cells. ERK2 phosphorylates RasGRP2 at Ser394 located in the linker region implicated in its autoinhibition. These studies identify RasGRP2 as a novel substrate of ERK1/2 and define a negative-feedback loop that regulates the BRaf–MEK–ERKmore » signaling cascade. This negative-feedback loop determines the amplitude and duration of active ERK1/2. -- Highlights: •ERK2 phosphorylates the guanine nucleotide exchange factor RasGRP2 at Ser394. •Phosphorylated RasGRP2 has decreased capacity to active Rap1b in vitro and in cells. •Phosphorylation of RasGRP2 by ERK1/2 introduces a negative-feedback loop into the BRaf-MEK-ERK pathway.« less

A PLC-γ1-independent, RasGRP1-ERK dependent pathway drives lymphoproliferative disease in LAT-Y136F mutant mice

PubMed Central

Kortum, Robert L.; Rouquette-Jazdanian, Alexandre K.; Miyaji, Michihiko; Merrill, Robert K.; Markegard, Evan; Pinski, John M.; Wesselink, Amelia; Nath, Nandan N.; Alexander, Clayton P.; Li, Wenmei; Kedei, Noemi; Roose, Jeroen P.; Blumberg, Peter M.; Samelson, Lawrence E.; Sommers, Connie L.

2012-01-01

Mice expressing a germline mutation in the PLC-γ1 binding site of LAT (linker for activation of T cells) show progressive lymphoproliferation and ultimately die at 4–6 months of age. The hyper-activated T cells in these mice show defective TCR-induced calcium flux, but enhanced Ras/ERK activation that is critical for disease progression. Despite the loss of LAT-dependent PLC-γ1 binding and activation, genetic analysis revealed RasGRP1, and not Sos1 or Sos2, to be the major RasGEF responsible for ERK activation and the lymphoproliferative phenotype in these mice. Analysis of isolated CD4+ T cells from LAT-Y136F mice showed altered proximal TCR-dependent kinase signaling, which activated a Zap70- and LAT-independent pathway. Moreover, LAT-Y136F T cells showed ERK activation that was dependent on Lck and/or Fyn, PKCθ, and RasGRP1. These data demonstrate a novel route to Ras activation in vivo in a pathological setting. PMID:23209318

Normal p21Ras/MAP kinase pathway expression and function in PBMC from patients with polycystic ovary disease.

PubMed

Buchs, A; Chagag, P; Weiss, M; Kish, E; Levinson, R; Aharoni, D; Rapoport, M J

2004-04-01

Polycystic ovary disease (PCOD) is associated with insulin resistance and increased prevalence of type II diabetes mellitus (T2DM). The p21Ras/MAP kinase is a major intracellular signaling pathway mediating insulin signaling in insulin responsive tissues. The expression, regulation and function of the p21Ras/MAP kinase pathway in PCOD patients were examined. Peripheral blood mononuclear cells (PBMC) were isolated from ten patients with PCOD and ten controls. The expression of p21Ras and its regulatory proteins; hSOS1 and p120GAP were studied. The basal and phytohemaglutinin (PHA) or insulin stimulated phosphorylation of MAP kinase was determined. Expression of p21Ras, and its regulatory proteins hSOS1 and p120GAP were similar in PCOD patients and controls. Basal, PHA and insulin stimulated phosphorylation of MAP kinase, were also comparable in the two groups as well as their PBMC proliferative response. These data indicate that the expression and overall function of the p21Ras/MAP kinase pathway remain intact in non-diabetic patients with PCOD.

Specific Activation of K-RasG12D Allele in the Bladder Urothelium Results in Lung Alveolar and Vascular Defects

PubMed Central

Kanasaki, Megumi; Vong, Sylvia; Rovira, Carlota; Kalluri, Raghu

2014-01-01

K-ras is essential for embryogenesis and its mutations are involved in human developmental syndromes and cancer. To determine the consequences of K-ras activation in urothelium, we used uroplakin-II (UPK II) promoter driven Cre recombinase mice and generated mice with mutated KrasG12D allele in the urothelium (UPK II-Cre;LSL-K-rasG12D). The UPK II-Cre;LSL-K-rasG12D mice died neonatally due to lung morphogenesis defects consisting of simplification with enlargement of terminal air spaces and dysmorphic pulmonary vasculature. A significant alteration in epithelial and vascular basement membranes, together with fragmentation of laminin, points to extracellular matrix degradation as the causative mechanism of alveolar and vascular defects. Our data also suggest that altered protease activity in amniotic fluid might be associated with matrix defects in lung of UPK II-Cre;LSL-K-rasG12. These defects resemble those observed in early stage human neonatal bronchopulmonary dysplasia (BPD), although the relevance of this new mouse model for BPD study needs further investigation. PMID:24760005

miR-11 regulates pupal size of Drosophila melanogaster via directly targeting Ras85D.

PubMed

Li, Yao; Li, Shengjie; Jin, Ping; Chen, Liming; Ma, Fei

2017-01-01

MicroRNAs play diverse roles in various physiological processes during Drosophila development. In the present study, we reported that miR-11 regulates pupal size during Drosophila metamorphosis via targeting Ras85D with the following evidences: pupal size was increased in the miR-11 deletion mutant; restoration of miR-11 in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant; ectopic expression of miR-11 in brain insulin-producing cells (IPCs) and whole body shows consistent alteration of pupal size; Dilps and Ras85D expressions were negatively regulated by miR-11 in vivo; miR-11 targets Ras85D through directly binding to Ras85D 3'-untranslated region in vitro; removal of one copy of Ras85D in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant. Thus, our current work provides a novel mechanism of pupal size determination by microRNAs during Drosophila melanogaster metamorphosis. Copyright © 2017 the American Physiological Society.

Interactions of Ras proteins with the plasma membrane and their roles in signaling.

PubMed

Eisenberg, Sharon; Henis, Yoav I

2008-01-01

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains.

PubMed

Nakamura, Yuki; Hibino, Kayo; Yanagida, Toshio; Sako, Yasushi

2016-01-01

Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS.

Discovery of Aminopiperidine Indoles That Activate the Guanine Nucleotide Exchange Factor SOS1 and Modulate RAS Signaling.

PubMed

Abbott, Jason R; Hodges, Timothy R; Daniels, R Nathan; Patel, Pratiq A; Kennedy, Jack Phillip; Howes, Jennifer E; Akan, Denis T; Burns, Michael C; Sai, Jiqing; Sobolik, Tammy; Beesetty, Yugandhar; Lee, Taekyu; Rossanese, Olivia W; Phan, Jason; Waterson, Alex G; Fesik, Stephen W

2018-06-01

Deregulated RAS activity, often the result of mutation, is implicated in approximately 30% of all human cancers. Despite this statistic, no clinically successful treatment for RAS-driven tumors has yet been developed. One approach for modulating RAS activity is to target and affect the activity of proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). Here, we report on structure-activity relationships (SAR) in an indole series of compounds. Using structure-based design, we systematically explored substitution patterns on the indole nucleus, the pendant amino acid moiety, and the linker unit that connects these two fragments. Best-in-class compounds activate the nucleotide exchange process at sub-micromolar concentrations in vitro, increase levels of active RAS-GTP in HeLa cells, and elicit signaling changes in the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway, resulting in a decrease in pERK1/2 T202/Y204 protein levels at higher compound concentrations.

Relationship between Gender Roles and Sexual Assertiveness in Married Women.

PubMed

Azmoude, Elham; Firoozi, Mahbobe; Sadeghi Sahebzad, Elahe; Asgharipour, Neghar

2016-10-01

Evidence indicates that sexual assertiveness is one of the important factors affecting sexual satisfaction. According to some studies, traditional gender norms conflict with women's capability in expressing sexual desires. This study examined the relationship between gender roles and sexual assertiveness in married women in Mashhad, Iran. This cross-sectional study was conducted on 120 women who referred to Mashhad health centers through convenient sampling in 2014-15. Data were collected using Bem Sex Role Inventory (BSRI) and Hulbert index of sexual assertiveness. Data were analyzed using SPSS 16 by Pearson and Spearman's correlation tests and linear Regression Analysis. The mean scores of sexual assertiveness was 54.93±13.20. According to the findings, there was non-significant correlation between Femininity and masculinity score with sexual assertiveness (P=0.069 and P=0.080 respectively). Linear regression analysis indicated that among the predictor variables, only Sexual function satisfaction was identified as the sexual assertiveness summary predictor variables (P=0.001). Based on the results, sexual assertiveness in married women does not comply with gender role, but it is related to Sexual function satisfaction. So, counseling psychologists need to consider this variable when designing intervention programs for modifying sexual assertiveness and find other variables that affect sexual assertiveness.