Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors.

PubMed

Anaka, Matthew; Hudson, Christopher; Lo, Pu-Han; Do, Hongdo; Caballero, Otavia L; Davis, Ian D; Dobrovic, Alexander; Cebon, Jonathan; Behren, Andreas

2013-10-11

Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma.

Ionizing irradiation not only inactivates clonogenic potential in primary normal human diploid lens epithelial cells but also stimulates cell proliferation in a subset of this population.

PubMed

Fujimichi, Yuki; Hamada, Nobuyuki

2014-01-01

Over the past century, ionizing radiation has been known to induce cataracts in the crystalline lens of the eye, but its mechanistic underpinnings remain incompletely understood. This study is the first to report the clonogenic survival of irradiated primary normal human lens epithelial cells and stimulation of its proliferation. Here we used two primary normal human cell strains: HLEC1 lens epithelial cells and WI-38 lung fibroblasts. Both strains were diploid, and a replicative lifespan was shorter in HLEC1 cells. The colony formation assay demonstrated that the clonogenic survival of both strains decreases similarly with increasing doses of X-rays. A difference in the survival between two strains was actually insignificant, although HLEC1 cells had the lower plating efficiency. This indicates that the same dose inactivates the same fraction of clonogenic cells in both strains. Intriguingly, irradiation enlarged the size of clonogenic colonies arising from HLEC1 cells in marked contrast to those from WI-38 cells. Such enhanced proliferation of clonogenic HLEC1 cells was significant at ≥2 Gy, and manifested as increments of ≤2.6 population doublings besides sham-irradiated controls. These results suggest that irradiation of HLEC1 cells not only inactivates clonogenic potential but also stimulates proliferation of surviving uniactivated clonogenic cells. Given that the lens is a closed system, the stimulated proliferation of lens epithelial cells may not be a homeostatic mechanism to compensate for their cell loss, but rather should be regarded as abnormal. This is because these findings are consistent with the early in vivo evidence documenting that irradiation induces excessive proliferation of rabbit lens epithelial cells and that suppression of lens epithelial cell divisions inhibits radiation cataractogenesis in frogs and rats. Thus, our in vitro model will be useful to evaluate the excessive proliferation of primary normal human lens epithelial cells that may underlie radiation cataractogenesis, warranting further investigations.

Anticancer activity of 7-epiclusianone, a benzophenone from Garcinia brasiliensis, in glioblastoma.

PubMed

Sales, Leilane; Pezuk, Julia Alejandra; Borges, Kleiton Silva; Brassesco, María Sol; Scrideli, Carlos Alberto; Tone, Luiz Gonzaga; dos Santos, Marcelo Henrique; Ionta, Marisa; de Oliveira, Jaqueline Carvalho

2015-10-30

Glioblastoma is the most common tumor of the central nervous system and one of the hardest tumors to treat. Consequently, the search for novel therapeutic options is imperative. 7-epiclusianone, a tetraprenylated benzophenone isolated from the epicarp of the native plant Garcinia brasiliensis, exhibits a range of biological activities but its prospect anticancer activity is underexplored. Thus, the aim of the present study was to evaluate the influence of 7-epiclusianone on proliferation, clonogenic capacity, cell cycle progression and induction of apoptosis in two glioblastoma cell lines (U251MG and U138MG). Cell viability was measured by the MTS assay; for the clonogenic assay, colonies were stained with Giemsa and counted by direct visual inspection; For cell cycle analysis, cells were stained with propidium iodide and analyzed by cytometry; Cyclin A expression was determined by immunoblotting; Apoptotic cell death was determined by annexin V fluorescein isothiocyanate labeling and Caspase-3 activity in living cells. Viability of both cell lines was drastically inhibited; moreover, the colony formation capacity was significantly reduced, demonstrating long-term effects even after removal of the drug. 7-epiclusianone treatment at low concentrations also altered cell cycle progression, decreased the S and G2/M populations and at higher concentrations increased the number of cells at sub-G1, in concordance with the increase of apoptotic cells. The present study demonstrates for the first time the anticancer potential of 7-epiclusianone against glioblastoma cells, thus meriting its further investigation as a potential therapeutic agent.

Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells

PubMed Central

Bao, Bin; Wang, Zhiwei; Ali, Shadan; Kong, Dejuan; Banerjee, Sanjeev; Ahmad, Aamir; Li, Yiwei; Azmi, Asfar S.; Miele, Lucio; Sarkar, Fazlul H.

2011-01-01

FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of Epithelial-to-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotypes are highly inter-related, and contributes to drug resistance, tumor recurrence and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self-renewal capacity is poorly understood. Therefore, we established FoxM1 over-expressing pancreatic cancer (AsPC-1) cells, which showed increased cell growth, clonogenicity and cell migration. Moreover, over-expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E-cadherin, and vimentin, which is consistent with increased sphere-forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over-expression of FoxM1 led to decreased expression of miRNAs (let-7a, let-7b, let-7c, miR-200b and miR-200c); however, re-expression of miR-200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E-cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo-preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over-expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR-200b and these processes, could be easily attenuated by genistein. PMID:21503965

Notch-1 induces Epithelial-mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells

PubMed Central

Bao, Bin; Wang, Zhiwei; Ali, Shadan; Kong, Dejuan; Li, Yiwei; Ahmad, Aamir; Banerjee, Sanjeev; Azmi, Asfar S.; Miele, Lucio; Sarkar, Fazlul H.

2011-01-01

Activation of Notch-1 is known to be associated with the development and progression of human malignancies including pancreatic cancer. Emerging evidence suggest that the acquisition of epithelial-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotype are interrelated and contributes to tumor recurrence and drug resistance. The molecular mechanism(s) by which Notch-1 contributes to the acquisition of EMT phenotype and CSC self-renewal capacity has not been fully elucidated. Here we show that forced over-expression of Notch-1 leads to increased cell growth, clonogenicity, migration and invasion of AsPC-1 cells. Moreover, over-expression of Notch-1 led to the induction of EMT phenotype by activation of mesenchymal cell markers such as ZEB1, CD44, EpCAM, and Hes 1. Here we also report, for the first time, that over-expression of Notch-1 leads to increased expression of miR-21, and decreased expression of miR-200b, miR-200c, let-7a, let-7b, and let-7c. Re-expression of miR-200b led to decreased expression of ZEB1, and vimentin, and increased expression of E-cadherin. Over-expression of Notch-1 also increased the formation of pancreatospheres consistent with expression of CSC surface markers CD44 and EpCAM. Finally, we found that genistein, a known natural anti-tumor agent inhibited cell growth, clonogenicity, migration, invasion, EMT phenotype, formation of pancreatospheres and expression of CD44 and EpCAM. These results suggest that the activation of Notch-1 signaling contributes to the acquisition of EMT phenotype, which is in part mediated through the regulation of miR-200b and CSC self-renewal capacity, and these processes could be attenuated by genistein treatment. PMID:21463919

Clonogenic assay: adherent cells.

PubMed

Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

2011-03-13

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

Enrichment of human bone marrow aspirates for low-density mononuclear cells using a haemonetics discontinuous blood cell separator.

PubMed

Raijmakers, R; de Witte, T; Koekman, E; Wessels, J; Haanen, C

1986-01-01

Isopycnic density floatation centrifugation has been proven to be a suitable technique to enrich bone marrow aspirates for clonogenic cells on a small scale. We have tested a Haemonetics semicontinuous blood cell separator in order to process large volumes of bone marrow with minimal bone marrow manipulation. The efficacy of isopycnic density floatation was tested in a one and a two-step procedure. Both procedures showed a recovery of about 20% of the nucleated cells and 1-2% of the erythrocytes. The enrichment of clonogenic cells in the one-step procedure appeared superior to the two-step enrichment, first separating buffy coat cells. The recovery of clonogenic cells was 70 and 50%, respectively. Repopulation capacity of the low-density cell fraction containing the clonogenic cells was excellent after autologous reinfusion (6 cases) and allogeneic bone marrow transplantation (3 cases). Fast enrichment of large volumes of bone marrow aspirates with low-density cells containing the clonogenic cells by isopycnic density floatation centrifugation can be done safely using a Haemonetics blood cell separator.

Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors

PubMed Central

2013-01-01

Background Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Methods Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. Results MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Conclusion Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma. PMID:24119551

Clonogenic Assay: Adherent Cells

PubMed Central

Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T.; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C.

2011-01-01

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation. PMID:21445039

Scribble Modulates the MAPK/Fra1 Pathway to Disrupt Luminal and Ductal Integrity and Suppress Tumour Formation in the Mammary Gland

PubMed Central

Godde, Nathan J.; Sheridan, Julie M.; Smith, Lorey K.; Pearson, Helen B.; Britt, Kara L.; Galea, Ryan C.; Yates, Laura L.; Visvader, Jane E.; Humbert, Patrick O.

2014-01-01

Polarity coordinates cell movement, differentiation, proliferation and apoptosis to build and maintain complex epithelial tissues such as the mammary gland. Loss of polarity and the deregulation of these processes are critical events in malignant progression but precisely how and at which stage polarity loss impacts on mammary development and tumourigenesis is unclear. Scrib is a core polarity regulator and tumour suppressor gene however to date our understanding of Scrib function in the mammary gland has been limited to cell culture and transplantation studies of cell lines. Utilizing a conditional mouse model of Scrib loss we report for the first time that Scrib is essential for mammary duct morphogenesis, mammary progenitor cell fate and maintenance, and we demonstrate a critical and specific role for Scribble in the control of the early steps of breast cancer progression. In particular, Scrib-deficiency significantly induced Fra1 expression and basal progenitor clonogenicity, which resulted in fully penetrant ductal hyperplasia characterized by high cell turnover, MAPK hyperactivity, frank polarity loss with mixing of apical and basolateral membrane constituents and expansion of atypical luminal cells. We also show for the first time a role for Scribble in mammalian spindle orientation with the onset of mammary hyperplasia being associated with aberrant luminal cell spindle orientation and a failure to apoptose during the final stage of duct tubulogenesis. Restoring MAPK/Fra1 to baseline levels prevented Scrib-hyperplasia, whereas persistent Scrib deficiency induced alveolar hyperplasia and increased the incidence, onset and grade of mammary tumours. These findings, based on a definitive genetic mouse model provide fundamental insights into mammary duct maturation and homeostasis and reveal that Scrib loss activates a MAPK/Fra1 pathway that alters mammary progenitor activity to drive premalignancy and accelerate tumour progression. PMID:24852022

Gallotannin-rich Caesalpinia spinosa fraction decreases the primary tumor and factors associated with poor prognosis in a murine breast cancer model

PubMed Central

2013-01-01

Background Several treatment alternatives are available for primary breast cancer, although those for metastatic disease or inflammation associated with tumor progression are ineffective. Therefore, there is a great need for new therapeutic alternatives capable of generating an immune response against residual tumor cells, thus contributing to eradication of micrometastases and cancer stem cells. The use of complex natural products is an excellent therapeutic alternative widely used by Chinese, Hindu, Egyptian, and ancestral Latin-American Indian populations. Methods The present study evaluated cytotoxic, antitumor, and tumor progression activities of a gallotannin-rich fraction derived from Caesalpinia spinosa (P2Et). The parameters evaluated in vitro were mitochondrial membrane depolarization, phosphatidylserine externalization, caspase 3 activation, DNA fragmentation, and clonogenic activity. The parameters evaluated in vivo were tumor growth, leukocyte number, metastatic cell number, and cytokine production by flow cytometry. Results The in vitro results showed that the P2Et fraction induced apoptosis with mitochondrial membrane potential loss, phosphatidylserine externalization, caspase 3 activation, DNA fragmentation, and decreased clonogenic capacity of 4T1 cells. In vivo, the P2Et fraction induced primary tumor reduction in terms of diameter and weight in BALB/c mice transplanted with 4T1 cells and decreased numbers of metastatic cells, mainly in the spleen. Furthermore, decreases in the number of peripheral blood leukocytes (leukemoid reaction) and interleukin 6 (IL-6) serum levels were found, which are events associated with a poor prognosis. The P2Et fraction exerts its activity on the primary tumor, reduces cell migration to distant organs, and decreases IL-6 serum levels, implying tumor microenvironment mechanisms. Conclusions Overall, the P2Et fraction lessens risk factors associated with tumor progression and diminishes primary tumor size, showing good potential for use as an adjuvant in breast cancer ER(+) treatment. PMID:23552194

Gallotannin-rich Caesalpinia spinosa fraction decreases the primary tumor and factors associated with poor prognosis in a murine breast cancer model.

PubMed

Urueña, Claudia; Mancipe, Juan; Hernandez, John; Castañeda, Diana; Pombo, Luis; Gomez, Alejandra; Asea, Alexzander; Fiorentino, Susana

2013-04-03

Several treatment alternatives are available for primary breast cancer, although those for metastatic disease or inflammation associated with tumor progression are ineffective. Therefore, there is a great need for new therapeutic alternatives capable of generating an immune response against residual tumor cells, thus contributing to eradication of micrometastases and cancer stem cells. The use of complex natural products is an excellent therapeutic alternative widely used by Chinese, Hindu, Egyptian, and ancestral Latin-American Indian populations. The present study evaluated cytotoxic, antitumor, and tumor progression activities of a gallotannin-rich fraction derived from Caesalpinia spinosa (P2Et). The parameters evaluated in vitro were mitochondrial membrane depolarization, phosphatidylserine externalization, caspase 3 activation, DNA fragmentation, and clonogenic activity. The parameters evaluated in vivo were tumor growth, leukocyte number, metastatic cell number, and cytokine production by flow cytometry. The in vitro results showed that the P2Et fraction induced apoptosis with mitochondrial membrane potential loss, phosphatidylserine externalization, caspase 3 activation, DNA fragmentation, and decreased clonogenic capacity of 4T1 cells. In vivo, the P2Et fraction induced primary tumor reduction in terms of diameter and weight in BALB/c mice transplanted with 4T1 cells and decreased numbers of metastatic cells, mainly in the spleen. Furthermore, decreases in the number of peripheral blood leukocytes (leukemoid reaction) and interleukin 6 (IL-6) serum levels were found, which are events associated with a poor prognosis. The P2Et fraction exerts its activity on the primary tumor, reduces cell migration to distant organs, and decreases IL-6 serum levels, implying tumor microenvironment mechanisms. Overall, the P2Et fraction lessens risk factors associated with tumor progression and diminishes primary tumor size, showing good potential for use as an adjuvant in breast cancer ER(+) treatment.

Miniaturization of the Clonogenic Assay Using Confluence Measurement

PubMed Central

Mayr, Christian; Beyreis, Marlena; Dobias, Heidemarie; Gaisberger, Martin; Pichler, Martin; Ritter, Markus; Jakab, Martin; Neureiter, Daniel; Kiesslich, Tobias

2018-01-01

The clonogenic assay is a widely used method to study the ability of cells to ‘infinitely’ produce progeny and is, therefore, used as a tool in tumor biology to measure tumor-initiating capacity and stem cell status. However, the standard protocol of using 6-well plates has several disadvantages. By miniaturizing the assay to a 96-well microplate format, as well as by utilizing the confluence detection function of a multimode reader, we here describe a new and modified protocol that allows comprehensive experimental setups and a non-endpoint, label-free semi-automatic analysis. Comparison of bright field images with confluence images demonstrated robust and reproducible detection of clones by the confluence detection function. Moreover, time-resolved non-endpoint confluence measurement of the same well showed that semi-automatic analysis was suitable for determining the mean size and colony number. By treating cells with an inhibitor of clonogenic growth (PTC-209), we show that our modified protocol is suitable for comprehensive (broad concentration range, addition of technical replicates) concentration- and time-resolved analysis of the effect of substances or treatments on clonogenic growth. In summary, this protocol represents a time- and cost-effective alternative to the commonly used 6-well protocol (with endpoint staining) and also provides additional information about the kinetics of clonogenic growth. PMID:29510509

Combination Treatment of Glioblastoma by Low-Dose Radiation and Genistein.

PubMed

Atefeh, Zamanian; Vahid, Changizi; Hasan, Nedaie; Saeed, Amanpour; Mahnaz, Haddadi

2016-01-01

Gioblastoma multiforme as a chemoresistant and radioresistant malignant cell line needs to novel strategies to treatment. Low-dose hyper-radiosensitivity (LDHRS) seems to be an effective phenomenon to irradiation that can save normal brain fibroblasts. Genistein which is a soy isoflavone can be cytotoxic in some tumor cell lines. So we determined to study the effect of combining these two treatment modalities. After 30 hours incubation with Genistein in different concentrations on U87MG cell line, proliferation and clonogenicity were conducted by both clonogenic and MTT assays. A conventional 2Gy radiation dose was compared with 10 doses of 0.2Gy gamma irradiation with 3 minutes and 1 hour intervals. Finally, concurrent effect of these modalities was assessed. Based on acquired cell doubling time (30 hours), one doubling time treatment by Genistein could decrease clonogenicity. U87MG cell line exhibited HRS at low dose irradiations. 2Gy irradiation was more effective than ultra-fractionation methods in comparison with control group. All groups with 50uM concentration of Genistein showed decrease in the survival. This decrease compared with control group, in 10x0.2Gy with 3 minutes intervals plus 50uM Genistein was significant and for groups with the same dose of Genistein but along with continuous 2Gy was more significant. In one day treatment regimen, 10x0.2Gy ultra-fractionation with 3 minutes and 1 hour intervals seems to be less effective than conventional 2Gy irradiation, however adding 50uM Genistein can decrease survival more. Although 2Gy conventional dose plus 50uM Genistein was the most effective regimen. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Progress in myeloma stem cells

PubMed Central

Cruz, Richard Dela; Tricot, Guido; Zangari, Maurizio; Zhan, Fenghuang

2011-01-01

Multiple myeloma (MM) is the second most common hematologic malignancy in the United States and affects about 4 in 100,000 Americans. Even though much progress has been made in MM therapy, MM remains an incurable disease for the vast majority of patients. The existence of MM stem cell is considered one of the major causes of MM drug-resistance, leading to relapse. This highlights the importance and urgency of developing approaches to target MM stem cells. However, very little is known about the molecular characteristics of the MM stem cells, which makes it difficult to target MM stem cells therapeutically. Evidence of the existence of a myeloma stem cell has been provided by Matsui et al. showing that the CD138- and CD20+ fraction, which is a minor population of the MM cells, has a greater clonogenic potential and has the phenotype of a memory B-cell (CD19+, CD27+). In this review, we report recent progress of cell surface markers in cancer stem cells, especially in myeloma and the molecular mechanisms related to drug resistance and myeloma disease progression. PMID:22432075

Identification of multipotent stem cells from adult dog periodontal ligament.

PubMed

Wang, Wen-Jun; Zhao, Yu-Ming; Lin, Bi-Chen; Yang, Jie; Ge, Li-Hong

2012-08-01

Periodontal diseases, which are characterized by destruction of the connective tissues responsible for restraining the teeth within the jaw, are the main cause of tooth loss. Periodontal regeneration mediated by human periodontal ligament stem cells (hPDLSCs) may offer an alternative strategy for the treatment of periodontal disease. Dogs are a widely used large-animal model for the study of periodontal-disease progression, tissue regeneration, and dental implants, but little attention has been paid to the identification of the cells involved in this species. This study aimed to characterize stem cells isolated from canine periodontal ligament (cPDLSCs). The cPDLSCs, like hPDLSCs, showed clonogenic capability and expressed the mesenchymal stem cell markers STRO-1, CD146, and CD105, but not CD34. After induction of osteogenesis, cPDLSCs showed calcium accumulation in vitro. Moreover, cPDLSCs also showed both adipogenic and chondrogenic potential. Compared with cell-free controls, more cementum/periodontal ligament-like structures were observed in CB-17/SCID mice into which cPDLSCs had been transplanted. These results suggest that cPDLSCs are clonogenic, highly proliferative, and have multidifferentiation potential, and that they could be used as a new cellular therapeutic approach to facilitate successful and more predictable regeneration of periodontal tissue using a canine model of periodontal disease. © 2012 Eur J Oral Sci.

Initial clonogenic potential of human endothelial progenitor cells is predictive of their further properties and establishes a functional hierarchy related to immaturity.

PubMed

Ferratge, Ségolène; Ha, Guillaume; Carpentier, Gilles; Arouche, Nassim; Bascetin, Rümeyza; Muller, Laurent; Germain, Stéphane; Uzan, Georges

2017-05-01

Endothelial progenitor cells (EPCs) generate in vitro Endothelial Colony Forming Cells (ECFCs) combining features of endothelial and stem/progenitor cells. Their angiogenic properties confer them a therapeutic potential for treating ischemic lesions. They may be isolated from umbilical cord blood (CB-ECFCs) or peripheral adult blood (AB-ECFCs). It is generally accepted that CB-ECFCs are more clonogenic, proliferative and angiogenic than AB-ECFCs. Nevertheless, only a few studies have focused on the functional heterogeneity of CB-ECFCs from different individuals. Moreover, AB-ECFC loss of function is yet to be precisely described. We have focused on these two issues that are critical for clinical perspectives. The detailed clonogenic profile of CB-ECFCs and AB-ECFCs was obtained and revealed a high inter individual heterogeneity and the absence of correlation with age. Most CB-ECFCs yielded initial colonies and had functional properties similar to those of AB-ECFCs. Conversely, a high clonogenicity was associated with an enhanced proliferative and angiogenic potential and stemness gene overexpression, confirming that immaturity, lost by AB-ECFCs, was a prerequisite to functionality. We thus demonstrated the importance of selecting CB-ECFCs according to specific criteria, and we propose using the initial clonogenicity as a relevant marker of their potential efficacy on vascular repair. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Influence of environmental pH on G2-phase arrest caused by ionizing radiation.

PubMed

Park, Heon Joo; Lee, Sang Hwa; Chung, HyunSook; Rhee, Yun Hee; Lim, Byung Uk; Ha, Sung Whan; Griffin, Robert J; Lee, Hyung Sik; Song, Chang Won; Choi, Eun Kyung

2003-01-01

We investigated the effects of an acidic environment on the G2/M-phase arrest, apoptosis, clonogenic death, and changes in cyclin B1-CDC2 kinase activity caused by a 4-Gy irradiation in RKO.C human colorectal cancer cells in vitro. The time to reach peak G2/M-phase arrest after irradiation was delayed in pH 6.6 medium compared to that in pH 7.5 medium. Furthermore, the radiation-induced G2/M-phase arrest decayed more slowly in pH 6.6 medium than in pH 7.5 medium. Finally, there was less radiation-induced apoptosis and clonogenic cell death in pH 6.6 medium than in pH 7.5 medium. It appeared that the prolongation of G2-phase arrest after irradiation in the acidic environment allowed for greater repair of radiation-induced DNA damage, thereby decreasing the radiation-induced cell death. The prolongation of G2-phase arrest after irradiation in the acidic pH environment appeared to be related at least in part to a prolongation of the phosphorylation of CDC2, which inhibited cyclin B1-CDC2 kinase activity.

miR-219-5p inhibits proliferation and clonogenicity in chordoma cells and is associated with tumor recurrence

PubMed Central

Wei, Wei; Zhang, Qiuhang; Wang, Zhenlin; Yan, Bo; Feng, Yanjun; Li, Pu

2016-01-01

Chordoma is a rare malignant bone tumor that is usually localized to the skull base, vertebral column and sacrum. The transcription factor brachyury, which is encoded by the T gene, has a critical role in the development and progression of chordoma, although the mechanisms underlying brachyury regulation remain unclear. The aim of the current study was to identify and characterize microRNAs (miRs) that regulate brachyury expression in chordoma. MicroRNAs that target brachyury were predicted using miRanda and TargetScan. Using reverse transcription-quantitative polymerase chain reaction, miR-219-5p was shown to be significantly downregulated in chordoma tissues and the U-CH2 chordoma cell lines. A dual-luciferase reporter assay was used to validate the inhibitory effect of miR-219-5p on brachyury mRNA expression. The expression level of brachyury was downregulated in U-CH2 cells following transfection with miR-219-5p mimics and upregulated following transfection with the miR-219-5p inhibitor. The effects of miR-219-5p on the proliferation and clonogenicity of chordoma cells were assessed using cell counting kit-8, EdU and clone formation assays. These in vitro results indicated that miR-219-5p may have an important role in regulating the cell proliferation and clonogenicity of human chordoma cells, potentially by targeting brachyury. Furthermore, the associations between the expression levels of miR-219-5p and various clinicopathological factors were analyzed, and miR-219-5p expression was shown to correlate with tumor extent and recurrence. These results suggested that miR-219-5p functions as a tumor suppressor in chordoma and, therefore, that miR-219-50 may be a potential target for therapeutic intervention. PMID:28105164

Novel poly (ADP-ribose) polymerase inhibitor, AZD2281, enhances radiosensitivity of both normoxic and hypoxic esophageal squamous cancer cells.

PubMed

Zhan, L; Qin, Q; Lu, J; Liu, J; Zhu, H; Yang, X; Zhang, C; Xu, L; Liu, Z; Cai, J; Ma, J; Dai, S; Tao, G; Cheng, H; Sun, X

2016-04-01

Radiotherapy plays an important role in the treatment of esophageal squamous cell carcinoma (ESCC). However, the outcome of radiotherapy in ESCC remains unsatisfactory because esophageal squamous cancer cells, particularly those under hypoxic condition, exhibit radioresistance. The aim of this study was to determine whether or not AZD2281, a potent poly (ADP-ribose) polymerase (PARP) inhibitor, could enhance the radiation sensitivity of two ESCC cell lines, namely ECA109 and TE13. The radiosensitizing effect of AZD2281 was evaluated on the basis of cell death, clonogenic survival and tumor xenograft progression. AZD2281 alone was slightly toxic to ESCC cell lines. Apoptosis was increased and clonogenic survival was decreased in both cell lines when AZD2281 was combined with ionizing radiation (IR) under normoxic condition. AZD2281 enhanced IR-induced apoptosis to a more significant level under chronic hypoxic condition (0.2% O(2), 48 hour) than under normoxic condition. AZD2281 also slightly enhanced clonogenic cell death under chronic hypoxic condition compared with that under normoxic condition. This result could be associated with increased radiation-induced DNA double-strand breaks (DSB), decreased DSB repair and increased apoptosis of ESCC cells. Furthermore, homologous recombination (HR) protein Rad51 expression and focus formation were decreased in ESCC cells exposed to moderate chronic hypoxic condition (0.2% O(2), 48 hour); this result indicated that chronic hypoxic ESCC cells were HR deficient, possibly causing contextual synthetic lethality with PARP inhibitor in radiation sensitization. AZD2281 was also a radiation sensitizer in ESCC tumor xenograft models. Hence, in vitro and in vivo findings provide evidence that AZD2281 potently sensitizes ESCC cells to X-ray irradiation. The selective cell killing of HR-defective hypoxic cells contributes to radiosensitization by PARP inhibitor in ESCC cells under hypoxic condition. © 2015 International Society for Diseases of the Esophagus.

Effects of silibinin on growth and invasive properties of human ovarian carcinoma cells through suppression of heregulin/HER3 pathway.

PubMed

Momeny, Majid; Ghasemi, Reza; Valenti, Giovanni; Miranda, Mariska; Zekri, Ali; Zarrinrad, Ghazaleh; Javadikooshesh, Sepehr; Yaghmaie, Marjan; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Ghaffari, Seyed H

2016-03-01

Epithelial ovarian cancer (EOC) is the most fatal gynecological malignancy due to its high proliferative and invasive capacities. A heregulin (HRG)/HER3 autocrine loop increases proliferative and metastatic properties of EOC cells, suggesting that modulators of this signaling pathway may prove effective to trammel growth and motility of these cells. This study aimed to evaluate the effects of multi-tyrosine kinase inhibitor silibinin on proliferative and invasive characteristics of EOC cell lines OVCAR8 and SKOV3 through suppression of the HRG/HER3 pathway. To achieve this, the effects of silibinin on proliferation, DNA synthesis, clonogenicity, cell cycle progression, cathepsin B enzymatic activity, and migration and invasion were explored in vitro. Silibinin suppressed proliferation, DNA synthesis, and clonogenic abilities of OVCAR8 and SKOV3 cells through inhibition of the autocrine HRG/HER3 circuit. Silibinin-mediated attenuation of the HER3 signaling disabled the HER3/AKT/survivin axis and thereby, induced G1/S cell cycle arrest. Furthermore, silibinin reduced invasive potentials of the EOC cells through quelling the HRG/HER3 pathway and suppression of cathepsin B activity. Altogether, these results suggest that silibinin is a potential anti-cancer drug to inhibit proliferative and invasive characteristics of the EOC cells that exhibit an autocrine HRG/HER3 pathway.

FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2

PubMed Central

Kim, Dong Geon; Cho, Hee Jin; Kim, Yeonghwan; Rheey, Jinguen; Shin, Kayoung; Seo, Yun Jee; Choi, Yeon-Sook; Lee, Jung-Il; Lee, Jeongwu; Joo, Kyeung Min; Nam, Do-Hyun

2015-01-01

Glioblastoma (GBM) is the most aggressive and most lethal brain tumor. As current standard therapy consisting of surgery and chemo-irradiation provides limited benefit for GBM patients, novel therapeutic options are urgently required. Forkhead box M1 (FoxM1) transcription factor is an oncogenic regulator that promotes the proliferation, survival, and treatment resistance of various human cancers. The roles of FoxM1 in GBM remain incompletely understood, due in part to pleotropic nature of the FoxM1 pathway. Here, we show the roles of FoxM1 in GBM stem cell maintenance and radioresistance. ShRNA-mediated FoxM1 inhibition significantly impeded clonogenic growth and survival of patient-derived primary GBM cells with marked downregulation of Sox2, a master regulator of stem cell phenotype. Ectopic expression of Sox2 partially rescued FoxM1 inhibition-mediated effects. Conversely, FoxM1 overexpression upregulated Sox2 expression and promoted clonogenic growth of GBM cells. These data, with a direct binding of FoxM1 in the Sox2 promoter region in GBM cells, suggest that FoxM1 regulates stemness of primary GBM cells via Sox2. We also found significant increases in FoxM1 and Sox2 expression in GBM cells after irradiation both in vitro and in vivo orthotopic tumor models. Notably, genetic or a small-molecule FoxM1 inhibitor-mediated FoxM1 targeting significantly sensitized GBM cells to irradiation, accompanying with Sox2 downregulation. Finally, FoxM1 inhibition combined with irradiation in a patient GBM-derived orthotopic model significantly impeded tumor growth and prolonged the survival of tumor bearing mice. Taken together, these results indicate that the FoxM1-Sox2 signaling axis promotes clonogenic growth and radiation resistance of GBM, and suggest that FoxM1 targeting combined with irradiation is a potentially effective therapeutic approach for GBM. PMID:26444992

FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2.

PubMed

Lee, Yeri; Kim, Kang Ho; Kim, Dong Geon; Cho, Hee Jin; Kim, Yeonghwan; Rheey, Jinguen; Shin, Kayoung; Seo, Yun Jee; Choi, Yeon-Sook; Lee, Jung-Il; Lee, Jeongwu; Joo, Kyeung Min; Nam, Do-Hyun

2015-01-01

Glioblastoma (GBM) is the most aggressive and most lethal brain tumor. As current standard therapy consisting of surgery and chemo-irradiation provides limited benefit for GBM patients, novel therapeutic options are urgently required. Forkhead box M1 (FoxM1) transcription factor is an oncogenic regulator that promotes the proliferation, survival, and treatment resistance of various human cancers. The roles of FoxM1 in GBM remain incompletely understood, due in part to pleotropic nature of the FoxM1 pathway. Here, we show the roles of FoxM1 in GBM stem cell maintenance and radioresistance. ShRNA-mediated FoxM1 inhibition significantly impeded clonogenic growth and survival of patient-derived primary GBM cells with marked downregulation of Sox2, a master regulator of stem cell phenotype. Ectopic expression of Sox2 partially rescued FoxM1 inhibition-mediated effects. Conversely, FoxM1 overexpression upregulated Sox2 expression and promoted clonogenic growth of GBM cells. These data, with a direct binding of FoxM1 in the Sox2 promoter region in GBM cells, suggest that FoxM1 regulates stemness of primary GBM cells via Sox2. We also found significant increases in FoxM1 and Sox2 expression in GBM cells after irradiation both in vitro and in vivo orthotopic tumor models. Notably, genetic or a small-molecule FoxM1 inhibitor-mediated FoxM1 targeting significantly sensitized GBM cells to irradiation, accompanying with Sox2 downregulation. Finally, FoxM1 inhibition combined with irradiation in a patient GBM-derived orthotopic model significantly impeded tumor growth and prolonged the survival of tumor bearing mice. Taken together, these results indicate that the FoxM1-Sox2 signaling axis promotes clonogenic growth and radiation resistance of GBM, and suggest that FoxM1 targeting combined with irradiation is a potentially effective therapeutic approach for GBM.

Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

PubMed

Subramaniam, Sindhu; Sreenivas, Prethish; Cheedipudi, Sirisha; Reddy, Vatrapu Rami; Shashidhara, Lingadahalli Subrahmanya; Chilukoti, Ravi Kumar; Mylavarapu, Madhavi; Dhawan, Jyotsna

2014-01-01

Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological states such as cancer and degenerative disease.

Glioma Cell Death Induced by Irradiation or Alkylating Agent Chemotherapy Is Independent of the Intrinsic Ceramide Pathway

PubMed Central

Gramatzki, Dorothee; Herrmann, Caroline; Happold, Caroline; Becker, Katrin Anne; Gulbins, Erich; Weller, Michael; Tabatabai, Ghazaleh

2013-01-01

Background/Aims Resistance to genotoxic therapy is a characteristic feature of glioma cells. Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and glucosylceramide synthase (GCS) catalyzes ceramide metabolism. Increased ceramide levels have been suggested to enhance chemotherapy-induced death of cancer cells. Methods Microarray and clinical data for ASM and GCS in astrocytomas WHO grade II–IV were acquired from the Rembrandt database. Moreover, the glioblastoma database of the Cancer Genome Atlas network (TCGA) was used for survival data of glioblastoma patients. For in vitro studies, increases in ceramide levels were achieved either by ASM overexpression or by the GCS inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) in human glioma cell lines. Combinations of alkylating chemotherapy or irradiation and ASM overexpression, PPMP or exogenous ceramide were applied in parental cells. The anti-glioma effects were investigated by assessing proliferation, metabolic activity, viability and clonogenicity. Finally, viability and clonogenicity were assessed in temozolomide (TMZ)-resistant cells upon treatment with PPMP, exogenous ceramide, alkylating chemotherapy, irradiation or their combinations. Results Interrogations from the Rembrandt and TCGA database showed a better survival of glioblastoma patients with low expression of ASM or GCS. ASM overexpression or PPMP treatment alone led to ceramide accumulation but did not enhance the anti-glioma activity of alkylating chemotherapy or irradiation. PPMP or exogenous ceramide induced acute cytotoxicity in glioblastoma cells. Combined treatments with chemotherapy or irradiation led to additive, but not synergistic effects. Finally, no synergy was found when TMZ-resistant cells were treated with exogenous ceramide or PPMP alone or in combination with TMZ or irradiation. Conclusion Modulation of intrinsic glioma cell ceramide levels by ASM overexpression or GCS inhibition does not enhance the anti-glioma activity of alkylating chemotherapy or irradiation. PMID:23667632

Blood and clonogenic hemopoietic cells of newts after the space flight

NASA Astrophysics Data System (ADS)

Michurina, T. V.; Domaratskaya, E. I.; Nikonova, T. M.; Khrushchov, N. G.

Ribbed newts were used for studying the effect of space flight on board of the biosatellite (Cosmos-2229) on blood and clonogenic hemopoietic cells. In blood of newts of the flight group, the relative proportion of neutrophils increased, whereas that of lymphocytes and eosinophils decreased. Space flight did not result in loss of the ability of newt blood cells to incorporate H^3-thymidine. Analysis of clonogenic hemopoietic cells was performed using the method of hemopoietic colony formation on cellulose acetate membranes implanted into the peritoneal cavity of irradiated newts. To analyze reconstitution of hemopoiesis after irradiation donor hemopoietic cells from flight or control newts were transplanted into irradiated newts whose hemopoietic organs were investigated. The newt can be considered an adequate model for studying hemopoiesis under the conditions of the space flight. Previous studies on rats subjected to 5- to 19-day space flights revealed a decrease in the number of clonogenic cells in their hemopoietic organs accompanied by specific changes in the precursor cell compartment and in blood /1,2/. Hence, it was interesting to analyze blood and hemopoietic tissue of lower vertebrates after a space flight and to compare the response to it of animals belonging to different taxonomic groups. We analyzed blood and clonogenic hemopoietic cells of ribbed newts, Pleurodeles waltl (age one year, weight 20-28 g) subjected to a 12-day space flight on board of a Cosmos-2229 biosatellite. The same animals were used in studies on limb and lens regeneration. The results were compared with those obtained with control groups of newts: (1) basic control, operated newts sacrificed on the day of biosatellite launching (BC); (2) synchronous control, operated newts kept in the laboratory under simulated space flight conditions (SC); and (3) intact newts (IC).

Correlation of Particle Traversals with Clonogenic Survival Using Cell-Fluorescent Ion Track Hybrid Detector.

PubMed

Dokic, Ivana; Niklas, Martin; Zimmermann, Ferdinand; Mairani, Andrea; Seidel, Philipp; Krunic, Damir; Jäkel, Oliver; Debus, Jürgen; Greilich, Steffen; Abdollahi, Amir

2015-01-01

Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD.

EG-03EXPRESSION OF PRMT5 CORRELATES WITH MALIGNANT GRADE IN GLIOMAS AND PLAYS A PIVOTAL ROLE IN TUMOR GROWTH

PubMed Central

Han, Xiaosi; Li, Rong; Zhang, Wenbin; Yang, Xiuhua; Fathallah-Shaykh, Hassan; Gillespie, Yancey; Nabors, Burt

2014-01-01

Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of ω-NG,N′G-symmetric dimethylarginine residues on histones as well as other proteins. The modification play an important role in cell differentiation and tumor cell growth. However, the role of PRMT5 in human glioma cells has not been characterized. In this study, we assessed protein expression profiles of PRMT5 in control brain, WHO grade II astrocytomas, anaplastic astrocytomas, and glioblastoma multiforme (GBM) by immunohistochemistry. PRMT5 was low in glial cells in control brain tissues and low grade astrocytomas. Its expression increased in parallel with malignant progression, and was highly expressed in GBM. Knockdown of PRMT5 by small hairpin RNA caused alterations of p-ERK1/2 and significantly repressed the clonogenic potential and viability of glioma cells. These findings indicate that PRMT5 is a marker of malignant progression in glioma tumors and plays a pivotal role in tumor growth.

RAD-ADAPT: Software for modelling clonogenic assay data in radiation biology.

PubMed

Zhang, Yaping; Hu, Kaiqiang; Beumer, Jan H; Bakkenist, Christopher J; D'Argenio, David Z

2017-04-01

We present a comprehensive software program, RAD-ADAPT, for the quantitative analysis of clonogenic assays in radiation biology. Two commonly used models for clonogenic assay analysis, the linear-quadratic model and single-hit multi-target model, are included in the software. RAD-ADAPT uses maximum likelihood estimation method to obtain parameter estimates with the assumption that cell colony count data follow a Poisson distribution. The program has an intuitive interface, generates model prediction plots, tabulates model parameter estimates, and allows automatic statistical comparison of parameters between different groups. The RAD-ADAPT interface is written using the statistical software R and the underlying computations are accomplished by the ADAPT software system for pharmacokinetic/pharmacodynamic systems analysis. The use of RAD-ADAPT is demonstrated using an example that examines the impact of pharmacologic ATM and ATR kinase inhibition on human lung cancer cell line A549 after ionizing radiation. Copyright © 2017 Elsevier B.V. All rights reserved.

USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance

PubMed Central

Lee, Jin-Ku; Chang, Nakho; Yoon, Yeup; Yang, Heekyoung; Cho, Heejin; Kim, Eunhee; Shin, Yongjae; Kang, Wonyoung; Oh, Young Taek; Mun, Gyeong In; Joo, Kyeung Min; Nam, Do-Hyun; Lee, Jeongwu

2016-01-01

Background Clinical benefits from standard therapies against glioblastoma (GBM) are limited in part due to intrinsic radio- and chemoresistance of GBM and inefficient targeting of GBM stem-like cells (GSCs). Novel therapeutic approaches that overcome treatment resistance and diminish stem-like properties of GBM are needed. Methods We determined the expression levels of ubiquitination-specific proteases (USPs) by transcriptome analysis and found that USP1 is highly expressed in GBM. Using the patient GBM-derived primary tumor cells, we inhibited USP1 by shRNA-mediated knockdown or its specific inhibitor pimozide and evaluated the effects on stem cell marker expression, proliferation, and clonogenic growth of tumor cells. Results USP1 was highly expressed in gliomas relative to normal brain tissues and more preferentially in GSC enrichment marker (CD133 or CD15) positive cells. USP1 positively regulated the protein stability of the ID1 and CHEK1, critical regulators of DNA damage response and stem cell maintenance. Targeting USP1 by RNA interference or treatment with a chemical USP1 inhibitor attenuated clonogenic growth and survival of GSCs and enhanced radiosensitivity of GBM cells. Finally, USP1 inhibition alone or in combination with radiation significantly prolonged the survival of tumor-bearing mice. Conclusion USP1-mediated protein stabilization promotes GSC maintenance and treatment resistance, thereby providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM. PMID:26032834

Identification of a role for the nuclear receptor EAR-2 in the maintenance of clonogenic status within the leukemia cell hierarchy

PubMed Central

Ichim, CV; Atkins, HL; Iscove, NN; Wells, RA

2016-01-01

Identification of genes that regulate clonogenicity of acute myelogenous leukemia (AML) cells is hindered by the difficulty of isolating pure populations of cells with defined proliferative abilities. By analyzing the growth of clonal siblings in low passage cultures of the cell line OCI/AML4 we resolved this heterogeneous population into strata of distinct clonogenic potential, permitting analysis of the transcriptional signature of single cells with defined proliferative abilities. By microarray analysis we showed that the expression of the orphan nuclear receptor EAR-2 (NR2F6) is greater in leukemia cells with extensive proliferative capacity than in those that have lost proliferative ability. EAR-2 is expressed highly in long-term hematopoietic stem cells, relative to short-term hematopoietic stem and progenitor cells, and is downregulated in AML cells after induction of differentiation. Exogenous expression of EAR-2 increased the growth of U937 cells and prevented the proliferative arrest associated with terminal differentiation, and blocked differentiation of U937 and 32Dcl3 cells. Conversely, silencing of EAR-2 by short-hairpin RNA initiated terminal differentiation of these cell lines. These data identify EAR-2 as an important factor in the regulation of clonogenicity and differentiation, and establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy. PMID:21637284

Effect of ultrasound sonication on clonogenic survival and mitochondria of ovarian cancer cells in the presence of methylene blue.

PubMed

Xiang, Junyan; Leung, Albert Wingnang; Xu, Chuanshan

2014-10-01

This study aimed to investigate the effect of ultrasound sonication in the presence of methylene blue on clonogenic survival and mitochondria of ovarian cancer cells. Human ovarian cancer HO-8910 cells, which were incubated with different concentrations of methylene blue for 1 hour, were exposed to an ultrasonic wave for 5 seconds with intensity of 0.46 W/cm(2). Clonogenic survival of HO-8910 cells after ultrasound sonication was measured by a colony-forming unit assay. Mitochondrial structural changes were observed on transmission electron microscopy, and the mitochondrial membrane potential was evaluated by confocal laser-scanning microscopy with rhodamine 123 staining. The colony-forming units of HO-8910 cells decreased considerably after ultrasound sonication in the presence of methylene blue. Transmission electron microscopy showed slightly enlarged mitochondria in the ultrasound-treated cells in the absence of methylene blue; however, seriously damaged mitochondria, even with almost complete disappearance of cristae, were found in the cells treated by ultrasound sonication in the presence of methylene blue. The mitochondrial membrane potential collapsed significantly when HO-8910 cells were treated by ultrasound sonication in the presence of methylene blue (P < .05). Ultrasound sonication in the presence of methylene blue markedly damaged mitochondrial structure and function and decreased clonogenic survival of HO-8910 cells. © 2014 by the American Institute of Ultrasound in Medicine.

1,25D3 potentiates cisplatin antitumor activity by p73 induction in a squamous cell carcinoma model

PubMed Central

Ma, Yingyu; Yu, Wei-Dong; Hershberger, Pamela A.; Flynn, Geraldine; Kong, Rui-Xian; Trump, Donald L.; Johnson, Candace S.

2008-01-01

1,25D3 exhibits anti-tumor activity in a variety of cancers including squamous cell carcinoma (SCC). Intrinsic resistance of SCC cells to cisplatin was observed and led to the investigation into whether 1,25D3 sensitizes SCC cells to cisplatin. Pretreatment with 1,25D3 followed by cisplatin enhanced growth inhibition in SCC cells compared with 1,25D3 alone, as assessed by cytotoxicity and in vitro clonogenic assays. In addition, 1,25D3 sensitized SCC cells to cisplatin-mediated apoptosis. Treatment of tumor-bearing C3H mice with 1,25D3 prior to cisplatin reduced clonogenic survival using in vivo excision clonogenic assay. These results were not observed in a 1,25D3-resistant SCC variant, indicating the critical role of 1,25D3 in sensitizing SCC cells to cisplatin. Further, a marked decrease in fractional tumor volume was observed when SCC tumor-bearing mice were treated with 1,25D3 prior to cisplatin as compared to either agent administered alone. Cisplatin has been shown to modulate p73 protein level in certain cancer cells. Our data showed that p73 level was not affected by cisplatin, but increased by 1,25D3 in SCC cells. Knocking down p73 by siRNA protected SCC cells against 1,25D3 and cisplatin-mediated clonogenic cell kill and apoptosis. Increasing p73 protein level by knocking down UFD2a, which mediates p73 degradation, promoted 1,25D3 and cisplatin-mediated clonogenic cell kill. These results suggest that 1,25D3 potentiates cisplatin anti-tumor activity in vitro and in vivo in a SCC model system, possibly through p73 induction and apoptosis. The combination treatment may provide a more effective therapeutic regimen in cancer treatment. PMID:18790784

/sup 125/I interstitial implants in the RIF-1 murine flank tumor: an animal model for brachytherapy

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

Bernstein, M.; Gutin, P.H.; Weaver, D.A.

1982-09-01

The development of a model for interstitial brachytherapy that uses high-activity, removable /sup 125/I sources in the RIF-1 murine flank tumor is reported. Experimental end points are clonogenic cell and tumor regrowth delay assays. For the clonogenic cell assay, interestitial radiation is delivered at total doses of 500-10,000 rad at dose rates of 0.9-2.7 rad/min to cells in annuli of tissue in the tumor. Dose-survival curves are characterized by an initial shoulder followed by a straight (exponential) portion, with D/sub 0/ similar to that of the curve obtained by external irradiation of the RIF-1 tumor in a self-contained cesium irradiatormore » at similar dose rates. Tumor regrowth curves have been obtained for minimum tumor doses of 500-5000 rad; marked tumor regression has been observed with minimum tumor doses as low as 2000 rad, but results are not as reproducible as the results obtained with the clonogenic cell assay.« less

Human Bone Marrow-Derived Mesenchymal Stem Cells Display Enhanced Clonogenicity but Impaired Differentiation With Hypoxic Preconditioning

PubMed Central

Boyette, Lisa B.; Creasey, Olivia A.; Guzik, Lynda; Lozito, Thomas

2014-01-01

Stem cells are promising candidate cells for regenerative applications because they possess high proliferative capacity and the potential to differentiate into other cell types. Mesenchymal stem cells (MSCs) are easily sourced but do not retain their proliferative and multilineage differentiative capabilities after prolonged ex vivo propagation. We investigated the use of hypoxia as a preconditioning agent and in differentiating cultures to enhance MSC function. Culture in 5% ambient O2 consistently enhanced clonogenic potential of primary MSCs from all donors tested. We determined that enhanced clonogenicity was attributable to increased proliferation, increased vascular endothelial growth factor secretion, and increased matrix turnover. Hypoxia did not impact the incidence of cell death. Application of hypoxia to osteogenic cultures resulted in enhanced total mineral deposition, although this effect was detected only in MSCs preconditioned in normoxic conditions. Osteogenesis-associated genes were upregulated in hypoxia, and alkaline phosphatase activity was enhanced. Adipogenic differentiation was inhibited by exposure to hypoxia during differentiation. Chondrogenesis in three-dimensional pellet cultures was inhibited by preconditioning with hypoxia. However, in cultures expanded under normoxia, hypoxia applied during subsequent pellet culture enhanced chondrogenesis. Whereas hypoxic preconditioning appears to be an excellent way to expand a highly clonogenic progenitor pool, our findings suggest that it may blunt the differentiation potential of MSCs, compromising their utility for regenerative tissue engineering. Exposure to hypoxia during differentiation (post-normoxic expansion), however, appears to result in a greater quantity of functional osteoblasts and chondrocytes and ultimately a larger quantity of high-quality differentiated tissue. PMID:24436440

The Delayed Effects of Acute Radiation Syndrome: Evidence of Long-Term Functional Changes in the Clonogenic Cells of the Small Intestine.

PubMed

Booth, Catherine; Tudor, Gregory L; Katz, Barry P; MacVittie, Thomas J

2015-11-01

Long term or residual damage post-irradiation has been described for many tissues. In hematopoietic stem cells (HSC), this is only revealed when the HSC are stressed and required to regenerate and repopulate a myeloablated host. Such an assay cannot be used to assess the recovery potential of previously irradiated intestinal stem cells (ISC) due to their incompatibility with transplantation. The best approximation to the HSC assay is the crypt microcolony assay, also based on clonogen survival. In the current study, the regenerative capacity of intestinal clonogenic cells in mice that had survived 13 Gy irradiation (with 5% bone marrow shielding to allow survival through the hematopoietic syndrome) and were then aged for 200 d was compared to previously unirradiated age-matched controls. Interestingly, at 200 d following 13 Gy, there remained a statistically significant reduction in crypts present in the various small intestinal regions (illustrating that the gastrointestinal epithelium had not fully recovered despite the 200-d interval). However, upon re-irradiation on day 196, those mice previously irradiated had improved crypt survival and regeneration compared to the age-matched controls. This was evident in all regions of the small intestine following 11-13 Gy re-exposure. Thus, there were either more clonogens per crypt within those previously irradiated and/or those that were present were more radioresistant (possibly because a subpopulation was more quiescent). This is contrary to the popular belief that previously irradiated animals may have an impaired/delayed regenerative response and be more radiosensitive.

Mutations in CIC and IDH1 cooperatively regulate 2-hydroxyglutarate levels and cell clonogenicity

PubMed Central

Chittaranjan, Suganthi; Chan, Susanna; Yang, Cindy; Yang, Kevin C.; Chen, Vincent; Moradian, Annie; Firme, Marlo; Song, Jungeun; Go, Nancy E.; Blough, Michael D.; Chan, Jennifer A.; Cairncross, J. Gregory; Gorski, Sharon M.; Morin, Gregg B.; Yip, Stephen; Marra, Marco A.

2014-01-01

The majority of oligodendrogliomas (ODGs) exhibit combined losses of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH1-R132H or IDH2-R172K). Approximately 70% of ODGs with 1p19q co-deletions harbor somatic mutations in the Capicua Transcriptional Repressor (CIC) gene on chromosome 19q13.2. Here we show that endogenous long (CIC-L) and short (CIC-S) CIC proteins are predominantly localized to the nucleus or cytoplasm, respectively. Cytoplasmic CIC-S is found in close proximity to the mitochondria. To study wild type and mutant CIC function and motivated by the paucity of 1p19q co-deleted ODG lines, we created HEK293 and HOG stable cell lines ectopically co-expressing CIC and IDH1. Non-mutant lines displayed increased clonogenicity, but cells co-expressing the mutant IDH1-R132H with either CIC-S-R201W or -R1515H showed reduced clonogenicity in an additive manner, demonstrating cooperative effects in our assays. Expression of mutant CIC-R1515H increased cellular 2-Hydroxyglutarate (2HG) levels compared to wild type CIC in IDH1-R132H background. Levels of phosphorylated ATP-citrate Lyase (ACLY) were lower in cell lines expressing mutant CIC-S proteins compared to cells expressing wild type CIC-S, supporting a cytosolic citrate metabolism-related mechanism of reduced clonogenicity in our in vitro model systems. ACLY or phospho-ACLY were similarly reduced in CIC-mutant 1p19q co-deleted oligodendroglioma patient samples. PMID:25277207

Self-assembling nanoparticles encapsulating zoledronic acid inhibit mesenchymal stromal cells differentiation, migration and secretion of proangiogenic factors and their interactions with prostate cancer cells

PubMed Central

Pivetta, Eliana; Colombatti, Alfonso; Boccellino, Mariarosaria; Amler, Evzen; Normanno, Nicola; Caraglia, Michele; De Rosa, Giuseppe; Aldinucci, Donatella

2017-01-01

Zoledronic Acid (ZA) rapidly concentrates into the bone and reduces skeletal-related events and pain in bone metastatic prostate cancer (PCa), but exerts only a limited or absent impact as anti-cancer activity. Recently, we developed self-assembling nanoparticles (NPS) encapsulating zoledronic acid (NZ) that allowed a higher intratumor delivery of the drug compared with free zoledronic acid (ZA) in in vivo cancer models of PCa. Increasing evidence suggests that Bone Marrow (BM) Mesenchymal stromal cells (BM-MSCs) are recruited into the stroma of developing tumors where they contribute to progression by enhancing tumor growth and metastasis. We demonstrated that treatment with NZ decreased migration and differentiation into adipocytes and osteoblasts of MSCs and inhibited osteoclastogenesis. Treatment with NZ reduced the capability of MSCs to promote the migration and the clonogenic growth of the prostate cancer cell lines PC3 and DU145. The levels of Interleukin-6 and of the pro-angiogenic factors VEGF and FGF-2 were significantly reduced in MSC-CM derived from MSCs treated with NZ, and CCL5 secretion was almost totally abolished. Moreover, treatment of MSCs with supernatants from PC3 cells, leading to tumor-educated MSCs (TE-MSCs), increased the secretion of IL-6, CCL5, VEGF and FGF-2 by MSCs and increased their capability to increase PC3 cells clonogenic growth. Treatment with NZ decreased cytokine secretion and the pro-tumorigenic effects also of TE-MSCS. In conclusion, demonstrating that NZ is capable to inhibit the cross talk between MSCs and PCa, this study provides a novel insight to explain the powerful anticancer activity of NZ on PCa. PMID:28477013

The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44.

PubMed

Liu, Can; Kelnar, Kevin; Liu, Bigang; Chen, Xin; Calhoun-Davis, Tammy; Li, Hangwen; Patrawala, Lubna; Yan, Hong; Jeter, Collene; Honorio, Sofia; Wiggins, Jason F; Bader, Andreas G; Fagin, Randy; Brown, David; Tang, Dean G

2011-02-01

Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has been implicated in tumorigenesis. CSCs in many tumors--including cancers of the breast, pancreas, head and neck, colon, small intestine, liver, stomach, bladder and ovary--have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic capacities are enriched in the CD44(+) cell population, but whether miRNAs regulate CD44(+) prostate cancer cells and prostate cancer metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target, was underexpressed in CD44(+) prostate cancer cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk or purified CD44(+) prostate cancer cells inhibited clonogenic expansion, tumor regeneration, and metastasis. In contrast, expression of miR-34a antagomirs in CD44(-) prostate cancer cells promoted tumor development and metastasis. Systemically delivered miR-34a inhibited prostate cancer metastasis and extended survival of tumor-bearing mice. We identified and validated CD44 as a direct and functional target of miR-34a and found that CD44 knockdown phenocopied miR-34a overexpression in inhibiting prostate cancer regeneration and metastasis. Our study shows that miR-34a is a key negative regulator of CD44(+) prostate cancer cells and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against prostate CSCs.

Low- and high-LET radiation drives clonal expansion of lung progenitor cells in vivo

PubMed Central

Farin, Alicia M.; Manzo, Nicholas D.; Kirsch, David G.; Stripp, Barry R.

2015-01-01

Abundant populations of epithelial progenitor cells maintain the epithelium along the proximal-to-distal axis of the airway. Exposure of lung tissue to ionizing radiation leads to tissue remodeling and potential cancer initiation or progression. However, little is known about the effects of ionizing radiation on airway epithelial progenitor cells. We hypothesized that ionizing radiation exposure will alter the behavior of airway epithelial progenitor cells in a radiation dose- and quality-dependent manner. To address this hypothesis, we cultured primary airway epithelial cells isolated from mice exposed to various doses of 320 kVp X-ray or 600 MeV/nucleon 56Fe ions in a 3D epithelial-fibroblast co-culture system. Colony-forming efficiency of the airway epithelial progenitor cells was assessed at culture day 14. In vivo clonogenic and proliferative potentials of airway epithelial progenitor cells were measured after exposure to ionizing radiation by lineage tracing and IdU incorporation. Exposure to both X-rays and 56Fe resulted in a dose dependent decrease in the ability of epithelial progenitors to form colonies in vitro. In vivo evidence for increased clonogenic expansion of epithelial progenitors was observed after exposure to both X-rays and 56Fe. Interestingly, we found no significant increase in the epithelial proliferative index, indicating that ionizing radiation does not promote increased turnover of the airway epithelium. Therefore, we propose a model in which radiation induces a dose-dependent decrease in the pool of available progenitor cells, leaving fewer progenitors able to maintain the airway long-term. This work provides novel insights into the effects of ionizing radiation exposure on airway epithelial progenitor cell behavior. PMID:25564721

Silibinin inhibits hypoxia-induced HIF-1α-mediated signaling, angiogenesis and lipogenesis in prostate cancer cells: In vitro evidence and in vivo functional imaging and metabolomics

PubMed Central

Deep, Gagan; Kumar, Rahul; Nambiar, Dhanya K.; Jain, Anil K.; Ramteke, Anand M.; Serkova, Natalie J.; Agarwal, Chapla; Agarwal, Rajesh

2017-01-01

Hypoxia is associated with aggressive phenotype and poor prognosis in prostate cancer (PCa) patients suggesting that PCa growth and progression could be controlled via targeting hypoxia-induced signaling and biological effects. Here, we analyzed silibinin (a natural flavonoid) efficacy to target cell growth, angiogenesis and metabolic changes in human PCa, LNCaP and 22Rv1 cells under hypoxic condition. Silibinin treatment inhibited the proliferation, clonogenicity and endothelial cells tube formation by hypoxic (1% O2) PCa cells. Interestingly, hypoxia promoted a lipogenic phenotype in PCa cells via activating acetyl-Co A carboxylase (ACC) and fatty acid synthase (FASN) that was inhibited by silibinin treatment. Importantly, silibinin treatment strongly decreased hypoxia-induced HIF-1α expression in PCa cells together with a strong reduction in hypoxia-induced NADPH oxidase (NOX) activity. HIF-1α overexpression in LNCaP cells significantly increased the lipid accumulation and NOX activity; however, silibinin treatment reduced HIF-1α expression, lipid levels, clonogenicity and NOX activity even in HIF-1α overexpressing LNCaP cells. In vivo, silibinin feeding (200 mg/kg body weight) to male nude mice with 22Rv1 tumors, specifically inhibited tumor vascularity (measured by dynamic contrast-enhanced MRI) resulting in tumor growth inhibition without directly inducing necrosis (as revealed by diffusion-weighted MRI). Silibinin feeding did not significantly affect tumor glucose uptake measured by FDG-PET; however, reduced the lipid synthesis measured by quantitative 1H-NMR metabolomics. IHC analyses of tumor tissues confirmed that silibinin feeding decreased proliferation and angiogenesis as well as reduced HIF-1α, FASN and ACC levels. Together, these findings further support silibinin usefulness against PCa through inhibiting hypoxia-induced signaling. PMID:27533043

Synergistic Tumor-Killing Effect of Radiation and Berberine Combined Treatment in Lung Cancer: The Contribution of Autophagic Cell Death

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

Peng Peiling; Division of Gastroenterology, Armed Forces Taichung Hospital, Taichung, Taiwan; Kuo, W.-H.

2008-02-01

Purpose: Radiotherapy is the most efficacious strategies for lung cancer. The radiation-enhancing effects and the underlying mechanisms of berberine were investigated both in vitro and in vivo. Methods and Materials: Clonogenic survival assays were used to evaluate the radio-sensitivity of berberine on non-small-cell lung cancer. Electron microscopic observation of the features of cell death, flow cytometry of acidic vascular organelles formation, mitochondria membrane potential and cell-cycle progression, and Western blotting of caspase 3, PARP, and LC3 were performed to identify the mechanisms underlying the enhancing effects. Lewis lung carcinoma model in mice was conducted to evaluate the possible application ofmore » berberine in synergistic treatment with irradiation. Results: Compared with radiation alone (SF2 = 0.423; D{sub 0} = 5.29 Gy), berberine at 5 and 10 {mu}M concentrations in combination with radiation showed significant enhancement on radiation-induced clonogenic inhibition (SF2 = 0.215: D{sub 0} = 2.70 Gy and SF2 = 0.099: D{sub 0} = 1.24 Gy) on A549 cells. The cellular ultrastructure showed the presence of autophagosome and an increased proportion of acridine orange stain-positive cells, demonstrating that berberine enhanced radiosensitivity via autophagy. The process involved LC3 modification and mitochondrial disruption. The animal model verified the synergistic cytotoxic effect of berberine and irradiation resulting in a substantial shrinkage of tumor volume. Conclusion: Supplement of berberine enhanced the cytotoxicity of radiation in both in vivo and in vitro models of lung cancer. The mechanisms underlying this synergistic effect involved the induction of autophagy. It suggests that berberine could be used as adjuvant therapy to treat lung cancer.« less

A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses

PubMed Central

Stevenson, Katrina; Gilmour, Lesley; Hamilton, Graham; Chalmers, Anthony J

2017-01-01

Abstract Background. Glioblastoma (GBM) is the most common primary brain tumor, with dismal prognosis. The failure of drug–radiation combinations with promising preclinical data to translate into effective clinical treatments may relate to the use of simplified 2-dimensional in vitro GBM cultures. Methods. We developed a customized 3D GBM culture system based on a polystyrene scaffold (Alvetex) that recapitulates key histological features of GBM and compared it with conventional 2D cultures with respect to their response to radiation and to molecular targeted agents for which clinical data are available. Results. In 3 patient-derived GBM lines, no difference in radiation sensitivity was observed between 2D and 3D cultures, as measured by clonogenic survival. Three different molecular targeted agents, for which robust clinical data are available were evaluated in 2D and 3D conditions: (i) temozolomide, which improves overall survival and is standard of care for GBM, exhibited statistically significant effects on clonogenic survival in both patient-derived cell lines when evaluated in the 3D model compared with only one cell line in 2D cells; (ii) bevacizumab, which has been shown to increase progression-free survival when added to standard chemoradiation in phase III clinical trials, exhibited marked radiosensitizing activity in our 3D model but had no effect on 2D cells; and (iii) erlotinib, which had no efficacy in clinical trials, displayed no activity in our 3D GBM model, but radiosensitized 2D cells. Conclusions. Our 3D model reliably predicted clinical efficacy, strongly supporting its clinical relevance and potential value in preclinical evaluation of drug–radiation combinations for GBM. PMID:27576873

Kinetics of depopulation, repopulation and host cell infiltration in the rhabdomyosarcoma R1H after 14 MeV neutron irradiation.

PubMed

Brammer, I; Zywietz, F; Beck-Bornholdt, H P; Jung, H

1992-05-01

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H in the rat was studied following irradiation with 5 Gy of 14 MeV neutrons. Several parameters were sequentially measured over a time period of 4 weeks after irradiation: the tumour volume was assessed by in situ caliper measurements; the numerical density of tumour cells was obtained by morphometry; the clonogenic fraction of tumour cells was derived from in vitro colony assay; and the numerical ratio of host to tumour cells was determined by flow cytometry. From these primary parameters the number of clonogenic tumour cells, non-clonogenic tumour cells, and nucleated host cells per tumour, as well as their variation with time, were derived. The results were compared with two sets of data obtained previously for the same tumour exposed to 15 Gy of 200 kVp X-rays. Survival of tumour cells was reduced to 5.5 +/- 0.5% by 5 Gy neutrons and to 4.5 +/- 0.5% by 15 Gy X-rays, i.e. an RBE of close to 3. There was a lag period before the onset of repopulation (4.9 +/- 0.4 days and 4.9 +/- 0.5 days, respectively), followed by a high initial rate of repopulation corresponding to a doubling time of 2.0 +/- 0.2 days for neutrons and 2.1 +/- 0.2 days for X-rays. The rate of depopulation was significantly different for the two treatment modalities; the halving time for the number of non-clonogenic tumour cells was 11 +/- 4 days for neutrons and 2.8 +/- 0.5 days for X-rays.(ABSTRACT TRUNCATED AT 250 WORDS)

USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance.

PubMed

Lee, Jin-Ku; Chang, Nakho; Yoon, Yeup; Yang, Heekyoung; Cho, Heejin; Kim, Eunhee; Shin, Yongjae; Kang, Wonyoung; Oh, Young Taek; Mun, Gyeong In; Joo, Kyeung Min; Nam, Do-Hyun; Lee, Jeongwu

2016-01-01

Clinical benefits from standard therapies against glioblastoma (GBM) are limited in part due to intrinsic radio- and chemoresistance of GBM and inefficient targeting of GBM stem-like cells (GSCs). Novel therapeutic approaches that overcome treatment resistance and diminish stem-like properties of GBM are needed. We determined the expression levels of ubiquitination-specific proteases (USPs) by transcriptome analysis and found that USP1 is highly expressed in GBM. Using the patient GBM-derived primary tumor cells, we inhibited USP1 by shRNA-mediated knockdown or its specific inhibitor pimozide and evaluated the effects on stem cell marker expression, proliferation, and clonogenic growth of tumor cells. USP1 was highly expressed in gliomas relative to normal brain tissues and more preferentially in GSC enrichment marker (CD133 or CD15) positive cells. USP1 positively regulated the protein stability of the ID1 and CHEK1, critical regulators of DNA damage response and stem cell maintenance. Targeting USP1 by RNA interference or treatment with a chemical USP1 inhibitor attenuated clonogenic growth and survival of GSCs and enhanced radiosensitivity of GBM cells. Finally, USP1 inhibition alone or in combination with radiation significantly prolonged the survival of tumor-bearing mice. USP1-mediated protein stabilization promotes GSC maintenance and treatment resistance, thereby providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The effect of short G-CSF administration on the numbers and clonogenic efficiency of hematopoietic progenitor cells in bone marrow and peripheral blood of normal donors.

PubMed

Zaucha, J M; Knopińska-Posłuszny, W; Bieniaszewska, M; Myśliwski, A; Hellmann, A

2000-01-01

We have analysed the cellularity, the number of clonogenic cells and their clonogenic efficiency (the number of clonogenic cells/2 x 10(5) MNC) in peripheral blood (PB) and bone marrow (BM) during and after filgrastim (rhG-CSF) mobilization of CD34+ cells in 12 healthy donors for allogeneic stem cell donation. G-CSF was administrated subcutaneously for 5 consecutive days at a dose of 10 micrograms/kg/day. WBC, MNC, CD34+ cell counts, CFU-GM and BFU-E assays in PB were performed at baseline and then daily 12 hours after each G-CSF dose. BM was assayed before start (day 1) and after the last dose (day 6) of G-CSF. Results are given as medians, with ranges in parentheses. In PB the total WBC and MNC increased 7.4-fold (6.0-12.3) and 3.3-fold (1.5-9.4), respectively, reaching a peak of 49.4 x 10(9)/l (32.5-66.6) on day 6 for WBC and 6.28 x 10(9)/l (4.7-13.3) for MNC on day 5. CD34+ cell number reached a peak value of 48.0 x 10(6)/l (45.6-285) on day 6 whereas CFU-GM and BFU-E reached their peaks on day 5, 0.95 x 10(4)/ml (0.05-6.08) and 1.04 x 10(4)/ml, respectively. CFU-MIX, not detectable at baseline, reached a peak of 0.95 x 10(4)/ml (0.006-0.51) on day 5 as well. This was accompanied by an increase in CFU-GM, BFU-E and CFU-MIX clonogenic efficiency: 23-fold (3-150), 9.75-fold (2.2-27.8) and 20-fold (2.5-210), respectively. In BM the total WBC number increased 2.5-fold (1.3-4.9) from the baseline value of 52.6 x 10(9)/l (7.9-137.0) whereas the MNC count increased 2.0-fold (0.81-3.7) from a baseline of 13.6 x 10(9)/l (3.5-54.8). This was, however, not significant. The number of CD34+ cells increased significantly 2.9-fold (0.8-8.3). In 8 donors CFU-MIX were detectable before but not after G-CSF treatment. A similar decrease in CFU-GM and BFU-E clonogenic efficiency occurred but was not significant. CFU-GM and BFU-E numbers did not change. We conclude that the total body numbers of lineage committed progenitors increased during G-CSF administration, which indicate their proliferation in addition to mobilization. The effect of G-CSF on the number of more primitive progenitors in BM is less clear and needs further investigation.

Thyroid hormone receptor interactor 13 (TRIP13) overexpression associated with tumor progression and poor prognosis in lung adenocarcinoma.

PubMed

Li, Wei; Zhang, Gengyan; Li, Xiaojun; Wang, Xiaojing; Li, Qing; Hong, Lei; Shen, Yuangbing; Zhao, Chenling; Gong, Xiaomeng; Chen, Yuqing; Zhou, Jihong

2018-05-15

Thyroid hormone receptor interactor 13 (TRIP13) is an AAA + -ATPase that plays a key role in mitotic checkpoint complex inactivation and is associated with the progression of several cancers. However, its role in lung adenocarcinogenesis remains unknown. Here, we report that TRIP13 is highly overexpressed in multiple lung adenocarcinoma cell lines and tumor tissues. Clinically, TRIP13 expression is positively associated with tumor size, T-stage, and N-stage, and Kaplan-Meier analysis revealed that heightened TRIP13 expression is associated with lower overall survival. TRIP13 promotes lung adenocarcinoma cell proliferation, clonogenicity, and migration while inhibiting apoptosis and G2/M phase shift in vitro. Accordingly, TRIP13-silenced xenograft tumors displayed significant growth inhibition in vivo. Bioinformatics analysis demonstrated that TRIP13 interacts with a protein network associated with dsDNA break repair and PI3K/Akt signaling. TRIP13 upregulatesAkt Ser473 and downregulatesAkt Thr308 /mTOR Ser2448 activity, which suppresses accurate dsDNA break repair. TRIP13 also downregulates pro-apoptotic Bad Ser136 and cleaved caspase-3 while upregulating survivin. In conclusion, heightened TRIP13 expression appears to promote lung adenocarcinoma tumor progression and displays potential as a therapeutic target or biomarker for lung adenocarcinoma. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification of Novel Targets for Lung Cancer Therapy Using an Induced Pluripotent Stem Cell Model.

PubMed

Shukla, Vivek; Rao, Mahadev; Zhang, Hongen; Beers, Jeanette; Wangsa, Darawalee; Wangsa, Danny; Buishand, Floryne O; Wang, Yonghong; Yu, Zhiya; Stevenson, Holly; Reardon, Emily; McLoughlin, Kaitlin C; Kaufman, Andrew; Payabyab, Eden; Hong, Julie A; Zhang, Mary; Davis, Sean R; Edelman, Daniel C; Chen, Guokai; Miettinen, Markku; Restifo, Nicholas; Ried, Thomas; Meltzer, Paul S; Schrump, David S

2018-04-01

Despite extensive studies, the genetic and epigenetic mechanisms that mediate initiation and progression of lung cancers have not been fully elucidated. Previously, we have demonstrated that via complementary mechanisms, including DNA methylation, polycomb repressive complexes, and noncoding RNAs, cigarette smoke induces stem-like phenotypes that coincide with progression to malignancy in normal respiratory epithelia as well as enhanced growth and metastatic potential of lung cancer cells. To further investigate epigenetic mechanisms contributing to stemness/pluripotency in lung cancers and potentially identify novel therapeutic targets in these malignancies, induced pluripotent stem cells were generated from normal human small airway epithelial cells. Lung induced pluripotent stem cells were generated by lentiviral transduction of small airway epithelial cells of OSKM (Yamanaka) factors (octamer-binding transcription factor 4 [Oct4], sex-determining region Y box 2 [SOX2], Kruppel-like factor 4 [KLF4], and MYC proto-oncogene, bHLH transcription factor [MYC]). Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation sequencing analysis were performed. The lung induced pluripotent stem cells exhibited hallmarks of pluripotency, including morphology, surface antigen and stem cell gene expression, in vitro proliferation, and teratoma formation. In addition, lung induced pluripotent stem cells exhibited no chromosomal aberrations, complete silencing of reprogramming transgenes, genomic hypermethylation, upregulation of genes encoding components of polycomb repressive complex 2, hypermethylation of stem cell polycomb targets, and modulation of more than 15,000 other genes relative to parental small airway epithelial cells. Additional sex combs like-3 (ASXL3), encoding a polycomb repressive complex 2-associated protein not previously described in reprogrammed cells, was markedly upregulated in lung induced pluripotent stem cell as well as human small cell lung cancer lines and specimens. Overexpression of the additional sex combs like-3 gene correlated with increased genomic copy number in small cell lung cancer lines. Knock-down of the additional sex combs like-3 gene inhibited proliferation, clonogenicity, and teratoma formation by lung induced pluripotent stem cells and significantly diminished in vitro clonogenicity and growth of small cell lung cancer cells in vivo. Collectively, these studies highlight the potential utility of this lung induced pluripotent stem cell model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and suggest that additional sex combs like-3 is a novel target for small cell lung cancer therapy.

[Effect of cryopreservation on umbilical blood cells and its mechanism].

PubMed

Li, Xin; Chen, Fangping; Jiang, Tiebin; Wang, Erhua; Liu, Jing

2013-07-01

To evaluate the effect of cryopreservation on clonogenic ability and apoptosis rate of mono-nuclear cells and CD34+ cells in umbilical blood (UB), and to choose the index to present the freezing injury and optimize the cryopreservation of UB. The mono-nuclear cells (MNC) and CD34+ cells were separated from UB and frozen.After 30 days, they were thawed in warm water. Clonogenic capacity and clonogenic recovery before and after the cryopreservation was compared. We also used Annexin V-FITC-PI to investigate the apoptosis rate of the cells before and after the cryopreservation of these 2 types of cells. The number of colony forming unit-granulocyte/monocyte (CFU-GMs) was not changed after freezing and thawing in both MNCs and CD34+ cells, while the number of colony forming unit-granulocyte, erythrocyte, monocyte and megakaryocyte (CFU-GEMM) was obviously reduced after freezing in CD34+ cells. The 2 types of cryopreserved cells had certain degree of apoptosis before the cryopreservation. MNC-type cryopreservation increased the cells apoptosis a little, while CD34+-type cryopreservation increased more. The cells have certain degree of apoptosis before the cryopreservation. The freezing and thawing procedure does affect the early stage progenitor cells-CFU-GEMM in the CD34+- type cryopreserved cells in UB. The damage may be induced by the cell apoptosis.

MicroRNA-451 regulates stemness of side population cells via PI3K/Akt/mTOR signaling pathway in multiple myeloma.

PubMed

Du, Juan; Liu, Shuyan; He, Jie; Liu, Xi; Qu, Ying; Yan, Wenqing; Fan, Jianling; Li, Rong; Xi, Hao; Fu, Weijun; Zhang, Chunyang; Yang, Jing; Hou, Jian

2015-06-20

Side population (SP) cells are an enriched source of cancer-initiating cells with stemness characteristics, generated by increased ABC transporter activity, which has served as a unique hallmark for multiple myeloma (MM) stem cell studies. Here we isolated and identified MM SP cells via Hoechst 33342 staining. Furthermore, we demonstrate that SP cells possess abnormal cell cycle, clonogenicity, and high drug efflux characteristics-all of which are features commonly seen in stem cells. Interestingly, we found that bortezomib, As2O3, and melphalan all affected apoptosis and clonogenicity in SP cells. We followed by characterizing the miRNA signature of MM SP cells and validated the specific miR-451 target tuberous sclerosis 1 (TSC1) gene to reveal that it activates the PI3K/Akt/mTOR signaling in MM SP cells. Inhibition of miR-451 enhanced anti-myeloma novel agents' effectiveness, through increasing cells apoptosis, decreasing clonogenicity, and reducing MDR1 mRNA expression. Moreover, the novel specific PI3K/Akt/mTOR signaling inhibitor S14161 displayed its prowess as a potential therapeutic agent by targeting MM SP cells. Our findings offer insights into the mechanisms regulating MM SP cells and provide a novel strategy to overcome resistance to existing therapies against myeloma.

The release of bystander factor(s) from tissue explant cultures of rainbow trout (Onchorhynchus mykiss) after exposure to gamma radiation.

PubMed

O'Dowd, Colm; Mothersill, Carmel E; Cairns, Michael T; Austin, Brian; McClean, Brendan; Lyng, Fiona M; Murphy, James E J

2006-10-01

The bystander response has been documented in cell lines and cell cultures derived from aquatic species over the past several years. However, little work has been undertaken to identify a similar bystander response in tissue explant cultures from fish. In this study, indirect effects of ionizing gamma radiation on tissue explant cultures of fish were investigated. Tissue explants in culture were exposed to 0.5 Gy and 5 Gy gamma radiation from a 60Co teletherapy unit. A bystander response in Epithelioma papulosum cyprini (EPC) cells exposed to gamma-irradiated tissue conditioned medium from rainbow trout explants was investigated, and the effects on cell survival were quantified by the clonogenic survival assay. Dichlorofluorescein and rhodamine 123 fluorescent dyes were used to identify alterations in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP), respectively. Results indicate a different response for the three tissue types investigated. Clonogenic assay results vary from a decrease in cell survival (gill) to no effect (skin) to a stimulatory effect (spleen). Results from fluorescence assays of ROS and MMP show similarities to clonogenic assay results. This study identifies a useful model for further studies relating to the bystander effect in aquatic organisms in vivo and ex vivo.

Elucidation of the EP defect in Diamond-Blackfan anemia by characterization and prospective isolation of human EPs.

PubMed

Iskander, Deena; Psaila, Bethan; Gerrard, Gareth; Chaidos, Aristeidis; En Foong, Hui; Harrington, Yvonne; Karnik, Leena C; Roberts, Irene; de la Fuente, Josu; Karadimitris, Anastasios

2015-04-16

Diamond-Blackfan anemia (DBA) is a disorder characterized by a selective defect in erythropoiesis. Delineation of the precise defect is hampered by a lack of markers that define cells giving rise to erythroid burst- and erythroid colony-forming unit (BFU-E and CFU-E) colonies, the clonogenic assays that quantify early and late erythroid progenitor (EEP and LEP) potential, respectively. By combining flow cytometry, cell-sorting, and single-cell clonogenic assays, we identified Lin(-)CD34(+)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(-)CD36(-) bone marrow cells as EEP giving rise to BFU-E, and Lin(-)CD34(+/-)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(+)CD36(+) cells as LEP giving rise to CFU-E, in a hierarchical fashion. We then applied these definitions to DBA and identified that, compared with controls, frequency, and clonogenicity of DBA, EEP and LEP are significantly decreased in transfusion-dependent but restored in corticosteroid-responsive patients. Thus, both quantitative and qualitative defects in erythroid progenitor (EP) contribute to defective erythropoiesis in DBA. Prospective isolation of defined EPs will facilitate more incisive study of normal and aberrant erythropoiesis. © 2015 by The American Society of Hematology.

Inhibition of human lung cancer cell proliferation and survival by wine

PubMed Central

2014-01-01

Background Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Wine contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt and extracellular signal-regulated kinase (Erk), and the tumor suppressor p53 are key modulators of cancer cell growth and survival. In this study, we examined the effects of wine on proliferation and survival of human Non-small cell lung cancer (NSCLC) cells and its effects on signaling events. Methods Human NSCLC adenocarcinoma A549 and H1299 cells were used. Cell proliferation was assessed by thymidine incorporation. Clonogenic assays were used to assess cell survival. Immunoblotting was used to examine total and phosphorylated levels of Akt, Erk and p53. Results In A549 cells red wine inhibited cell proliferation and reduced clonogenic survival at doses as low as 0.02%. Red wine significantly reduced basal and EGF-stimulated Akt and Erk phosphorylation while it increased the levels of total and phosphorylated p53 (Ser15). Control experiments indicated that the anti-proliferative effects of wine were not mediated by the associated contents of ethanol or the polyphenol resveratrol and were independent of glucose transport into cancer cells. White wine also inhibited clonogenic survival, albeit at a higher doses (0.5-2%), and reduced Akt phosphorylation. The effects of both red and white wine on Akt phosphorylation were also verified in H1299 cells. Conclusions Red wine inhibits proliferation of lung cancer cells and blocks clonogenic survival at low concentrations. This is associated with inhibition of basal and EGF-stimulated Akt and Erk signals and enhancement of total and phosphorylated levels of p53. White wine mediates similar effects albeit at higher concentrations. Our data suggest that wine may have considerable anti-tumour and chemoprevention properties in lung cancer and deserves further systematic investigation in animal models of lung cancer. PMID:24456610

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells.

PubMed

Jayakumar, Sundarraj; Bhilwade, Hari N; Pandey, Badri N; Sandur, Santosh K; Chaubey, Ramesh C

2012-10-09

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation. © 2012 Elsevier B.V. All rights reserved.

SU-E-T-352: Why Is the Survival Rate Low in Oropharyngeal Squamous Cell Carcinoma?

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

Huang, Z; Feng, Y; Rasmussen, K

2014-06-01

Purpose: Tumors are composed of a large number of clonogens that have the capability of indefinite reproduction. Even when there is complete clinical or radiographic regression of the gross tumor mass after treatment, tumor recurrence can occur if the clonogens are not completely eradicated by radiotherapy. This study was to investigate the colonogen number and its association with the tumor control probability (TCP) in oropharyngeal squamous cell carcinoma (OSCCA). Methods: A literature search was conducted to collect clinical information of patients with OSCCA, including the prescription dose, tumor volume and survival rate. The linear-quadratic (LQ) model was incorporated into TCPmore » model for clinical data analysis. The total dose ranged from 60 to 70 Gy and tumor volume ranged from 10 to 50 cc. The TCP was calculated for each group according to tumor size and dose. The least χ{sup 2} method was used to fit the TCP calculation to clinical data while other LQ model parameters (α, β) were adopted from the literature, due to the limited patient data. Results: A total of 190 patients with T2–T4 OSCCA were included. The association with HPV was not available for all the patients. The 3-year survival rate was about 82% for T2 squamous cell carcinoma and 40% for advanced tumors. Fitting the TCP model to the survival data, the average clonogen number was 1.56×10{sup 12}. For the prescription dose of 70 Gy, the calculated TCP ranged from 40% to 90% when the tumor volume varied from 10 to 50 cc. Conclusion: Our data suggests variation between the clonogen number and TCP in OSCCA. Tumors with larger colonogen number tend to have lower TCP and therefore dose escalation above 70 Gy may be indicated in order to improve the TCP and survival rate. Our result will require future confirmation with a large number of patients.« less

The impact of the IGF-1 system of cancer cells on radiation response - An in vitro study.

PubMed

Venkatachalam, Senthiladipan; Mettler, Esther; Fottner, Christian; Miederer, Matthias; Kaina, Bernd; Weber, Matthias M

2017-12-01

Overexpression of the insulin-like growth factor-1 receptor (IGF-1R) is associated with increased cell proliferation, differentiation, transformation, and tumorigenicity. Additionally, signaling involved in the resistance of cancer cells to radiotherapy originates from IGF-1R. The purpose of this study was to investigate the role of the IGF-1 system in the radiation response and further evaluate its effect on the expression of DNA repair pathway genes. To inhibit the IGF-1 system, we stably transfected the Caco-2 cell line to express a kinase-deficient IGF-1R mutant. We then studied the effects of this mutation on cell growth, the response to radiation, and clonogenic survival, as well as using a cell viability assay to examine DNA damage and repair. Finally, we performed immunofluorescence for γ-H2AX to examine double-strand DNA breaks and evaluated the expression of 84 key genes involved in DNA repair with a real-time PCR array. Mutant IGF-1R cells exhibited significantly blunted cell growth and viability, compared to wild-type cells, as well as reduced clonogenic survival after γ-irradiation. However, mutant IGF-1R cells did not show any significant delays in the repair of radiation-induced DNA double-strand breaks. Furthermore, expression of mutant IGF-1R significantly down-regulated the mRNA levels of BRCA2, a major protein involved in homologous recombination DNA repair. These results indicate that blocking the IGF-1R-mediated signaling cascade, through the expression of a kinase-deficient IGF-1R mutant, reduces cell growth and sensitizes cancer cells to ionizing radiation. Therefore, the IGF-1R system could be a potential target to enhance radio-sensitivity and the efficacy of cancer treatments.

Heat shock protein 90 (HSP90) is overexpressed in p16-negative oropharyngeal squamous cell carcinoma, and its inhibition in vitro potentiates the effects of chemoradiation.

PubMed

Patel, Kirtesh; Wen, Jing; Magliocca, Kelly; Muller, Susan; Liu, Yuan; Chen, Zhuo Georgia; Saba, Nabil; Diaz, Roberto

2014-11-01

Cisplatin and radiation therapy remain the current standard for treating locally advanced SCCHN. Novel treatment approaches are needed, especially in patients with human papilloma virus (HPV)-negative disease who have worse outcomes despite multimodality therapy. Using our institutional review board approved database, we obtained twenty oropharyngeal squamous cell carcinoma (SCC) tissue samples: ten p16 positive, ten p16-negative. Because p16 expression is strongly associated with HPV positivity in oropharyngeal SCC, p16 status was used as a marker of HPV. We subsequently analyzed, via immunohistochemistry, heat shock protein 90 (HSP90) protein levels. Using HPV-positive and HPV-negative SCC cell lines, we compared baseline HSP90 expression levels and the effect of the HSP90 inhibitor ganetespib on viability and apoptosis. Clonogenic survival of HPV-negative cells treated with ganetespib, radiation therapy, and/or cisplatin was then investigated. We characterize the effects of ganetespib on proteins that are thought to drive DNA damage resistance in HPV-negative cells. HSP90 expression was significantly higher in p16-negative compared with p16-positive samples (p = 0.016) and in HPV-negative cell lines compared with positive cells. Ganetespib increased cytotoxicity and induced apoptosis in HPV-negative more than positive cells. Adding ganetespib to cisplatin and/or radiation therapy in HPV-negative cells further decreased clonogenic survival. Finally, ganetespib downregulated expressions of EGFR, ERK, AKT, p53, and HIF-1α. Ganetespib inhibited HPV-negative SCCHN viability and potentiated cell kill when combined with cisplatin or radiation therapy in vitro. With HSP90 expression higher in HPV-negative cells and in p16-negative patients, further exploration of the clinical activity of HSP90 inhibitors in SCCHN is warranted.

Estrogen and progesterone promote breast cancer cell proliferation by inducing cyclin G1 expression.

PubMed

Tian, J-M; Ran, B; Zhang, C-L; Yan, D-M; Li, X-H

2018-01-23

Breast cancer is the most common cause of cancer among women in most countries (WHO). Ovarian hormone disorder is thought to be associated with breast tumorigenesis. The present study investigated the effects of estrogen and progesterone administration on cell proliferation and underlying mechanisms in breast cancer MCF-7 cells. It was found that a single administration of estradiol (E2) or progesterone increased MCF-7 cell viability in a dose-dependent manner and promoted cell cycle progression by increasing the percentage of cells in the G2/M phase. A combination of E2 and progesterone led to a stronger effect than single treatment. Moreover, cyclin G1 was up-regulated by E2 and/or progesterone in MCF-7 cells. After knockdown of cyclin G1 in MCF-7 cells using a specific shRNA, estradiol- and progesterone-mediated cell viability and clonogenic ability were significantly limited. Additionally, estradiol- and progesterone-promoted cell accumulation in the G2/M phase was reversed after knockdown of cyclin G1. These data indicated that estrogen and progesterone promoted breast cancer cell proliferation by inducing the expression of cyclin G1. Our data indicated that novel therapeutics against cyclin G1 are promising for the treatment of estrogen- and progesterone-mediated breast cancer progression.

Characterization of bone marrow mesenchymal stromal cells in aplastic anaemia.

PubMed

Hamzic, Edita; Whiting, Karen; Gordon Smith, Edward; Pettengell, Ruth

2015-06-01

In aplastic anaemia (AA), haemopoietic activity is significantly reduced and generally attributed to failure of haemopoietic stem cells (HSC) within the bone marrow (BM). The regulation of haemopoiesis depends on the interaction between HSC and various cells of the BM microenvironment, including mesenchymal stromal cells (MSC). MSC involvement in the functional restriction of HSC in AA is largely unknown and therefore, the physical and functional properties of AA MSC were studied in vitro. MSC were characterized by their phenotype and ability to form adherent stromal layers. The functional properties of AA MSC were assessed through proliferative, clonogenic and cross-over culture assays. Results indicate that although AA MSC presented typical morphology and distinctive mesenchymal markers, stromal formation was reduced, with 50% of BM samples failing to produce adherent layers. Furthermore, their proliferative and clonogenic capacity was markedly decreased (P = 0·03 and P = 0·04 respectively) and the ability to sustain haemopoiesis was significantly reduced, as assessed by total cell proliferation (P = 0·032 and P = 0·019 at Week 5 and 6, respectively) and clonogenic potential of HSC (P = 0·02 at Week 6). It was concluded that the biological characteristics of AA MSC are different from those of control MSC and their in vitro haemopoiesis-supporting ability is significantly reduced. © 2015 John Wiley & Sons Ltd.

Targeting PYK2 mediates microenvironment-specific cell death in multiple myeloma

PubMed Central

Meads, MB; Fang, B; Mathews, L; Gemmer, J; Nong, L; Rosado-Lopez, I; Nguyen, T; Ring, JE; Matsui, W; MacLeod, AR; Pachter, JA; Hazlehurst, LA; Koomen, JM; Shain, KH

2015-01-01

Multiple myeloma (MM) remains an incurable malignancy due, in part, to the influence of the bone marrow microenvironment on survival and drug response. Identification of microenvironment-specific survival signaling determinants is critical for the rational design of therapy and elimination of MM. Previously, we have shown that collaborative signaling between β1 integrin-mediated adhesion to fibronectin and interleukin-6 confers a more malignant phenotype via amplification of signal transducer and activator of transcription 3 (STAT3) activation. Further characterization of the events modulated under these conditions with quantitative phosphotyrosine profiling identified 193 differentially phosphorylated peptides. Seventy-seven phosphorylations were upregulated upon adhesion, including PYK2/FAK2, Paxillin, CASL and p130CAS consistent with focal adhesion (FA) formation. We hypothesized that the collaborative signaling between β1 integrin and gp130 (IL-6 beta receptor, IL-6 signal transducer) was mediated by FA formation and proline-rich tyrosine kinase 2 (PYK2) activity. Both pharmacological and molecular targeting of PYK2 attenuated the amplification of STAT3 phosphorylation under co-stimulatory conditions. Co-culture of MM cells with patient bone marrow stromal cells (BMSC) showed similar β1 integrin-specific enhancement of PYK2 and STAT3 signaling. Molecular and pharmacological targeting of PYK2 specifically induced cell death and reduced clonogenic growth in BMSC-adherent myeloma cell lines, aldehyde dehydrogenase-positive MM cancer stem cells and patient specimens. Finally, PYK2 inhibition similarly attenuated MM progression in vivo. These data identify a novel PYK2-mediated survival pathway in MM cells and MM cancer stem cells within the context of microenvironmental cues, providing preclinical support for the use of the clinical stage FAK/PYK2 inhibitors for treatment of MM, especially in a minimal residual disease setting. PMID:26387544

Chemopreventive effect of chalcone derivative, L2H17, in colon cancer development.

PubMed

Xu, Shanmei; Chen, Minxiao; Chen, Wenbo; Hui, Junguo; Ji, Jiansong; Hu, Shuping; Zhou, Jianmin; Wang, Yi; Liang, Guang

2015-11-09

Colon cancer is the third most commonly diagnosed cancer and the second leading cause of cancer mortality worldwide. Chalcone and its derivatives are reported to exhibit anti-cancer effects in several cancer cell lines, including colon cancer cells. In addition, chalcones have advantages such as poor interaction with DNA and low risk of mutagenesity. In our previous study, a group of chalcone derivatives were synthesized and exhibited strong anti-inflammatory activities. In this study, we evaluated the anti-cancer effects of the chalcone derivative, L2H17, in colon cancer cells. The cytotoxicities of L2H17 on various colon cancer cell lines were investigated by MTT and clonogenic assay. Cell cycle and apoptosis analysis were performed to evaluate the molecular mechanism of L2H17-mediated inhibition of tumor growth. Also, scratch wound and matrigel invasion experiments were performed to estimate the cell migration and invasion after L2H17 treatment. Finally, we observed the anti-colon cancer effects of L2H17 in vivo. Our data show that compound L2H17 exhibited selective cytotoxic effect on colon cancer cells, via inducing G0/G1 cell cycle arrest and apoptosis in CT26.WT cells. Furthermore, L2H17 treatment decreased cell migration and invasion of CT26.WT cells. In addition, L2H17 possessed marked anti-tumor activity in vivo. The molecular mechanism of L2H17-mediated inhibition of tumor promotion and progression were function through inactivated NF-κB and Akt signaling pathways. All these findings show that L2H17 might be a potential growth inhibitory chalcones derivative for colon cancer cells.

The Formation of Tight Tumor Clusters Affects the Efficacy of Cell Cycle Inhibitors: A Hybrid Model Study

PubMed Central

Kim, MunJu; Reed, Damon; Rejniak, Katarzyna A.

2014-01-01

Cyclin-dependent kinases (CDKs) are vital in regulating cell cycle progression, and, thus, in highly proliferating tumor cells CDK inhibitors are gaining interest as potential anticancer agents. Clonogenic assay experiments are frequently used to determine drug efficacy against the survival and proliferation of cancer cells. While the anticancer mechanisms of drugs are usually described at the intracellular single-cell level, the experimental measurements are sampled from the entire cancer cell population. This approach may lead to discrepancies between the experimental observations and theoretical explanations of anticipated drug mechanisms. To determine how individual cell responses to drugs that inhibit CDKs affect the growth of cancer cell populations, we developed a spatially explicit hybrid agent-based model. In this model, each cell is equipped with internal cell cycle regulation mechanisms, but it is also able to interact physically with its neighbors. We model cell cycle progression, focusing on the G1 and G2/M cell cycle checkpoints, as well as on related essential components, such as CDK1, CDK2, cell size, and DNA damage. We present detailed studies of how the emergent properties (e.g., cluster formation) of an entire cell population depend on altered physical and physiological parameters. We analyze the effects of CDK1 and CKD2 inhibitors on population growth, time-dependent changes in cell cycle distributions, and the dynamic evolution of spatial cell patterns. We show that cell cycle inhibitors that cause cell arrest at different cell cycle phases are not necessarily synergistically super-additive. Finally, we demonstrate that the physical aspects of cell population growth, such as the formation of tight cell clusters versus dispersed colonies, alter the efficacy of cell cycle inhibitors, both in 2D and 3D simulations. This finding may have implications for interpreting the treatment efficacy results of in vitro experiments, in which treatment is applied before the cells can grow to produce clusters, especially because in vivo tumors, in contrast, form large masses before they are detected and treated. PMID:24607745

Silibinin inhibits hypoxia-induced HIF-1α-mediated signaling, angiogenesis and lipogenesis in prostate cancer cells: In vitro evidence and in vivo functional imaging and metabolomics.

PubMed

Deep, Gagan; Kumar, Rahul; Nambiar, Dhanya K; Jain, Anil K; Ramteke, Anand M; Serkova, Natalie J; Agarwal, Chapla; Agarwal, Rajesh

2017-03-01

Hypoxia is associated with aggressive phenotype and poor prognosis in prostate cancer (PCa) patients suggesting that PCa growth and progression could be controlled via targeting hypoxia-induced signaling and biological effects. Here, we analyzed silibinin (a natural flavonoid) efficacy to target cell growth, angiogenesis, and metabolic changes in human PCa, LNCaP, and 22Rv1 cells under hypoxic condition. Silibinin treatment inhibited the proliferation, clonogenicity, and endothelial cells tube formation by hypoxic (1% O 2 ) PCa cells. Interestingly, hypoxia promoted a lipogenic phenotype in PCa cells via activating acetyl-Co A carboxylase (ACC) and fatty acid synthase (FASN) that was inhibited by silibinin treatment. Importantly, silibinin treatment strongly decreased hypoxia-induced HIF-1α expression in PCa cells together with a strong reduction in hypoxia-induced NADPH oxidase (NOX) activity. HIF-1α overexpression in LNCaP cells significantly increased the lipid accumulation and NOX activity; however, silibinin treatment reduced HIF-1α expression, lipid levels, clonogenicity, and NOX activity even in HIF-1α overexpressing LNCaP cells. In vivo, silibinin feeding (200 mg/kg body weight) to male nude mice with 22Rv1 tumors, specifically inhibited tumor vascularity (measured by dynamic contrast-enhanced MRI) resulting in tumor growth inhibition without directly inducing necrosis (as revealed by diffusion-weighted MRI). Silibinin feeding did not significantly affect tumor glucose uptake measured by FDG-PET; however, reduced the lipid synthesis measured by quantitative 1 H-NMR metabolomics. IHC analyses of tumor tissues confirmed that silibinin feeding decreased proliferation and angiogenesis as well as reduced HIF-1α, FASN, and ACC levels. Together, these findings further support silibinin usefulness against PCa through inhibiting hypoxia-induced signaling. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A survival model for fractionated radiotherapy with an application to prostate cancer

NASA Astrophysics Data System (ADS)

Zaider, Marco; Zelefsky, Michael J.; Hanin, Leonid G.; Tsodikov, Alexander D.; Yakovlev, Andrei Y.; Leibel, Steven A.

2001-10-01

This paper explores the applicability of a mechanistic survival model, based on the distribution of clonogens surviving a course of fractionated radiation therapy, to clinical data on patients with prostate cancer. The study was carried out using data on 1100 patients with clinically localized prostate cancer who were treated with three-dimensional conformal radiation therapy. The patients were stratified by radiation dose (group 1: <67.5 Gy; group 2: 67.5-72.5 Gy; group 3: 72.5-77.5 Gy; group 4: 77.5-87.5 Gy) and prognosis category (favourable, intermediate and unfavourable as defined by pre-treatment PSA and Gleason score). A relapse was recorded when tumour recurrence was diagnosed or when three successive prostate specific antigen (PSA) elevations were observed from a post-treatment nadir PSA level. PSA relapse-free survival was used as the primary end point. The model, which is based on an iterated Yule process, is specified in terms of three parameters: the mean number of tumour clonogens that survive the treatment, the mean of the progression time of post-treatment tumour development and its standard deviation. The model parameters were estimated by the maximum likelihood method. The fact that the proposed model provides an excellent description both of the survivor function and of the hazard rate is prima facie evidence of the validity of the model because closeness of the two survivor functions (empirical and model-based) does not generally imply closeness of the corresponding hazard rates. The estimated cure probabilities for the favourable group are 0.80, 0.74 and 0.87 (for dose groups 1-3, respectively); for the intermediate group: 0.25, 0.51, 0.58 and 0.78 (for dose groups 1-4, respectively) and for the unfavourable group: 0.0, 0.27, 0.33 and 0.64 (for dose groups 1-4, respectively). The distribution of progression time to tumour relapse was found to be independent of prognosis group but dependent on dose. As the dose increases the mean progression time decreases (41, 28.5, 26.2 and 14.7 months for dose groups 1-4, respectively). This analysis confirms that, in terms of cure rate, dose escalation has a significant positive effect only in the intermediate and unfavourable groups. It was found that progression time is inversely proportional to dose, which means that patients recurring in higher dose groups have shorter recurrence times, yet these groups have better survival, particularly long-term. The explanation for this seemingly illogical observation lies in the fact that less aggressive tumours, potentially recurring after a long period of time, are cured by higher doses and do not contribute to the recurrence pattern. As a result, patients in higher dose groups are less likely to recur; however, if they do, they tend to recur earlier. The estimated hazard rates for prostate cancer pass through a clear-cut maximum, thus revealing a time period with especially high values of instantaneous cancer-specific risk; the estimates appear to be nonproportional across dose strata.

Radiobiological effectiveness of laser accelerated electrons in comparison to electron beams from a conventional linear accelerator.

PubMed

Laschinsky, Lydia; Baumann, Michael; Beyreuther, Elke; Enghardt, Wolfgang; Kaluza, Malte; Karsch, Leonhard; Lessmann, Elisabeth; Naumburger, Doreen; Nicolai, Maria; Richter, Christian; Sauerbrey, Roland; Schlenvoigt, Hans-Peter; Pawelke, Jörg

2012-01-01

The notable progress in laser particle acceleration technology promises potential medical application in cancer therapy through compact and cost effective laser devices that are suitable for already existing clinics. Previously, consequences on the radiobiological response by laser driven particle beams characterised by an ultra high peak dose rate have to be investigated. Therefore, tumour and non-malignant cells were irradiated with pulsed laser accelerated electrons at the JETI facility for the comparison with continuous electrons of a conventional therapy LINAC. Dose response curves were measured for the biological endpoints clonogenic survival and residual DNA double strand breaks. The overall results show no significant differences in radiobiological response for in vitro cell experiments between laser accelerated pulsed and clinical used electron beams. These first systematic in vitro cell response studies with precise dosimetry to laser driven electron beams represent a first step toward the long term aim of the application of laser accelerated particles in radiotherapy.

23 CFR 140.609 - Progress and final vouchers.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 23 Highways 1 2011-04-01 2011-04-01 false Progress and final vouchers. 140.609 Section 140.609 Highways FEDERAL HIGHWAY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION PAYMENT PROCEDURES REIMBURSEMENT Reimbursement for Bond Issue Projects § 140.609 Progress and final vouchers. (a) Progress vouchers may be...

23 CFR 140.609 - Progress and final vouchers.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 23 Highways 1 2013-04-01 2013-04-01 false Progress and final vouchers. 140.609 Section 140.609 Highways FEDERAL HIGHWAY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION PAYMENT PROCEDURES REIMBURSEMENT Reimbursement for Bond Issue Projects § 140.609 Progress and final vouchers. (a) Progress vouchers may be...

23 CFR 140.609 - Progress and final vouchers.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 23 Highways 1 2014-04-01 2014-04-01 false Progress and final vouchers. 140.609 Section 140.609 Highways FEDERAL HIGHWAY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION PAYMENT PROCEDURES REIMBURSEMENT Reimbursement for Bond Issue Projects § 140.609 Progress and final vouchers. (a) Progress vouchers may be...

23 CFR 140.609 - Progress and final vouchers.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 23 Highways 1 2010-04-01 2010-04-01 false Progress and final vouchers. 140.609 Section 140.609 Highways FEDERAL HIGHWAY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION PAYMENT PROCEDURES REIMBURSEMENT Reimbursement for Bond Issue Projects § 140.609 Progress and final vouchers. (a) Progress vouchers may be...

Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

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

Kang, Khong Bee, E-mail: dmskkb@nccs.com.sg; Zhu Congju; Wong Yinling

Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, {gamma}-H{sub 2}AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival,more » {gamma}-H{sub 2}AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G{sub 2}/M arrest and increased {gamma}-H{sub 2}AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased {gamma}-H{sub 2}AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are resistant to irradiation-induced cytotoxicity, G{sub 2}/M arrest, and DNA DSBs, compared with nonstem glioma cells. Gefitinib differentially enhances radiosensitivity of stem-like gliomaspheres by reducing EGFR-Akt activation and DNA-PKcs expression, accompanied by enhanced irradiation-induced DNA DSBs and inhibition of DSB repair.« less

Gefitinib radiosensitizes stem-like glioma cells: inhibition of epidermal growth factor receptor-Akt-DNA-PK signaling, accompanied by inhibition of DNA double-strand break repair.

PubMed

Kang, Khong Bee; Zhu, Congju; Wong, Yin Ling; Gao, Qiuhan; Ty, Albert; Wong, Meng Cheong

2012-05-01

We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, γ-H(2)AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, γ-H(2)AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G(2)/M arrest and increased γ-H(2)AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased γ-H(2)AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Stem-like gliomaspheres are resistant to irradiation-induced cytotoxicity, G(2)/M arrest, and DNA DSBs, compared with nonstem glioma cells. Gefitinib differentially enhances radiosensitivity of stem-like gliomaspheres by reducing EGFR-Akt activation and DNA-PKcs expression, accompanied by enhanced irradiation-induced DNA DSBs and inhibition of DSB repair. Copyright © 2012 Elsevier Inc. All rights reserved.

Propofol inhibits gap junctions by attenuating sevoflurane-induced cytotoxicity against rat liver cells in vitro.

PubMed

Huang, Fei; Li, Shangrong; Gan, Xiaoliang; Wang, Ren; Chen, Zhonggang

2014-04-01

Liver abnormalities are seen in a small proportion of patients following anaesthesia with sevoflurane. To investigate whether the cytotoxicity of sevoflurane against rat liver cells was mediated by gap junction intercellular communications, and the effect of propofol on sevoflurane-induced cytotoxicity. Experimental study. The study was carried out in the central laboratory of The Third Affiliated Hospital, Sun Yat-sen University. BRL-3A rat liver cells. Immortal rat liver cells BRL-3A were grown at low and high density. Colony-forming assays were performed to determine clonogenic growth of these cells. To investigate the effect of oleamide and propofol on gap junction function, we measured fluorescence transmission between cells using parachute dye-coupling assays. Immunoblotting assays were performed to determine connexin32 and connexin43 expression. Our colony formation assays revealed that, in low-density culture, sevoflurane caused no apparent inhibition of clonogenic growth of BRL-3A cells. In high-density culture, 2.2 to 4.4% sevoflurane markedly inhibited clonogenic growth of BRL-3A cells with 67.6 (0.34)% and 61.2 (0.17)% of the cells being viable, respectively (P = 0.003 vs. low-density culture), suggesting cell density dependency of sevoflurane-induced cytotoxicity. Our colony formation assays revealed that propofol markedly attenuated the suppression by sevoflurane of the clonogenic growth of BRL-3A cells (viability: propofol and sevoflurane, 91.5 (0.014)% vs. sevoflurane, 56.6 (0.019)%; P <0.01). Blocking gap junctions with 10 μmol l oleamide significantly attenuated 4.4% sevoflurane-induced suppression with a viability of 83.6 ± 0.138% (oleamide and sevoflurane vs. sevoflurane, P < 0.01). Immunoblotting assays further showed that propofol (3.2 μg ml) markedly reduced CX32 levels and significantly inhibited gap junctional intercellular communications as revealed by parachute dye-coupling assays. Values are mean (SD). This study provides the first direct evidence that sevoflurane-induced cytotoxicity, which is mediated through gap junctions, is attenuated by propofol, possibly by its action on Cx32 homomeric or heteromeric complexes.

Radiation sensitivity of Merkel cell carcinoma cell lines

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

Leonard, J.H.; Ramsay, J.R.; Birrell, G.W.

1995-07-30

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT)more » assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.« less

Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers.

PubMed Central

Sutherland, H J; Lansdorp, P M; Henkelman, D H; Eaves, A C; Eaves, C J

1990-01-01

A major goal of current hematopoiesis research is to develop in vitro methods suitable for the measurement and characterization of stem cells with long-term in vivo repopulating potential. Previous studies from several centers have suggested the presence in normal human or murine marrow of a population of very primitive cells that are biologically, physically, and pharmacologically different from cells detectable by short-term colony assays and that can give rise to the latter in long-term cultures (LTCs) containing a competent stromal cell layer. In this report, we show that such cultures can be used to provide a quantitative assay for human "LTC-initiating cells" based on an assessment of the number of clonogenic cells present after 5-8 weeks. Production of derivative clonogenic cells is shown to be absolutely dependent on the presence of a stromal cell feeder. When this requirement is met, the clonogenic cell output (determined by assessment of 5-week-old cultures) is linearly related to the input cell number over a wide range of cell concentrations. Using limiting dilution analysis techniques, we have established the frequency of LTC-initiating cells in normal human marrow to be approximately 1 per 2 X 10(4) cells and in a highly purified CD34-positive subpopulation to be approximately 1 per 50-100 cells. The proliferative capacity exhibited by individual LTC-initiating cells cultured under apparently identical culture conditions was found to be highly variable. Values for the number of clonogenic cells per LTC-initiating cell in 5-week-old cultures ranged from 1 to 30 (the average being 4) with similar levels being detected in positive 8-week-old cultures. Some LTC-initiating cells are multipotent as evidenced by their generation of erythroid as well as granulopoietic progeny. The availability of a system for quantitative analysis of the proliferative and differentiative behavior of this newly defined compartment of primitive human hematopoietic cells should facilitate future studies of specific genetic or microenvironmental parameters involved in the regulation of these cells. Images PMID:2333304

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells.

PubMed

Fedr, Radek; Pernicová, Zuzana; Slabáková, Eva; Straková, Nicol; Bouchal, Jan; Grepl, Michal; Kozubík, Alois; Souček, Karel

2013-05-01

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. Copyright © 2013 International Society for Advancement of Cytometry.

A decrease in miR-150 regulates the malignancy of pancreatic cancer by targeting c-Myb and MUC4.

PubMed

Yang, Ke; He, Miaoxia; Cai, Zailong; Ni, Canrong; Deng, Jingjing; Ta, Na; Xu, Jingjing; Zheng, Jianming

2015-04-01

Pancreatic cancer is an aggressive cancer with high mortality. Conventional treatments have little impact on its progression. Limited research investigating the role of oncogene miR-150 specifically in pancreatic cancer has been published. The purpose of this study was to determine the tumorigenesis of miR-150 in pancreatic cancer. One hundred six pancreatic ductal adenocarcinomas were analyzed together with their adjacent benign pancreatic tissues. The associations of miR-150, c-Myb, and MUC4 expression with survival rates were determined. Functional studies on miR-150 in pancreatic cancer were used to assess its effect on proliferation and malignancy in several pancreatic cell lines. miR-150 expression was significantly down-regulated in pancreatic ductal adenocarcinoma tissues compared with adjacent benign pancreatic tissues. Patients with low miR-150 expression had significantly higher mortality rates than those with high miR-150 expression. The in vitro and in vivo assays of pancreatic cancer cells showed that miR-150 overexpression leads to reduced cell growth, clonogenicity, migration, invasion, modular cell cycles, and induced apoptosis. Moreover, miR-150 expression was inversely correlated with c-Myb and MUC4 activities in pancreatic tissue, cell lines, and nude mouse model. miR-150 is an important suppressor of pancreatic ductal carcinoma and acts as a regulator of c-Myb and MUC4 in aggressive progress.

SHBG Is an Important Factor in Stemness Induction of Cells by DHT In Vitro and Associated with Poor Clinical Features of Prostate Carcinomas

PubMed Central

Ma, Yuanyuan; Liang, Dongming; Liu, Jian; Wen, Jian-Guo; Servoll, Einar; Waaler, Gudmund; Sæter, Thorstein; Axcrona, Karol; Vlatkovic, Ljiljana; Axcrona, Ulrika; Paus, Elisabeth; Yang, Yue; Zhang, Zhiqian; Kvalheim, Gunnar; Nesland, Jahn M.; Suo, Zhenhe

2013-01-01

Androgen plays a vital role in prostate cancer development. However, it is not clear whether androgens influence stem-like properties of prostate cancer, a feature important for prostate cancer progression. In this study, we show that upon DHT treatment in vitro, prostate cancer cell lines LNCaP and PC-3 were revealed with higher clonogenic potential and higher expression levels of stemness related factors CD44, CD90, Oct3/4 and Nanog. Moreover, sex hormone binding globulin (SHBG) was also simultaneously upregulated in these cells. When the SHBG gene was blocked by SHBG siRNA knock-down, the induction of Oct3/4, Nanog, CD44 and CD90 by DHT was also correspondingly blocked in these cells. Immunohistochemical evaluation of clinical samples disclosed weakly positive, and areas negative for SHBG expression in the benign prostate tissues, while most of the prostate carcinomas were strongly positive for SHBG. In addition, higher levels of SHBG expression were significantly associated with higher Gleason score, more seminal vesicle invasions and lymph node metastases. Collectively, our results show a role of SHBG in upregulating stemness of prostate cancer cells upon DHT exposure in vitro, and SHBG expression in prostate cancer samples is significantly associated with poor clinicopathological features, indicating a role of SHBG in prostate cancer progression. PMID:23936228

The flavonoid quercetin transiently inhibits the activity of taxol and nocodazole through interference with the cell cycle

PubMed Central

Samuel, Temesgen; Fadlalla, Khalda; Turner, Timothy; Yehualaeshet, Teshome E.

2010-01-01

Quercetin is a flavonoid with anticancer properties. In this study, we examined the effects of quercetin on cell cycle, viability and proliferation of cancer cells, either singly or in combination with the microtubule-targeting drugs taxol and nocodazole. Although quercetin induced cell death in a dose dependent manner, 12.5-50μM quercetin inhibited the activity of both taxol and nocodazole to induce G2/M arrest in various cell lines. Quercetin also partially restored drug-induced loss in viability of treated cells for up to 72 hours. This antagonism of microtubule-targeting drugs was accompanied by a delay in cell cycle progression and inhibition of the buildup of cyclin-B1 at the microtubule organizing center of treated cells. However, quercetin did not inhibit the microtubule targeting of taxol or nocodazole. Despite the short-term protection of cells by quercetin, colony formation and clonogenicity of HCT116 cells were still suppressed by quercetin or quercetin-taxol combination. The status of cell adherence to growth matrix was critical in determining the sensitivity of HCT116 cells to quercetin. We conclude that while long-term exposure of cancer cells to quercetin may prevent cell proliferation and survival, the interference of quercetin with cell cycle progression diminishes the efficacy of microtubule-targeting drugs to arrest cells at G2/M. PMID:21058190

New mouse tumor model system (RIF-1) for comparison of end-point studies

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

Twentyman, P.R.; Brown, J.M.; Gray, J.W.

1980-03-01

A new tumor model system (RIF-1) was developed that is very suitable for studies in which clonogenic survival is compared with growth delay and control probability following various forms of treatment. The tumor was a radiation-induced sarcoma in the inbred female C3H/Km mouse. It had a low median tumor dose, had a satisfactory plating efficiency direct from in vivo to in vitro, was nonimmunogenic or minimally immunogenic, and metastasized only at a relatively advanced stage of growth. The cell line grew either as a monolayer on plastic dishes, as tumor spheroids in spinner culture, as lung nodules following injection ofmore » a single-cell suspension into the tall veins of syngeneic mice, or as a solid tumor. Both diploid and tetraploid clonogenic cells were found in monolayer cultures of the RIF-1 line.« less

LDHA in neuroblastoma is associated with poor outcome and its depletion decreases neuroblastoma growth independent of aerobic glycolysis.

PubMed

Dorneburg, Carmen; Fischer, Matthias; Barth, Thomas F E; Mueller-Klieser, Wolfgang; Hero, Barbara; Gecht, Judith; Carter, Daniel R; De Preter, Katleen; Mayer, Benjamin; Christner, Lisa; Speleman, Frank; Marshall, Glenn M; Debatin, Klaus-Michael; Beltinger, Christian

2018-06-20

To investigate whether lactate dehydrogenase A (LDHA), an important component of the LDH tetramer crucial for aerobic glycolysis, is associated with patient outcome and constitutes a therapeutic target in neuroblastoma (NB). Expression of LDHA mRNA and protein was determined in 709 and 110 NB patient samples, respectively, and correlated to survival and risk factors. LDHA and LDHB were depleted in human NB cell lines by CRISPR/Cas9 and shRNA, respectively, and aerobic glycolysis, clonogenicity and tumorigenicity were determined. Expression of LDHA in relation to MYCN was measured in NB cell lines and in the TH-MYCN NB mouse model. Expression of LDHA, both on the mRNA and the protein level, was significantly and independently associated with decreased patient survival. Predominant cytoplasmic localization of LDHA protein was associated with poor outcome. Amplification and expression of MYCN did not correlate with expression of LDHA in NB cell lines or TH-MYCN mice, respectively. Knockout of LDHA inhibited clonogenicity, tumorigenicity and tumor growth without abolishing LDH activity or significantly decreasing aerobic glycolysis. Concomitant depletion of LDHA and the isoform LDHB ablated clonogenicity while not abrogating LDH activity or decreasing aerobic glycolysis. The isoform LDHC was not expressed. High expression of LDHA is independently associated with poor outcome of NB and NB cells can be inhibited by depletion of LDHA or LDHB. This inhibition appears to be unrelated to LDH activity and aerobic glycolysis. Thus, investigations of inhibitory mechanisms beyond attenuation of aerobic glycolysis are warranted, both in NB and normal cells. Copyright ©2018, American Association for Cancer Research.

Hematopoietic Stem Cells from Ts65Dn Mice Are Deficient in the Repair of DNA Double-Strand Breaks.

PubMed

Wang, Yingying; Chang, Jianhui; Shao, Lijian; Feng, Wei; Luo, Yi; Chow, Marie; Du, Wei; Meng, Aimin; Zhou, Daohong

2016-06-01

Down syndrome (DS) is a genetic disorder caused by the presence of an extra partial or whole copy of chromosome 21. In addition to musculoskeletal and neurodevelopmental abnormalities, children with DS exhibit various hematologic disorders and have an increased risk of developing acute lymphoblastic leukemia and acute megakaryocytic leukemia. Using the Ts65Dn mouse model, we investigated bone marrow defects caused by trisomy for 132 orthologs of the genes on human chromosome 21. The results showed that, although the total bone marrow cellularity as well as the frequency of hematopoietic progenitor cells (HPCs) was comparable between Ts65Dn mice and their age-matched euploid wild-type (WT) control littermates, human chromosome 21 trisomy led to a significant reduction in hematopoietic stem cell (HSC) numbers and clonogenic function in Ts65Dn mice. We also found that spontaneous DNA double-strand breaks (DSBs) were significantly increased in HSCs from the Ts65Dn mice, which was correlated with the significant reduction in HSC clonogenic activity compared to those from WT controls. Moreover, analysis of the repair kinetics of radiation-induced DSBs revealed that HSCs from Ts65Dn mice were less proficient in DSB repair than the cells from WT controls. This deficiency was associated with a higher sensitivity of Ts65Dn HSCs to radiation-induced suppression of HSC clonogenic activity than that of euploid HSCs. These findings suggest that an additional copy of genes on human chromosome 21 may selectively impair the ability of HSCs to repair DSBs, which may contribute to DS-associated hematological abnormalities and malignancies.

A comparative assessment of cartilage and joint fat pad as a potential source of cells for autologous therapy development in knee osteoarthritis.

PubMed

English, A; Jones, E A; Corscadden, D; Henshaw, K; Chapman, T; Emery, P; McGonagle, D

2007-11-01

The utility of autologous chondrocytes for cartilage repair strategies in older subjects with osteoarthritis (OA) may be limited by both age-related and disease-associated decline in chondrogenesis. The aim of this work was to assess OA Hoffa's fat pad as an alternative source of autologous chondroprogenitor cells and to compare it with OA chondrocytes derived from different areas of cartilage. Cartilage and fat pad tissue digests were obtained from 26 subjects with knee OA and compared with normal bone marrow (BM) mesenchymal stem cells (MSCs) with respect to their in vitro colony-forming potential, growth kinetics, multipotentiality and clonogenicity. Flow cytometry was used to investigate their MSC marker phenotype. Expanded cultures derived from eroded areas of cartilage were slightly more chondrogenic than those derived from macroscopically normal cartilage or chondro-osteophytes; however, all cartilage-derived cultures failed to maintain their chondrogenic potency following extended expansion. In contrast, OA fat pads contained highly clonogenic and multipotential cells with stable chondrogenic potency in vitro, even after 16 population doublings. Standard colony-forming assays failed to reflect the observed functional differences between the studied tissues whereas flow cytometry revealed higher levels of a putative MSC marker low-affinity growth factor receptor (LNGFR) on culture expanded fat pad-derived, but not cartilage-derived, MSCs. In contrast to OA cartilage from three different sites, OA Hoffa's fat pad contains clonogenic cells that meet the criteria for MSCs and produce multipotential cultures that maintain their chondrogenesis long term. These findings have broad implications for future strategies aimed at cartilage repair in OA.

Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells

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

Chow, Paik Wah; Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur; Abdul Hamid, Zariyantey, E-mail: zyantey@ukm.edu.my

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are sensitive targets for benzene-induced hematotoxicity and leukemogenesis. The impact of benzene exposure on the complex microenvironment of HSCs and HPCs remains elusive. This study aims to investigate the mechanism linking benzene exposure to targeting HSCs and HPCs using phenotypic and clonogenic analyses. Mouse bone marrow (BM) cells were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), for 24 h. Expression of cellular surface antigens for HSC (Sca-1), myeloid (Gr-1, CD11b), and lymphoid (CD45, CD3e) populations were confirmed by flow cytometry. The clonogenicity of cells was studied using the colony-formingmore » unit (CFU) assay for multilineage (CFU-GM and CFU-GEMM) and single-lineage (CFU-E, BFU-E, CFU-G, and CFU-M) progenitors. 1,4-BQ demonstrated concentration-dependent cytotoxicity in mouse BM cells. The percentage of apoptotic cells increased (p < 0.05) following 1,4-BQ exposure. Exposure to 1,4-BQ showed no significant effect on CD3e{sup +} cells but reduced the total counts of Sca-1{sup +}, CD11b{sup +}, Gr-1{sup +}, and CD45{sup +} cells at 7 and 12 μM (p < 0.05). Furthermore, the CFU assay showed reduced (p < 0.05) clonogenicity in 1,4-BQ-treated cells. 1,4-BQ induced CFU-dependent cytotoxicity by significantly inhibiting colony growth for CFU-E, BFU-E, CFU-G, and CFU-M starting at a low concentration of exposure (5 μM); whereas for the CFU-GM and CFU-GEMM, the inhibition of colony growth was remarkable only at 7 and 12 μM of 1,4-BQ, respectively. Taken together, 1,4-BQ caused lineage-related cytotoxicity in mouse HPCs, demonstrating greater toxicity in single-lineage progenitors than in those of multi-lineage. - Highlights: • We examine 1,4-BQ toxicity targeting mouse hematopoietic cell lineages. • 1,4-BQ induces concentration-dependent cytotoxicity in bone marrow (BM) cells. • 1,4-BQ shows lineage-related toxicity on hematopoietic stem and progenitors. • 1,4-BQ toxicity is greater in single- than multilineage committed progenitors.« less

A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition

PubMed Central

Shi, Hubing; Hong, Aayoung; Kong, Xiangju; Koya, Richard C.; Song, Chunying; Moriceau, Gatien; Hugo, Willy; Yu, Clarissa C.; Ng, Charles; Chodon, Thinle; Scolyer, Richard A.; Kefford, Richard F.; Ribas, Antoni; Long, Georgina V.; Lo, Roger S.

2013-01-01

BRAF inhibitor (BRAFi) therapy leads to remarkable anti-melanoma responses, but the initial tumor shrinkage is commonly incomplete, providing a nidus for subsequent disease progression. Adaptive signaling may underlie early BRAFi resistance and influence the selection pattern for genetic variants causing late, acquired resistance. We show here that BRAFi (or BRAFi+MEKi) therapy in patients frequently led to rebound p-AKT levels in their melanomas early on treatment. In cell lines, BRAFi treatment led to rebound levels of RTKs (including PDGFRβ), PIP3, pleckstrin homology domain (PHD) recruitment, and p-AKT. PTEN expression limited this BRAFi-elicited PI3K-AKT signaling, which could be rescued by introduction of a mutant AKT1 (Q79K) kown to confer acquired BRAFi resistance. Functionally, AKT1 Q79K conferred BRAFi resistance via amplifying BRAFi-elicited PI3K-AKT signaling. Additionally, MAPK pathway inhibition enhanced clonogenic growth dependency on PI3K or AKT. Thus, adaptive or genetic upregulation of AKT critically participates in melanoma survival during BRAFi therapy. PMID:24265152

Bortezomib sensitizes esophageal squamous cancer cells to radiotherapy by suppressing the expression of HIF-1α and apoptosis proteins.

PubMed

Wang, Di; Qin, Qin; Jiang, Qin-Juan; Wang, Da-Fei

2016-04-13

Radiation therapy is a typical treatment for esophageal squamous cell carcinoma (ESCC), especially middle and upper segment esophagus, and inoperable patients. However, how to promote radiation sensitivity in radio-resistant cancer cells is a conundrum. Here, our study investigated the radiosensitizing effect of bortezomib, a specific and reversible dipeptide boronic acid analog, in ESCC cells. Human esophageal squamous carcinoma cell lines Eca109 and TE-13 were exposed to hypoxia and/or ionizing radiation (IR) with or without treatment of bortezomib. Cell proliferation assay was performed with CCK8. Cell apoptosis and cell cycle assay were performed with flow cytometry. The radiosensitization effect of was assessed by clonogenic survival and progression of tumor xenograft. The expression of HIF-1α, VEGF, and apoptosis proteins was evaluated by Western blot. Radiation-induced DNA double strand break and homologous recombination repair were assessed by immunofluorescence. Our results show that bortezomib efficiently radiosensitizes ESCC cells by decreasing the expression of HIF- 1α and VEGF, inducing apoptosis by activating caspase, and delaying DNA damage repair after radiation.

Grant Closeout Requirements and Reports

Cancer.gov

Requirements and reports to comply with grant closeout, including Final Federal Financial Report (FFR, SF425); Final Research Performance Progress Report (FRPPR); Interim Research Performance Progress Report (IRPPR); Final Invention Statement (FIS, HHS

Long noncoding RNA CCAT1 promotes cell proliferation and metastasis in human medulloblastoma via MAPK pathway.

PubMed

Gao, Ran; Zhang, Rui; Zhang, Cuicui; Zhao, Li; Zhang, Yue

2018-01-01

Medulloblastoma is the most common posterior fossa tumor in children and one that easily metastasizes. The mechanisms of how the medulloblastoma develops and progresses remain to be elucidated. The present study aimed to assess the role of long noncoding colon cancer-associated transcript-1 (lncRNA CCAT1) in cell proliferation and metastasis in human medulloblastoma. Levels of CCAT1 were measured in samples and cell lines of medulloblastoma. Cell cycle progression, cell viability assay, colony formation assay, wound-healing and Transwell assays Corning, Cambridge, MA, USA were used to investigate the viability and motility of cells. Western blot assay was used to investigate the levels of CCAT1 and other proteins. The initial findings indicated that CCAT1 was significantly up-regulated in clinical cancerous tissues and expressed differently in a series of medulloblastoma cell lines. CCAT1 knockdown significantly slowed cell proliferation rates and inhibited cell clonogenic potential in Daoy cells and D283 cells. Cell cycle progression was disrupted with cell proportions in the G0/G1 phase decreased and the proportion in the S phase and G2/M phases increased, in Daoy cells and D283 cells. Concordantly, medulloblastoma tumor cell growth rates were found to be impaired in xenotransplanted mice. After CCAT1 knockdown, cell wound recovery ability was significantly inhibited. Furthermore, the phosphorylated levels of MAPK, ERK and MEK, but not their total levels decreased after the down-regulation of CCAT1 in Daoy and D283 cells. Our results suggested that the lncRNA CCAT1 promotes cell proliferation and metastasis in human medulloblastoma by possibly regulating the MAPK pathway.

Improvement of cardiac function by placenta-derived mesenchymal stem cells does not require permanent engraftment and is independent of the insulin signaling pathway.

PubMed

Passipieri, Juliana A; Kasai-Brunswick, Tais H; Suhett, Grazielle; Martins, Andreza B; Brasil, Guilherme V; Campos, Dilza B; Rocha, Nazareth N; Ramos, Isalira P; Mello, Debora B; Rodrigues, Deivid C; Christie, Beatriz B; Silva-Mendes, Bernardo J; Balduíno, Alex; Sá, Renato M; Lopes, Laudelino M; Goldenberg, Regina C; Campos de Carvalho, Antonio C; Carvalho, Adriana B

2014-08-21

The objective of this work was to evaluate the efficacy of placenta-derived mesenchymal stem cell (MSC) therapy in a mouse model of myocardial infarction (MI). Since MSCs can be obtained from two different regions of the human term placenta (chorionic plate or villi), cells obtained from both these regions were compared so that the best candidate for cell therapy could be selected. For the in vitro studies, chorionic plate MSCs (cp-MSCs) and chorionic villi MSCs (cv-MSCs) were extensively characterized for their genetic stability, clonogenic and differentiation potential, gene expression, and immunophenotype. For the in vivo studies, C57Bl/6 mice were submitted to MI and, after 21 days, received weekly intramyocardial injections of cp-MSCs for 3 weeks. Cells were also stably transduced with a viral construct expressing luciferase, under the control of the murine stem cell virus (MSCV) promoter, and were used in a bioluminescence assay. The expression of genes associated with the insulin signaling pathway was analyzed in the cardiac tissue from cp-MSCs and placebo groups. Morphology, differentiation, immunophenotype, and proliferation were quite similar between these cells. However, cp-MSCs had a greater clonogenic potential and higher expression of genes related to cell cycle progression and genome stability. Therefore, we considered that the chorionic plate was preferable to the chorionic villi for the isolation of MSCs. Sixty days after MI, cell-treated mice had a significant increase in ejection fraction and a reduction in end-systolic volume. This improvement was not caused by a reduction in infarct size. In addition, tracking of cp-MSCs transduced with luciferase revealed that cells remained in the heart for 4 days after the first injection but that the survival period was reduced after the second and third injections. Quantitative reverse transcription-polymerase chain reaction revealed similar expression of genes involved in the insulin signaling pathway when comparing cell-treated and placebo groups. Improvement of cardiac function by cp-MSCs did not require permanent engraftment and was not mediated by the insulin signaling pathway.

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

Cheng, Ya-Hsin; Huang, Su-Chin; Lin, Chun-Ju

Environmental cigarette smoke has been suggested to promote lung adenocarcinoma progression through aryl hydrocarbon receptor (AhR)-signaled metabolism. However, whether AhR facilitates metabolic activation or detoxification in exposed adenocarcinoma cells remains ambiguous. To address this question, we have modified the expression level of AhR in two human lung adenocarcinoma cell lines and examined their response to an extract of cigarette sidestream smoke particulates (CSSP). We found that overexpression of AhR in the CL1-5 cell line reduced CSSP-induced ROS production and oxidative DNA damage, whereas knockdown of AhR expression increased ROS level in CSSP-exposed H1355 cells. Oxidative stress sensor Nrf2 and itsmore » target gene NQO1 were insensitive to AhR expression level and CSSP treatment in human lung adenocarcinoma cells. In contrast, induction of AhR expression concurrently increased mRNA expression of xenobiotic-metabolizing genes CYP1B1, UGT1A8, and UGT1A10 in a ligand-independent manner. It appeared that AhR accelerated xenobiotic clearing and diminished associated oxidative stress by coordinate regulation of a set of phase I and II metabolizing genes. However, the AhR-signaled protection could not shield cells from constant oxidative stress. Prolonged exposure to high concentrations of CSSP induced G0/G1 cell cycle arrest via the p53–p21–Rb1 signaling pathway. Despite no effect on DNA repair rate, AhR facilitated the recovery of cells from growth arrest when CSSP exposure ended. AhR-overexpressing lung adenocarcinoma cells exhibited an increased anchorage-dependent and independent proliferation when recovery from exposure. In summary, our data demonstrated that AhR protected lung adenocarcinoma cells against CSSP-induced oxidative stress and promoted post-exposure clonogenicity. -- Highlights: ► AhR expression level influences cigarette sidestream smoke-induced ROS production. ► AhR reduces oxidative stress by coordinate regulation of metabolizing genes. ► Constant exposure to cigarette smoke arrests cell cycle via p53–p21–Rb1 signaling. ► AhR increases post-exposure clonogenicity of lung adenocarcinoma cells.« less

HAb18G/CD147 Promotes pSTAT3-Mediated Pancreatic Cancer Development via CD44s †, ‡

PubMed Central

Li, Ling; Tang, Wenhua; Wu, Xiaoqing; Karnak, David; Meng, Xiaojie; Thompson, Rachel; Hao, Xinbao; Li, Yongmin; Qiao, Xiaotan T.; Lin, Jiayuh; Fuchs, James; Simeone, Diane M.; Chen, Zhi-Nan; Lawrence, Theodore S.; Xu, Liang

2013-01-01

Purpose STAT3 plays a critical role in initiation and progression of pancreatic cancer. However, therapeutically targeting STAT3 is failure in clinic. We previously identified HAb18G/CD147 as an effective target for cancer treatment. In this study, we aimed to investigate potential role of HAb18G/CD147 in STAT3-involved pancreatic tumorigenesis in vitro and in vivo. Experimental Design The expression of HAb18G/CD147, pSTAT3 and CD44s were determined in tissue microarrays. The tumorigenic function and molecular signaling mechanism of HAb18G/CD147 was assessed by in vitro cellular and clonogenic growth, reporter assay, immunoblot, immunofluorescence staining, immunoprecipitation, and in vivo tumor formationusing loss or gain-of-function strategies. Results Highly expressed HAb18G/CD147 promoted cellular and clonogenic growth in vitro and tumorigenicity in vivo. CyPA, a ligand of CD147, stimulated STAT3 phosphorylation and its downstream genes cyclin D1/survivin through HAb18G/CD147 dependent mechanisms. HAb18G/CD147 was associated and co-localized with cancer stem cell marker CD44s in lipid rafts. The inhibitors of STAT3 and survivin, as well as CD44s neutralizing antibodies suppressed the HAb18G/CD147-induced cell growth. High HAb18G/CD147 expression in pancreatic cancer was significantly correlated with the poor tumor differentiation, and the high co-expression of HAb18G/CD147-CD44s-STAT3 associated with poor survival of patients with pancreatic cancer. Conclusions We identified HAb18G/CD147 as a novel upstream activator of STAT3 via interacts with CD44s and plays a critical role in the development of pancreatic cancer. The data suggest HAb18G/CD147 could be a promising therapeutic target for highly aggressive pancreatic cancer and a surrogate marker in the STAT3-targeted molecular therapies. PMID:24132924

Effects of osteopontin inhibition on radiosensitivity of MDA-MB-231 breast cancer cells.

PubMed

Hahnel, Antje; Wichmann, Henri; Kappler, Matthias; Kotzsch, Matthias; Vordermark, Dirk; Taubert, Helge; Bache, Matthias

2010-09-17

Osteopontin (OPN) is a secreted glycophosphoprotein that is overexpressed in various tumors, and high levels of OPN have been associated with poor prognosis of cancer patients. In patients with head and neck cancer, high OPN plasma levels have been associated with poor prognosis following radiotherapy. Since little is known about the relationship between OPN expression and radiosensitivity, we investigated the cellular and radiation induced effects of OPN siRNA in human MDA-MB-231 breast cancer cells. MDA-MB-231 cells were transfected with OPN-specific siRNAs and irradiated after 24 h. To verify the OPN knockdown, we measured the OPN mRNA and protein levels using qRT-PCR and Western blot analysis. Furthermore, the functional effects of OPN siRNAs were studied by assays to assess clonogenic survival, migration and induction of apoptosis. Treatment of MDA-MB-231 cells with OPN siRNAs resulted in an 80% decrease in the OPN mRNA level and in a decrease in extracellular OPN protein level. Transfection reduced clonogenic survival to 42% (p = 0.008), decreased the migration rate to 60% (p = 0.15) and increased apoptosis from 0.3% to 1.7% (p = 0.04). Combination of OPN siRNA and irradiation at 2 Gy resulted in a further reduction of clonogenic survival to 27% (p < 0.001), decreased the migration rate to 40% (p = 0.03) and increased apoptosis to 4% (p < 0.005). Furthermore, OPN knockdown caused a weak radiosensitization with an enhancement factor of 1.5 at 6 Gy (p = 0.09) and a dose modifying factor (DMF10) of 1.1. Our results suggest that an OPN knockdown improves radiobiological effects in MDA-MB-231 cells. Therefore, OPN seems to be an attractive target to improve the effectiveness of radiotherapy.

Three-dimensional Invasion of Human Glioblastoma Cells Remains Unchanged by X-ray and Carbon Ion Irradiation In Vitro

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

Eke, Iris; Storch, Katja; Kaestner, Ina

Purpose: Cell invasion represents one of the major determinants that treatment has failed for patients suffering from glioblastoma. Contrary findings have been reported for cell migration upon exposure to ionizing radiation. Here, the migration and invasion capability of glioblastoma cells on and in collagen type I were evaluated upon irradiation with X-rays or carbon ions. Methods and Materials: Migration on and invasion in collagen type I were evaluated in four established human glioblastoma cell lines exposed to either X-rays or carbon ions. Furthermore, clonogenic radiation survival, proliferation (5-bromo-2-deoxyuridine positivity), DNA double-strand breaks ({gamma}H2AX/53BP1-positive foci), and expression of invasion-relevant proteins (eg,more » {beta}1 integrin, FAK, MMP2, and MMP9) were explored. Migration and invasion assays for primary glioblastoma cells also were carried out with X-ray irradiation. Results: Neither X-ray nor carbon ion irradiation affected glioblastoma cell migration and invasion, a finding similarly observed in primary glioblastoma cells. Intriguingly, irradiated cells migrated unhampered, despite DNA double-strand breaks and reduced proliferation. Clonogenic radiation survival was increased when cells had contact with extracellular matrix. Specific inhibition of the {beta}1 integrin or proliferation-associated signaling molecules revealed a critical function of JNK, PI3K, and p38 MAPK in glioblastoma cell invasion. Conclusions: These findings indicate that X-rays and carbon ion irradiation effectively reduce proliferation and clonogenic survival without modifying the migration and invasion ability of glioblastoma cells in a collagen type I environment. Addition of targeted agents against members of the MAPK and PI3K signaling axis to conventional chemoradiation therapy seems potentially useful to optimize glioblastoma therapy.« less

The importance of serum serotonin levels in the measurement of radiation-induced bystander cell death in HaCaT cells.

PubMed

Lyng, Fiona M; Desplanques, Maxime; Jella, Kishore Kumar; Garcia, Amaya; McClean, Brendan

2012-10-01

The aim of this study was to investigate the importance of serum serotonin levels in the measurement of bystander cell death. The study was undertaken as part of an intercomparison exercise involving seven European laboratories funded under the European Union Sixth Framework Programme (FP6) Non-Targeted Effects (NOTE) integrated project. Three batches of foetal bovine serum were tested; serum with high and low serotonin content from the intercomparison exercise as well as serum from the home laboratory. Three sets of human keratinocytes (HaCaT cell line) were cultured in DMEM:F12 medium supplemented with serum with high or low serotonin content or serum from the home laboratory and both donor and recipient HaCaT cells were plated. The donor HaCaT cells were irradiated (0.5 Gy) using a cobalt 60 teletherapy unit, the medium was harvested 1 hour post irradiation and transferred to the recipient HaCaT cells. Bystander induced cell death was measured by the clonogenic survival assay and the Alamar blue viability assay. A significant reduction in cell survival, as measured by the clonogenic assay, and in cell viability, as measured by the Alamar blue assay, was observed in the recipient HaCaT cells treated with medium from irradiated cells compared to the cells treated with medium from unirradiated cells. No significant difference was found between the three batches of serum. The data suggest that in our cell system and with our endpoints (clonogenic assay and Alamar blue assay), serum serotonin levels do not play a role in bystander-induced cell death.

Indirect radio-chemo-beta therapy: a targeted approach to increase biological efficiency of x-rays based on energy

NASA Astrophysics Data System (ADS)

Oktaria, Sianne; Corde, Stéphanie; Lerch, Michael L. F.; Konstantinov, Konstantin; Rosenfeld, Anatoly B.; Tehei, Moeava

2015-10-01

Despite the use of multimodal treatments incorporating surgery, chemotherapy and radiotherapy, local control of gliomas remains a major challenge. The potential of a new treatment approach called indirect radio-chemo-beta therapy using the synergy created by combining methotrexate (MTX) with bromodeoxyuridine (BrUdR) under optimum energy x-ray irradiation is assessed. 9L rat gliosarcoma cells pre-treated with 0.01 μM MTX and/or 10 μM BrUdR were irradiated in vitro with 50 kVp, 125 kVp, 250 kVp, 6 MV and 10 MV x-rays. The cytotoxicity was assessed using clonogenic survival as the radiobiological endpoint. The photon energy with maximum effect was determined using radiation sensitization enhancement factors at 10% clonogenic survival (SER10%). The cell cycle distribution was investigated using flow cytometric analysis with propidium iodide staining. Incorporation of BrUdR in the DNA was detected by the fluorescence of labelled anti-BrUdR antibodies. The radiation sensitization enhancement exhibits energy dependence with a maximum of 2.3 at 125 kVp for the combined drug treated cells. At this energy, the shape of the clonogenic survival curve of the pharmacological agents treated cells changes substantially. This change is interpreted as an increased lethality of the local radiation environment and is attributed to supplemented inhibition of DNA repair. Radiation induced chemo-beta therapy was demonstrated in vitro by the targeted activation of combined pharmacological agents with optimized energy tuning of x-ray beams on 9 L cells. Our results show that this is a highly effective form of chemo-radiation therapy.

Critical role of zinc finger protein 521 in the control of growth, clonogenicity and tumorigenic potential of medulloblastoma cells

PubMed Central

Iaccino, Enrico; Scicchitano, Stefania; Lupia, Michela; Chiarella, Emanuela; Mega, Tiziana; Bernaudo, Francesca; Pelaggi, Daniela; Mesuraca, Maria; Pazzaglia, Simonetta; Semenkow, Samantha; Bar, Eli E.; Kool, Marcel; Pfister, Stefan; Bond, Heather M.; Eberhart, Charles G.; Steinkühler, Christian; Morrone, Giovanni

2013-01-01

The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of hematopoietic, osteo-adipogenic and neural progenitor cells. ZNF521 is highly expressed in cerebellum and in particular in the neonatal external granule layer that contains candidate medulloblastoma cells-of-origin, and in the majority of human medulloblastomas. Here we have explored its involvement in the control of human and murine medulloblastoma cells. The effect of ZNF521 on growth and tumorigenic potential of human medulloblastoma cell lines as well as primary Ptc1−/+ mouse medulloblastoma cells was investigated in a variety of in vitro and in vivo assays, by modulating its expression using lentiviral vectors carrying the ZNF521 cDNA, or shRNAs that silence its expression. Enforced overexpression of ZNF521 in DAOY medulloblastoma cells significantly increased their proliferation, growth as spheroids and ability to generate clones in single-cell cultures and semisolid media, and enhanced their migratory ability in wound-healing assays. Importantly, ZNF521-expressing cells displayed a greatly enhanced tumorigenic potential in nude mice. All these activities required the ZNF521 N-terminal motif that recruits the nucleosome remodeling and histone deacetylase complex, which might therefore represent an appealing therapeutic target. Conversely, silencing of ZNF521 in human UW228 medulloblastoma cells that display high baseline expression decreased their proliferation, clonogenicity, sphere formation and wound-healing ability. Similarly, Zfp521 silencing in mouse Ptc1−/+ medulloblastoma cells drastically reduced their growth and tumorigenic potential. Our data strongly support the notion that ZNF521, through the recruitment of the NuRD complex, contributes to the clonogenic growth, migration and tumorigenicity of medulloblastoma cells. PMID:23907569

Critical role of zinc finger protein 521 in the control of growth, clonogenicity and tumorigenic potential of medulloblastoma cells.

PubMed

Spina, Raffaella; Filocamo, Gessica; Iaccino, Enrico; Scicchitano, Stefania; Lupia, Michela; Chiarella, Emanuela; Mega, Tiziana; Bernaudo, Francesca; Pelaggi, Daniela; Mesuraca, Maria; Pazzaglia, Simonetta; Semenkow, Samantha; Bar, Eli E; Kool, Marcel; Pfister, Stefan; Bond, Heather M; Eberhart, Charles G; Steinkühler, Christian; Morrone, Giovanni

2013-08-01

The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of hematopoietic, osteo-adipogenic and neural progenitor cells. ZNF521 is highly expressed in cerebellum and in particular in the neonatal external granule layer that contains candidate medulloblastoma cells-of-origin, and in the majority of human medulloblastomas. Here we have explored its involvement in the control of human and murine medulloblastoma cells. The effect of ZNF521 on growth and tumorigenic potential of human medulloblastoma cell lines as well as primary Ptc1-/+ mouse medulloblastoma cells was investigated in a variety of in vitro and in vivo assays, by modulating its expression using lentiviral vectors carrying the ZNF521 cDNA, or shRNAs that silence its expression. Enforced overexpression of ZNF521 in DAOY medulloblastoma cells significantly increased their proliferation, growth as spheroids and ability to generate clones in single-cell cultures and semisolid media, and enhanced their migratory ability in wound-healing assays. Importantly, ZNF521-expressing cells displayed a greatly enhanced tumorigenic potential in nude mice. All these activities required the ZNF521 N-terminal motif that recruits the nucleosome remodeling and histone deacetylase complex, which might therefore represent an appealing therapeutic target. Conversely, silencing of ZNF521 in human UW228 medulloblastoma cells that display high baseline expression decreased their proliferation, clonogenicity, sphere formation and wound-healing ability. Similarly, Zfp521 silencing in mouse Ptc1-/+ medulloblastoma cells drastically reduced their growth and tumorigenic potential. Our data strongly support the notion that ZNF521, through the recruitment of the NuRD complex, contributes to the clonogenic growth, migration and tumorigenicity of medulloblastoma cells.

Autocrine CSF-1R signaling drives mesothelioma chemoresistance via AKT activation

PubMed Central

Cioce, M; Canino, C; Goparaju, C; Yang, H; Carbone, M; Pass, H I

2014-01-01

Clinical management of malignant pleural mesothelioma (MPM) is very challenging because of the uncommon resistance of this tumor to chemotherapy. We report here increased expression of macrophage colony-stimulating-factor-1-receptor (M-CSF/CSF-1R) mRNA in mesothelioma versus normal tissue specimens and demonstrate that CSF-1R expression identifies chemoresistant cells of mesothelial nature in both primary cultures and mesothelioma cell lines. By using RNAi or ligand trapping, we demonstrate that the chemoresistance properties of those cells depend on autocrine CSF-1R signaling. At the single-cell level, the isolated CSF-1Rpos cells exhibit a complex repertoire of pluripotency, epithelial–mesenchymal transition and detoxifying factors, which define a clonogenic, chemoresistant, precursor-like cell sub-population. The simple activation of CSF-1R in untransformed mesothelial cells is sufficient to confer clonogenicity and resistance to pemetrexed, hallmarks of mesothelioma. In addition, this induced a gene expression profile highly mimicking that observed in the MPM cells endogenously expressing the receptor and the ligands, suggesting that CSF-1R expression is mainly responsible for the phenotype of the identified cell sub-populations. The survival of CSF1Rpos cells requires active AKT (v-akt murine thymoma viral oncogene homolog 1) signaling, which contributed to increased levels of nuclear, transcriptionally competent β-catenin. Inhibition of AKT reduced the transcriptional activity of β-catenin-dependent reporters and sensitized the cells to senescence-induced clonogenic death after pemetrexed treatment. This work expands what is known on the non-macrophage functions of CSF-1R and its role in solid tumors, and suggests that CSF-1R signaling may have a critical pathogenic role in a prototypical, inflammation-related cancer such as MPM and therefore may represent a promising target for therapeutic intervention. PMID:24722292

Transient elevation of glycolysis confers radio-resistance by facilitating DNA repair in cells.

PubMed

Bhatt, Anant Narayan; Chauhan, Ankit; Khanna, Suchit; Rai, Yogesh; Singh, Saurabh; Soni, Ravi; Kalra, Namita; Dwarakanath, Bilikere S

2015-05-01

Cancer cells exhibit increased glycolysis for ATP production (the Warburg effect) and macromolecular biosynthesis; it is also linked with therapeutic resistance that is generally associated with compromised respiratory metabolism. Molecular mechanisms underlying radio-resistance linked to elevated glycolysis remain incompletely understood. We stimulated glycolysis using mitochondrial respiratory modifiers (MRMs viz. di-nitro phenol, DNP; Photosan-3, PS3; Methylene blue, MB) in established human cell lines (HEK293, BMG-1 and OCT-1). Glucose utilization and lactate production, levels of glucose transporters and glycolytic enzymes were investigated as indices of glycolysis. Clonogenic survival, DNA repair and cytogenetic damage were studied as parameters of radiation response. MRMs induced the glycolysis by enhancing the levels of two important regulators of glucose metabolism GLUT-1 and HK-II and resulted in 2 fold increase in glucose consumption and lactate production. This increase in glycolysis resulted in resistance against radiation-induced cell death (clonogenic survival) in different cell lines at an absorbed dose of 5 Gy. Inhibition of glucose uptake and glycolysis (using fasentin, 2-deoxy-D-glucose and 3-bromopyruvate) in DNP treated cells failed to increase the clonogenic survival of irradiated cells, suggesting that radio-resistance linked to inhibition of mitochondrial respiration is glycolysis dependent. Elevated glycolysis also facilitated rejoining of radiation-induced DNA strand breaks by activating both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways of DNA double strand break repair leading to a reduction in radiation-induced cytogenetic damage (micronuclei formation) in these cells. These findings suggest that enhanced glycolysis generally observed in cancer cells may be responsible for the radio-resistance, partly by enhancing the repair of DNA damage.

Hematopoietic Stem Cells from Ts65Dn Mice Are Deficient in the Repair of DNA Double-Strand Breaks

PubMed Central

Wang, Yingying; Chang, Jianhui; Shao, Lijian; Feng, Wei; Luo, Yi; Chow, Marie; Du, Wei; Meng, Aimin; Zhou, Daohong

2016-01-01

Down syndrome (DS) is a genetic disorder caused by the presence of an extra partial or whole copy of chromosome 21. In addition to musculoskeletal and neurodevelopmental abnormalities, children with DS exhibit various hematologic disorders and have an increased risk of developing acute lymphoblastic leukemia and acute megakaryocytic leukemia. Using the Ts65Dn mouse model, we investigated bone marrow defects caused by trisomy for 132 orthologs of the genes on human chromosome 21. The results showed that, although the total bone marrow cellularity as well as the frequency of hematopoietic progenitor cells (HPCs) was comparable between Ts65Dn mice and their age-matched euploid wild-type (WT) control littermates, human chromosome 21 trisomy led to a significant reduction in hematopoietic stem cell (HSC) numbers and clonogenic function in Ts65Dn mice. We also found that spontaneous DNA double-strand breaks (DSBs) were significantly increased in HSCs from the Ts65Dn mice, which was correlated with the significant reduction in HSC clonogenic activity compared to those from WT controls. Moreover, analysis of the repair kinetics of radiation-induced DSBs revealed that HSCs from Ts65Dn mice were less proficient in DSB repair than the cells from WT controls. This deficiency was associated with a higher sensitivity of Ts65Dn HSCs to radiation-induced suppression of HSC clonogenic activity than that of euploid HSCs. These findings suggest that an additional copy of genes on human chromosome 21 may selectively impair the ability of HSCs to repair DSBs, which may contribute to DS-associated hematological abnormalities and malignancies. PMID:27243896

Notch3 marks clonogenic mammary luminal progenitor cells in vivo.

PubMed

Lafkas, Daniel; Rodilla, Veronica; Huyghe, Mathilde; Mourao, Larissa; Kiaris, Hippokratis; Fre, Silvia

2013-10-14

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive "triple negative" human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2(SAT) transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells.

Enhanced effects by 4-phenylbutyrate in combination with RTK inhibitors on proliferation in brain tumor cell models

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

Marino, Ana-Maria; Center for Molecular Medicine CMM, Karolinska University Hospital, Stockholm; Sofiadis, Anastasios

2011-07-22

Highlights: {yields} The histone deacetylase inhibitor 4-phenylbutyrate substantially enhance efficacy of the receptor tyrosine kinase inhibitors gefitinib or vandetanib in glioma and medulloblastoma cell lines. {yields} Cell death increases and clonogenic survival is reduced in the combination treatments, over mono-therapy. {yields} Combination treatments with these drugs may improve clinical outcome for cancer therapy. -- Abstract: We have investigated in vitro effects of anticancer therapy with the histone deacetylase inhibitor (HDACi) 4-phenylbutyrate (4-PB) combined with receptor tyrosine kinase inhibitors (RTKi) gefitinib or vandetanib on the survival of glioblastoma (U343MGa) and medulloblastoma (D324Med) cells. In comparison with individual effects of these drugs,more » combined treatment with gefitinib/4-PB or vandetanib/4-PB resulted in enhanced cell killing and reduced clonogenic survival in both cell lines. Our results suggest that combined treatment using HDACi and RTKi may beneficially affect the outcome of cancer therapy.« less

Free radical production by high energy shock waves--comparison with ionizing irradiation.

PubMed

Morgan, T R; Laudone, V P; Heston, W D; Zeitz, L; Fair, W R

1988-01-01

Fricke chemical dosimetry is used as an indirect measure of the free radical production of ionizing irradiation. We adapted the Fricke ferrous sulfate radiation dosimeter to examine the chemical effects of high energy shock waves. Significant free radical production was documented. The reaction was dose dependent, predictably increased by acoustic impedance, but curvilinear. A thousand shocks at 18 kilovolts induced the same free radical oxidation as 1100 rad cobalt-60 gamma ionizing irradiation, increasing to 2900 rad in the presence of an air-fluid zone of acoustic impedance. The biological effect of these free radicals was compared to that of cobalt-60 ionizing irradiation by measuring the affect on Chinese hamster cells by clonogenic assay. While cobalt-60 irradiation produced a marked decrease in clonogenic survivors, little effect was noted with high energy shock waves. This suggested that the chemical effects produced by shock waves were either absent or attenuated in the cells, or were inherently less toxic than those of ionizing irradiation.

Clonogenic colony-forming ability of flow cytometrically isolated hepatic progenitor cells in the murine fetal liver.

PubMed

Taniguchi, H; Kondo, R; Suzuki, A; Zheng, Y W; Takada, Y; Fukunaga, K; Seino, K; Yuzawa, K; Otsuka, M; Fukao, K; Nakauchi, H

2000-01-01

Stem cells are defined as cells having multilineage differentiation potential and self-renewal capability. Hepatic stem cells have aroused considerable interest not only because of their developmental importance but also for their therapeutic potential. However, their presence in the liver has not yet been demonstrated. With the use of a fluorescence-activated cell sorter (FACS) and monoclonal antibodies, we attempted to ascertain whether hepatic stem cells are present in the murine fetal liver. For this purpose, we optimized a cell isolation technique for FACS sorting of fetal liver cells. When isolated CD45 TER119 cells (the non-blood cell fraction in the fetal liver) were tested for their clonogenic colony-forming ability, mechanical dissociation (pipetting) was the most suitable cell isolation technique for FACS sorting. We confirmed that these colonies contained not only cells expressing hepatocyte markers but also cells expressing cholangiocyte markers. To identify hepatic stem cells, studies must focus on CD45TER119- cells in the murine fetal liver.

CWP232228 targets liver cancer stem cells through Wnt/β-catenin signaling: a novel therapeutic approach for liver cancer treatment.

PubMed

Kim, Ji-Young; Lee, Hwa-Yong; Park, Kwan-Kyu; Choi, Yang-Kyu; Nam, Jeong-Seok; Hong, In-Sun

2016-04-12

Liver cancer stem cells (CSCs) are resistant to conventional chemotherapy and radiation, which may destroy tumor masses, but not all liver CSCs contribute to tumor initiation, metastasis, and relapse. In the present study, we showed that liver CSCs with elevated Wnt/β-catenin signaling possess much greater self-renewal and clonogenic potential. We further documented that the increased clonogenic potential of liver CSCs is highly associated with changes in Wnt/β-catenin signaling and that Wnt/β-catenin signaling activity is positively correlated with CD133 expression and aldehyde dehydrogenase (ALDH) enzymatic activity. Notably, the small molecule inhibitor CWP232228, which antagonizes the binding of β-catenin to TCF in the nucleus, inhibits Wnt/β-catenin signaling and depletes CD133+/ALDH+ liver CSCs, thus ultimately diminishing the self-renewal capacity of CSCs and decreasing tumorigenicity in vitro and in vivo. Taken together, our findings suggest that CWP232228 acts as a candidate therapeutic agent for liver cancer by preferentially targeting liver CSCs.

Radiosensitivity of cancer-initiating cells and normal stem cells (or what the Heisenberg uncertainly principle has to do with biology).

PubMed

Woodward, Wendy Ann; Bristow, Robert Glen

2009-04-01

Mounting evidence suggests that parallels between normal stem cell biology and cancer biology may provide new targets for cancer therapy. Prospective identification and isolation of cancer-initiating cells from solid tumors has promoted the descriptive and functional identification of these cells allowing for characterization of their response to contemporary cancer therapies, including chemotherapy and radiation. In clinical radiation therapy, the failure to clinically eradicate all tumor cells (eg, a lack of response, partial response, or nonpermanent complete response by imaging) is considered a treatment failure. As such, biologists have explored the characteristics of the small population of clonogenic cancer cells that can survive and are capable of repopulating the tumor after subcurative therapy. Herein, we discuss the convergence of these clonogenic studies with contemporary radiosensitivity studies that use cell surface markers to identify cancer-initiating cells. Implications for and uncertainties regarding incorporation of these concepts into the practice of modern radiation oncology are discussed.

Notch3 marks clonogenic mammary luminal progenitor cells in vivo

PubMed Central

Lafkas, Daniel; Rodilla, Veronica; Huyghe, Mathilde; Mourao, Larissa; Kiaris, Hippokratis

2013-01-01

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive “triple negative” human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2SAT transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells. PMID:24100291

TAZ Expression as a Prognostic Indicator in Colorectal Cancer

PubMed Central

Tham, Jill M.; Zhang, Xiaoqian; Zeng, Qi; Zhang, Shu-Dong; Hong, WanJin

2013-01-01

The Hippo pathway restricts the activity of transcriptional coactivators TAZ (WWTR1) and YAP. TAZ and YAP are reported to be overexpressed in various cancers, however, their prognostic significance in colorectal cancers remains unstudied. The expression levels of TAZ and YAP, and their downstream transcriptional targets, AXL and CTGF, were extracted from two independent colon cancer patient datasets available in the Gene Expression Omnibus database, totaling 522 patients. We found that mRNA expressions of both TAZ and YAP were positively correlated with those of AXL and CTGF (p<0.05). High level mRNA expression of TAZ, AXL or CTGF significantly correlated with shorter survival. Importantly, patients co-overexpressing all 3 genes had a significantly shorter survival time, and combinatorial expression of these 3 genes was an independent predictor for survival. The downstream target genes for TAZ-AXL-CTGF overexpression were identified by Java application MyStats. Interestingly, genes that are associated with colon cancer progression (ANTXR1, EFEMP2, SULF1, TAGLN, VCAN, ZEB1 and ZEB2) were upregulated in patients co-overexpressing TAZ-AXL-CTGF. This TAZ-AXL-CTGF gene expression signature (GES) was then applied to Connectivity Map to identify small molecules that could potentially be utilized to reverse this GES. Of the top 20 small molecules identified by connectivity map, amiloride (a potassium sparing diuretic,) and tretinoin (all-trans retinoic acid) have shown therapeutic promise in inhibition of colon cancer cell growth. Using MyStats, we found that low level expression of either ANO1 or SQLE were associated with a better prognosis in patients who co-overexpressed TAZ-AXL-CTGF, and that ANO1 was an independent predictor of survival together with TAZ-AXL-CTGF. Finally, we confirmed that TAZ regulates Axl, and plays an important role in clonogenicity and non-adherent growth in vitro and tumor formation in vivo. These data suggest that TAZ could be a therapeutic target for the treatment of colon cancer. PMID:23372686

Inhibition of cell growth and induction of apoptosis in ovarian carcinoma cell lines CaOV3 and SKOV3 by natural withanolide Withaferin A.

PubMed

Zhang, Xuan; Samadi, Abbas K; Roby, Katherine F; Timmermann, Barbara; Cohen, Mark S

2012-03-01

Withaferin A, a natural withanolide, has shown anti-cancer properties in various cancers including breast cancer, but its effects in ovarian cancer remain unexplored. Notch 1 and Notch3 are critically involved in ovarian cancer progression. We decided to examine the effects of Withaferin A in ovarian carcinoma cell lines and its molecular mechanism of action including its regulation of Notch. The effects of Withaferin A were examined in CaOV3 and SKOV3 ovarian carcinoma cell lines using MTS assay, clonogenic assay, annexin V/propidium iodide flow cytometry, and cell cycle analysis. Western analysis was conducted to examine the molecular mechanisms of action. Withaferin A inhibited the growth and colony formation of CaOV3 and SKOV3 cells by inducing apoptosis and cell cycle arrest. These changes correlated with down-regulation of Notch1, Notch3, cdc25C, total and phosphorylated Akt, and bcl-2 proteins. Withaferin A inhibits CaOV3 and SKOV3 ovarian carcinoma cell growth, at least in part by targeting Notch1 and Notch3. Copyright © 2011 Elsevier Inc. All rights reserved.

Ibrutinib improves the development of acute lymphoblastic leukemia by activating endoplasmic reticulum stress-induced cell death.

PubMed

Li, Zhaohui; Wu, Jia; Sheng, Lei

2018-05-01

The current study mainly aims to evaluate the effects of ibrutinib on endoplasmic reticulum stress (ERS)-induced apoptosis in Reh cells, which may shed light on the treatment of acute lymphoblastic leukemia (ALL) among children. In line with previous studies, our data show that ibrutinib significantly suppressed Reh cell viability in a time- and dose-dependent manner. We further evaluated the role of ibrutinib on Reh cell colony formation and apoptosis. Ibrutinib inhibited clonogenic capacity and induced Reh cell apoptosis, suggesting an anti-tumor effects of ibrutinib in the progression of ALL. Further study showed that ibrutinib treatment increased ERS-related protein expression, including Bip, ATF4 and CHOP, suggesting the induction of ER-stress in Reh cells. More importantly, once ER-stress was suppressed by tauroursodeoxycholic acid (TUDCA), an ER-stress inhibitor, the upregulation of Bip, ATF4, CHOP, cleaved-caspase3 and cleaved-PARP after ibrutinib treatment was partially reversed, suggesting that induction of ALL cell apoptosis by ibrutinib was partially attributed to activation of ER stress. In summary, we showed novel data that ER-stress induced cell apoptosis plays a key role in the therapeutic effects of ibrutinib on ALL cell malignancies.

Targeted Nanoparticles for Kidney Cancer Therapy

DTIC Science & Technology

2012-10-01

24 hrs. Clonogenic survival was assessed 7-10 days after CNT incubation after plating 200-300 cells/well (Figure 7). Similar trends were seen using... ferritin on angiogenesis. Proc Natl Acad Sci U S A. 2009;106(2):570-5. PMCID: 2626744. 4. Burke A, Ding X, Singh R, Kraft RA, Levi-Polyachenko N, Rylander

Comprehensive Profiling of Radiosensitive Human Cell Lines with DNA Damage Response Assays Identifies the Neutral Comet Assay as a Potential Surrogate for Clonogenic Survival

PubMed Central

Nahas, Shareef A.; Davies, Robert; Fike, Francesca; Nakamura, Kotoka; Du, Liutao; Kayali, Refik; Martin, Nathan T.; Concannon, Patrick; Gatti, Richard A.

2015-01-01

In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We characterized a unique panel of 19 “radiosensitive” human lymphoblastoid cell lines from individuals with undiagnosed diseases suggestive of a DNA repair disorder. Radiosensitivity was defined by reduced cellular survival using a clonogenic survival assay. Each assay identified cell lines with defects in DNA damage response functions. The highest concordance rate observed, 89% (17/19), was between an abnormal neutral comet assay and reduced survival by the colony survival assay. Our data also suggested that the neutral comet assay would be a more rapid surrogate for analyzing DNA repair/processing disorders. PMID:21962002

Clonogenicity of human leukemic cells protected from cell-lethal agents by heat shock protein 70

PubMed Central

Bases, Robert

2005-01-01

Pretreatment of human leukemia THP-1 cells with heat shock protein Hsp70 (Hsp70) protected them from the cell-lethal effects of the topoisomerase II inhibitor, lucanthone and from ionizing radiation. Cell viability was scored in clonogenic assays of single cells grown in liquid medium containing 0.5% methyl cellulose. Colonies were observed and rapidly scored after staining with the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide. The frequency of abasic sites in the deoxyribonucleic acid (DNA) of THP-1 cells was reduced when these cells were treated with Hsp70. Hsp70 is presumed to have protected the cells by promoting repair of cell DNA, in agreement with previous studies that showed that Hsp70 enhanced base excision repair by purified enzymes. The shoulders of radiation dose-response curves were enhanced by pretreatment of cells with Hsp70 and, importantly, were reduced when cells were transfected with ribonucleic acid designed to silence Hsp70. Hsp70 influenced repair of sublethal damage after radiation. PMID:15832946

Nanoscale liposomal formulation of a SYK P-site inhibitor against B-precursor leukemia

PubMed Central

Qazi, Sanjive; Cely, Ingrid; Sahin, Kazim; Shahidzadeh, Anoush; Ozercan, Ibrahim; Yin, Qian; Gaynon, Paul; Termuhlen, Amanda; Cheng, Jianjun

2013-01-01

We report preclinical proof of principle for effective treatment of B-precursor acute lymphoblastic leukemia (ALL) by targeting the spleen tyrosine kinase (SYK)–dependent antiapoptotic blast cell survival machinery with a unique nanoscale pharmaceutical composition. This nanoscale liposomal formulation (NLF) contains the pentapeptide mimic 1,4-Bis (9-O dihydroquinidinyl) phthalazine/hydroquinidine 1,4-phathalazinediyl diether (C61) as the first and only selective inhibitor of the substrate binding P-site of SYK. The C61 NLF exhibited a very favorable pharmacokinetic and safety profile in mice, induced apoptosis in primary B-precursor ALL blast cells taken directly from patients as well as in vivo clonogenic ALL xenograft cells, destroyed the in vivo clonogenic fraction of ALL blast cells, and, at nontoxic dose levels, exhibited potent in vivo antileukemic activity against patient-derived ALL cells in xenograft models of aggressive B-precursor ALL. Our findings establish SYK as an attractive molecular target for therapy of B-precursor ALL. Further development of the C61 NLF may provide the foundation for therapeutic innovation against therapy-refractory B-precursor ALL. PMID:23568490

Ca(2+)-mediated prostaglandin E2 induction reduces haematoporphyrin-derivative-induced cytotoxicity of T24 human bladder transitional carcinoma cells in vitro.

PubMed Central

Penning, L C; Keirse, M J; VanSteveninck, J; Dubbelman, T M

1993-01-01

The effects of haematoporphyrin-derivative-mediated photodynamic treatment on arachidonic acid metabolism and its relation to clonogenicity have been studied in human bladder-tumour cells. Photodynamic treatment resulted in a transient release of arachidonic acid-derived compounds; prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) especially were strongly increased. This release was reduced by chelation of intracellular Ca2+ with Quin-2 or by lowering the extracellular Ca2+ concentration in the medium with EGTA, presumably resulting in inhibition of phospholipase A2. A similar reduction was obtained when indomethacin, an inhibitor of the cyclo-oxygenase pathway, was added prior to light exposure. These three treatments enhanced the photosensitivity, as revealed by the clonogenicity assay. Incubation with PGE2 prior to light exposure, but not with TXB2, protected against reproductive-cell death. The results of these experiments suggest that Ca(2+)-mediated activation of cyclo-oxygenase, resulting in increased levels of PGE2, participates in a cellular-defence mechanism against photodynamic cell killing. PMID:8503851

Flow-induced protein kinase A–CREB pathway acts via BMP signaling to promote HSC emergence

PubMed Central

Kim, Peter Geon; Nakano, Haruko; Das, Partha P.; Chen, Michael J.; Rowe, R. Grant; Chou, Stephanie S.; Ross, Samantha J.; Sakamoto, Kathleen M.; Zon, Leonard I.; Schlaeger, Thorsten M.; Orkin, Stuart H.; Nakano, Atsushi

2015-01-01

Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta–gonad–mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)–cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA–CREB and BMP pathways in isolated AGM VE-cadherin+ cells from mid-gestation embryos, we demonstrate that PKA–CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA–CREB–BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition. PMID:25870201

PW1 gene/paternally expressed gene 3 (PW1/Peg3) identifies multiple adult stem and progenitor cell populations

PubMed Central

Besson, Vanessa; Smeriglio, Piera; Wegener, Amélie; Relaix, Frédéric; Nait Oumesmar, Brahim; Sassoon, David A.; Marazzi, Giovanna

2011-01-01

A variety of markers are invaluable for identifying and purifying stem/progenitor cells. Here we report the generation of a murine reporter line driven by Pw1 that reveals cycling and quiescent progenitor/stem cells in all adult tissues thus far examined, including the intestine, blood, testis, central nervous system, bone, skeletal muscle, and skin. Neurospheres generated from the adult PW1-reporter mouse show near 100% reporter-gene expression following a single passage. Furthermore, epidermal stem cells can be purified solely on the basis of reporter-gene expression. These cells are clonogenic, repopulate the epidermal stem-cell niches, and give rise to new hair follicles. Finally, we demonstrate that only PW1 reporter-expressing epidermal cells give rise to follicles that are capable of self-renewal following injury. Our data demonstrate that PW1 serves as an invaluable marker for competent self-renewing stem cells in a wide array of adult tissues, and the PW1-reporter mouse serves as a tool for rapid stem cell isolation and characterization. PMID:21709251

A Rapid Survival Assay to Measure Drug-Induced Cytotoxicity and Cell Cycle Effects

PubMed Central

Valiathan, Chandni; McFaline, Jose L.

2012-01-01

We describe a rapid method to accurately measure the cytotoxicity of mammalian cells upon exposure to various drugs. Using this assay, we obtain survival data in a fraction of the time required to perform the traditional clonogenic survival assay, considered the gold standard. The dynamic range of the assay allows sensitivity measurements on a multi-log scale allowing better resolution of comparative sensitivities. Moreover, the results obtained contain additional information on cell cycle effects of the drug treatment. Cell survival is obtained from a quantitative comparison of proliferation between drug-treated and untreated cells. During the assay, cells are treated with a drug and, following a recovery period, allowed to proliferate in the presence of BrdU. Cells that synthesize DNA in the presence of bromodeoxyuridine (BrdU) exhibit quenched Hoechst fluorescence easily detected by flow cytometry; quenching is used to determine relative proliferation in treated versus untreated cells. Finally, the multi-well setup of this assay allows the simultaneous screening of multiple cell lines, multiple doses, or multiple drugs to accurately measure cell survival and cell cycle changes after drug treatment. PMID:22133811

Molecular Validation of PACE4 as a Target in Prostate Cancer12

PubMed Central

D'Anjou, François; Routhier, Sophie; Perreault, Jean-Pierre; Latil, Alain; Bonnel, David; Fournier, Isabelle; Salzet, Michel; Day, Robert

2011-01-01

Prostate cancer remains the single most prevalent cancer in men. Standard therapies are still limited and include androgen ablation that initially causes tumor regression. However, tumor cells eventually relapse and develop into a hormone-refractory prostate cancer. One of the current challenges in this disease is to define new therapeutic targets, which have been virtually unchanged in the past 30 years. Recent studies have suggested that the family of enzymes known as the proprotein convertases (PCs) is involved in various types of cancers and their progression. The present study examined PC expression in prostate cancer and validates one PC, namely PACE4, as a target. The evidence includes the observed high expression of PACE4 in all different clinical stages of human prostate tumor tissues. Gene silencing studies targeting PACE4 in the DU145 prostate cancer cell line produced cells (cell line 4-2) with slower proliferation rates, reduced clonogenic activity, and inability to grow as xenografts in nude mice. Gene expression and proteomic profiling of the 4-2 cell line reveals an increased expression of known cancer-related genes (e.g., GJA1, CD44, IGFBP6) that are downregulated in prostate cancer. Similarly, cancer genes whose expression is decreased in the 4-2 cell line were upregulated in prostate cancer (e.g., MUC1, IL6). The direct role of PACE4 in prostate cancer is most likely through the upregulated processing of growth factors or through the aberrant processing of growth factors leading to sustained cancer progression, suggesting that PACE4 holds a central role in prostate cancer. PMID:21633671

Radiosensitivity of Patient-Derived Glioma Stem Cell 3-Dimensional Cultures to Photon, Proton, and Carbon Irradiation

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

Chiblak, Sara; Tang, Zili; Molecular and Translational Radiation Oncology, Heidelberg Ion Therapy Center, Heidelberg Institute of Radiation Oncology, University of Heidelberg Medical School and National Center for Tumor Diseases, German Cancer Research Center, Heidelberg

Purpose: To investigate the radiosensitivity of primary glioma stem cell (GSC) cultures with different CD133 status in a 3-dimensional (3D) model after photon versus proton versus carbon irradiation. Methods and Materials: Human primary GSC spheroid cultures were established from tumor specimens of six consented glioblastoma patients. Human U87MG was used as a classical glioblastoma radioresistant cell line. Cell suspensions were generated by mechanical dissociation of GSC spheroids and embedded in a semi-solid 3D matrix before irradiation. Spheroid-like colonies were manually counted by microscopy. Cells were also recovered and quantified by fluorescence. CD133 expression and DNA damage were evaluated by flow cytometry.more » Results: The fraction of CD133{sup +} cells varied between 0.014% and 96% in the six GSC cultures and showed a nonsignificant correlation with plating efficiency and survival fractions. The 4 most photon-radioresistant GSC cultures were NCH644, NCH421k, NCH441, and NCH636. Clonogenic survival for proton irradiation revealed relative biologic effectiveness (RBE) in the range of 0.7-1.20. However, carbon irradiation rendered the photon-resistant GSC cultures sensitive, with average RBE of 1.87-3.44. This effect was partly attributed to impaired capability of GSC to repair carbon ion–induced DNA double-strand breaks as determined by residual DNA repair foci. Interestingly, radiosensitivity of U87 cells was comparable to GSC cultures using clonogenic survival as the standard readout. Conclusions: Carbon irradiation is effective in GSC eradication with similar RBE ranges approximately 2-3 as compared with non-stem GSC cultures (U87). Our data strongly suggest further exploration of GSC using classic radiobiology endpoints such as the here-used 3D clonogenic survival assay and integration of additional GSC-specific markers.« less

Protective Properties of Radio-Chemoresistant Glioblastoma Stem Cell Clones Are Associated with Metabolic Adaptation to Reduced Glucose Dependence

PubMed Central

Yamada, Kazunari; Tso, Jonathan L.; Menjivar, Jimmy C.; Tian, Jane Y.; Yong, William H.; Schaue, Dörthe; Mischel, Paul S.; Cloughesy, Timothy F.; Nelson, Stanley F.; Liau, Linda M.; McBride, William; Tso, Cho-Lea

2013-01-01

Glioblastoma stem cells (GSC) are a significant cell model for explaining brain tumor recurrence. However, mechanisms underlying their radiochemoresistance remain obscure. Here we show that most clonogenic cells in GSC cultures are sensitive to radiation treatment (RT) with or without temozolomide (TMZ). Only a few single cells survive treatment and regain their self-repopulating capacity. Cells re-populated from treatment-resistant GSC clones contain more clonogenic cells compared to those grown from treatment-sensitive GSC clones, and repeated treatment cycles rapidly enriched clonogenic survival. When compared to sensitive clones, resistant clones exhibited slower tumor development in animals. Upregulated genes identified in resistant clones via comparative expression microarray analysis characterized cells under metabolic stress, including blocked glucose uptake, impaired insulin/Akt signaling, enhanced lipid catabolism and oxidative stress, and suppressed growth and inflammation. Moreover, many upregulated genes highlighted maintenance and repair activities, including detoxifying lipid peroxidation products, activating lysosomal autophagy/ubiquitin-proteasome pathways, and enhancing telomere maintenance and DNA repair, closely resembling the anti-aging effects of caloric/glucose restriction (CR/GR), a nutritional intervention that is known to increase lifespan and stress resistance in model organisms. Although treatment–introduced genetic mutations were detected in resistant clones, all resistant and sensitive clones were subclassified to either proneural (PN) or mesenchymal (MES) glioblastoma subtype based on their expression profiles. Functional assays demonstrated the association of treatment resistance with energy stress, including reduced glucose uptake, fatty acid oxidation (FAO)-dependent ATP maintenance, elevated reactive oxygen species (ROS) production and autophagic activity, and increased AMPK activity and NAD+ levels accompanied by upregulated mRNA levels of SIRT1/PGC-1α axis and DNA repair genes. These data support the view that treatment resistance may arise from quiescent GSC exhibiting a GR-like phenotype, and suggest that targeting stress response pathways of resistant GSC may provide a novel strategy in combination with standard treatment for glioblastoma. PMID:24260384

Metabolic profiling and correlation analysis for the determination of killer compounds of proliferating and clonogenic HRT-18 colon cancer cells from Lafoensia pacari.

PubMed

Reichert, Cristiane Loiva; da Silva, Denise Brentan; Carollo, Carlos Alexandre; Weffort-Santos, Almeriane Maria; de Moraes Santos, Cid Aimbiré

2018-06-18

Lafoensia pacari A. St.-Hil., belonging to the family Lythraceae and popularly known as 'dedaleira' and 'mangava-brava,' is a native tree of the Brazilian Cerrado, and its barks have been traditionally used as a tonic to treat inflammatory conditions, particularly related to gastric ulcers, wounds or fevers and various types of cancer. We have previously demonstrated the apoptogenic effects of the methanolic extract of L. pacari using various cancer cell lines. In the present study, this extract has been partitioned into fractions to identify the components that might be responsible for the apoptogenic effects using HRT-18 cells, which have been previously demonstrated to be sensitive to this extract. A standard methanolic extract was prepared and fractionated by centrifugal partition chromatography. The fractions were submitted to cytotoxicity and clonogenic assays to monitor the effects in parallel with LC-DAD-MS and statistical analyses to suggest the potential bioactive compounds. Besides ellagic acid, the primary constituent of the plant and also the biomarker of the species, one punicalin isomer, three pedunculagin I isomers, two castalagin isomers, three punicalagin HHDP-gallagyl-hexoside isomers, one ellagic acid deoxyhexose conjugate and one methyl ellagic acid deoxyhexose conjugate were putatively identified. The barks of L. pacari are rich in ellagic acid and various hydrolysable tannins, some of which were reported for the first time in this species, such as punicalagin and ellagitannins. This mixture of substances had the ability to kill proliferating cells and abrogate the growth of clonogenic cells in a similar manner shown by the methanolic extract of our previous study. The collective data reported herein suggest that the biological activities of the L. pacari barks used by the Cerrado's population to treat cancer conditions are due to the apoptogenic effects promoted by a mixed content of ellagitannins. Copyright © 2018. Published by Elsevier B.V.

WE-G-BRE-08: Radiosensitization by Olaparib Eluting Nanospheres

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

Tangutoori, S; Kumar, R; Sridhar, S

2014-06-15

Purpose: Permanent prostate brachytherapy often uses inert bio-absorbable spacers to achieve the desired geometric distribution of sources within the prostate. Transforming these spacers into implantable nanoplatforms for chemo-radiation therapy (INCeRT) provides a means of providing sustained in-situ release of radiosensitizers in the prostate to enhance the therapeutic ratio of the procedure. Olaparib, a PARP inhibitor, suppresses DNA repair processes present during low dose rate continuous irradiation. This work investigates the radiosensitizing/DNA damage repair inhibition by NanoOlaparib eluting nanospheres. Methods: Human cell line PC3 (from ATCC), was maintained in F12-k medium supplemented with fetal bovine serum. Clonogenic assay kit (from Fischermore » Scientific) was used to fix and stain the cells to determine the long term effects of irradiation. Nanoparticle size and zeta potential of nanospheres were determined using a Zeta particle size analyzer. The incorporation of Olaparib in nanospheres was evaluated by HPLC. Irradiation was performed in a small animal irradiator operating at 220 KeV.The long term effects of radio-sensitization with olaparib and nanoolaparib was determined using the clonogenic assay at 2 Gy and 4 Gy doses. The cells were allowed to grow for around 10 doubling cycles, The colonies were fixed and stained using clonogenic assay kit. The excess stain was washed off using DI water and the images were taken using a digital camera. Results: Radiosensitization studies were carried out in prostate cancer cell line, PC3 radiation at 0, 2 and 4Gy doses. Strongest dose response was observed with nanoolaparib treated cells compared to untreated cells. Conclusion: A two stage drug release of drug eluting nanospheres from a biodegradable spacer has been suggested for sustained in-situ release of Olaparib to suppress DNA repair processes during prostate brachytherapy. The Olaparib eluting nanospheres had the same in-vitro radiosensitizing effect as free olaparib. DOD 1R21CA16977501, A. David Mazzone Awards Program 2012PD164.« less

A Caspase-Resistant Form of Bcl-XL, but Not Wild Type Bcl-XL, Promotes Clonogenic Survival After Ionizing Radiation

PubMed Central

Rehemtulla, Alnawaz; Hamilton, A Christin; Taneja, Neelam; Fridman, Jordan; Juan, Todd SC; Maybaum, Jonathan; Chinnaiyan, Arul

1999-01-01

Abstract Bcl-2 and Bcl-XL belong to a family of proteins overexpressed in a variety of human cancers which inhibit apoptosis in response to a number of stimuli including chemotherapeutic agents and ionizing radiation. To better understand the role of these polypeptides in modulating the response of cancer cells to ionizing radiation we used cell lines that were engineered to overexpress the two polypeptides. Although Bcl-2 and Bcl-XL overexpression resulted in inhibition of radiation-induced apoptosis, it did not result in enhanced clonogenic survival. Consistent with this was the observation that Bcl-2 and Bcl-XL protected cells from DNA fragmentation, loss of mitochondrial membrane potential, and caspase activation for up to 72 hours after irradiation. Beyond 72 hours, there was a rapid loss in the ability of Bcl-2 and Bcl-XL to inhibit these markers of apoptosis. When Bcl-XL was analyzed at 72 hours after irradiation and beyond, a rapid accumulation of a 16-kDa form of Bcl-XL was observed. To test the hypothesis that cleavage of the 29-kDa form of Bcl-XL by caspases to a 16-kDa polypeptide results in its inability to inhibit apoptosis beyond 72 hours, we constructed a cell line that overexpressed a caspase-resistant form of Bcl-XL Bcl-XLΔloop. Cells overexpressing Bcl-XL-Δloop were resistant to apoptosis beyond 72 hours after irradiation and did not contain the 16-kDa form at these time points. In addition, Bcl-XL-Δloop overexpression resulted in enhanced clonogenic survival compared with control or Bcl-XL overexpressing cells. These results provide a molecular basis for the observation that expression of Bcl-2 or Bcl-XL is not a prognostic marker of tumor response to cancer therapy. PMID:10935471

1,25D3 enhances antitumor activity of gemcitabine and cisplatin in human bladder cancer models

PubMed Central

Ma, Yingyu; Yu, Wei-Dong; Trump, Donald L.; Johnson, Candace S.

2010-01-01

Background 1,25 dihydroxyvitamin D3 (1,25D3) potentiates the cytotoxic effects of several common chemotherapeutic agents. The combination of gemcitabine and cisplatin (GC) is a current standard chemotherapy regimen for bladder cancer. We investigated whether 1,25D3 could enhance the antitumor activity of GC in bladder cancer model systems. Methods Human bladder cancer T24 and UMUC3 cells were pretreated with 1,25D3 followed by GC. Apoptosis were assessed by annexin V staining. Caspase activation was examined by immunoblot analysis and substrate-based caspase activity assay. The cytotoxic effects were examined using MTT and in vitro clonogenic assay. p73 protein levels were assessed by immunoblot analysis. Knockdown of p73 was achieved by siRNA. The in vivo antitumor activity was assessed by in vivo excision clonogenic assay and tumor regrowth delay in the T24 xenograft model. Results 1,25D3 pretreatment enhanced GC-induced apoptosis and the activities of caspases- 8, 9 and 3 in T24 and UMUC3 cells. 1,25D3 synergistically reduced GC-suppressed surviving fraction in T24 cells. 1,25D3, gemcitabine, or cisplatin induced p73 accumulation, which was enhanced by GC or 1,25D3 and GC. p73 expression was lower in human primary bladder tumor tissue compared with adjacent normal tissue. Knockdown of p73 increased clonogenic capacity of T24 cells treated with 1,25D3, GC or 1,25D3 and GC. 1,25D3 and GC combination enhanced tumor regression compared with 1,25D3 or GC alone. Conclusions 1,25D3 potentiates GC-mediated growth inhibition in human bladder cancer models in vitro and in vivo, which involves p73 induction and apoptosis. PMID:20564622

Combinatorial cytotoxic effects of Curcuma longa and Zingiber officinale on the PC-3M prostate cancer cell line

PubMed Central

Kurapati, Kesava Rao V.; Samikkannu, Thangavel; Kadiyala, Dakshayani B.; Zainulabedin, Saiyed M.; Gandhi, Nimisha; Sathaye, Sadhana S.; Indap, Manohar A.; Boukli, Nawal; Rodriguez, Jose W.; Nair, Madhavan P.N.

2015-01-01

Background Many plant-derived products exhibit potent chemopreventive activity against animal tumor models as well as rodent and human cancer cell lines. They have low side effects and toxicity and presumably modulate the factors that are critical for cell proliferation, differentiation, senescence and apoptosis. The present study investigates the effects of some medicinal plant extracts from generally recognized as safe plants that may be useful in the prevention and treatment of cancer. Methods Clonogenic assays using logarithmically-growing cells were performed to test the effect. The cytotoxic effects of Curcuma longa and Zingiber officinale were studied using sulforhodamine B assay, tetrazolium dye assay, colony morphology and microscopic analysis. Results Out of the 13 lyophilized plant-derived extracts evaluated for growth-inhibitory effects on the PC-3M prostate cancer cell line, two extracts derived from C. longa and Z. officinale showed significant inhibitory effects on colony-forming ability. The individual and augmentative effects of these two extracts were tested for their narrow range effective lower concentration on PC-3M in clonogenic assays. At relatively lower concentrations, C. longa showed significant inhibition of colony formation in clonogenic assays; whereas at same concentrations Z. officinale showed only moderate inhibitory effects. However, when both the agents were tested together at the same concentrations, the combined effects were much more significant than their individual ones. On normal prostate epithelial cells both C. longa and Z. officinale had similar effects but at a lower magnitude. These observations were confirmed by several cytotoxicity assays involving the morphological appearance of the colonies, microscopic observations, per cent inhibition in comparison to control by sulforhodamine B and tetrazolium dye assay. Conclusions From these observations, it was concluded that the combined effects of C. longa and Z. officinale are much greater than their individual effects, suggesting the role of multiple components and their synergistic mode of actions to elicit stronger beneficial effects. PMID:23072849

Predicting the 5-Year Risk of Biochemical Relapse After Postprostatectomy Radiation Therapy in ≥PT2, pN0 Patients With a Comprehensive Tumor Control Probability Model

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

Fiorino, Claudio, E-mail: fiorino.claudio@hsr.it; Broggi, Sara; Fossati, Nicola

Purpose: To fit the individual biochemical recurrence-free survival (bRFS) data from patients treated with postprostatectomy radiation therapy (RT) with a comprehensive tumor control probability (TCP) model. Methods and Materials: Considering pre-RT prostate-specific antigen (PSA) as a surrogate of the number of clonogens, bRFS may be expressed as a function of dose-per-fraction–dependent radiosensitivity (α{sub eff}), the number of clonogens for pre-RT PSA = 1 ng/mL (C), and the fraction of patients who relapse because of clonogens outside the treated volume (K), assumed to depend (linearly or exponentially) on pre-RT PSA and Gleason score (GS). Data from 894 node-negative, ≥pT2, pN0 hormone-naive patients treated withmore » adjuvant (n=331) or salvage (n=563) intent were available: 5-year bRFS data were fitted grouping patients according to GS (<7:392, =7:383, >7:119). Results: The median follow-up time, pre-RT PSA, and dose were 72 months, 0.25 ng/mL, and 66.6 Gy (range 59.4-77.4 Gy), respectively. The best-fit values were 0.23 to 0.26 Gy{sup −1} and 10{sup 7} for α{sub eff} and C for the model considering a linear dependence between K and PSA. Calibration plots showed good agreement between expected and observed incidences (slope: 0.90-0.93) and moderately high discriminative power (area under the curve [AUC]: 0.68-0.69). Cross-validation showed satisfactory results (average AUCs in the training/validation groups: 0.66-0.70). The resulting dose-effect curves strongly depend on pre-RT PSA and GS. bRFS rapidly decreases with PSA: the maximum obtainable bRFS (defined as 95% of the maximum) declined by about 2.7% and 4.5% for each increment of 0.1 ng/mL for GS <7 and ≥7, respectively. Conclusions: Individual data were fitted by a TCP model, and the resulting best-fit parameters were radiobiologically consistent. The model suggests that relapses frequently result from clonogens outside the irradiated volume, supporting the choice of lymph-node irradiation, systemic therapy, or both for specific subgroups (GS <7: PSA >0.8-1.0 ng/mL; GS ≥7: PSA >0.3 ng/mL). Early RT should be preferred over delayed RT; the detrimental effect of PSA increase can never be fully compensated by increasing the dose, especially for patients with GS ≥7.« less

Final Progress Report for Ionospheric Dusty Plasma In the Laboratory [Smokey Plasma

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

Robertson, Scott

2010-07-31

“Ionospheric Dusty Plasma in the Laboratory” is a research project with the purpose of finding and reproducing the characteristics of plasma in the polar mesosphere that is unusually cold (down to 140 K) and contains nanometer-sized dust particles. This final progress report summarizes results from four years of effort that include a final year with a no-cost extension.

DNA Topoisomerase I-Targeted Therapy for Breast Cancer

DTIC Science & Technology

1997-06-01

Fort Detrick, Frederick, MD 21702-5012. 13. ABSTRACT (Maximum 200 words] Camptothecin analogues have been developed that show enhanced pre-clinical...activity against breast cancer cells. These analogues have been synthesized with two fundamental modifications that aid in their effectiveness against...activity. The new analogues , in both growth inhibition and clonogenic assays, are substantially more effective against breast cancer cells than those

Augmentation of the Differentiation Response to Antitumor Antimalarials

DTIC Science & Technology

2004-07-01

Release; Distribution Unlimited 13. ABSTRACT (Maximum 200 Words) We have shown that the quinoline antimalarials chloroquine (CQ) and hydroxychloroquine (HCQ...Introduction: Preliminary studies showed that two of the quinoline antimalarials, chloroquine (CQ) and hydroxychloroquine (HCQ), displayed selective... hydroxychloroquine upon pretreatment with ATRA or Aza on tumor cell survival (Figures 1 and 2, respectively). Clonogenic survival of MDA-MB-231 cells exposed to

MiR-593 mediates curcumin-induced radiosensitization of nasopharyngeal carcinoma cells via MDR1.

PubMed

Fan, Haoning; Shao, Meng; Huang, Shaohui; Liu, Ying; Liu, Jie; Wang, Zhiyuan; Diao, Jianxin; Liu, Yuanliang; Tong, L I; Fan, Qin

2016-06-01

Curcumin (Cur) exhibits radiosensitization effects to a variety of malignant tumors. The present study investigates the radiosensitizing effect of Cur on nasopharyngeal carcinoma (NPC) cells and whether its mechanism is associated with microRNA-593 (miR-593) and multidrug resistance gene 1 (MDR1). A clonogenic assay was performed to measure the radiosensitizing effect. The expression of miR-593 and MDR1 was analyzed by quantitative polymerase chain reaction (qPCR) or western blot assay. A transplanted tumor model was established to identify the radiosensitizing effect in vivo . A luciferase-based reporter was constructed to evaluate the effect of direct binding of miR-593 to the putative target site on the 3' UTR of MDR1. The clonogenic assay showed that Cur enhanced the radiosensitivity of cells. Cur (100 mg/kg) combined with 4 Gy irradiation inhibited the growth of a transplanted tumor model in vivo , resulting in the higher inhibition ratio compared with the radiotherapy-alone group. These results demonstrated that Cur had a radiosensitizing effect on NPC cells in vivo and in vitro ; Cur-mediated upregulation of miR-593 resulted in reduced MDR1 expression, which may promote radiosensitivity of NPC cells.

Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines.

PubMed

Yu, Vicky; Rahimy, Mehran; Korrapati, Avinaash; Xuan, Yinan; Zou, Angela E; Krishnan, Aswini R; Tsui, Tzuhan; Aguilera, Joseph A; Advani, Sunil; Crotty Alexander, Laura E; Brumund, Kevin T; Wang-Rodriguez, Jessica; Ongkeko, Weg M

2016-01-01

Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 h to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cafestol, a diterpene molecule found in coffee, induces leukemia cell death.

PubMed

Lima, Cauê S; Spindola, Daniel G; Bechara, Alexandre; Garcia, Daniel M; Palmeira-Dos-Santos, Caroline; Peixoto-da-Silva, Janaina; Erustes, Adolfo G; Michelin, Luis F G; Pereira, Gustavo J S; Smaili, Soraya S; Paredes-Gamero, Edgar; Calgarotto, Andrana K; Oliveira, Carlos R; Bincoletto, Claudia

2017-08-01

To evaluate the antitumor properties of Cafestol four leukemia cell lines were used (NB4, K562, HL60 and KG1). Cafestol exhibited the highest cytotoxicity against HL60 and KG1 cells, as evidenced by the accumulation of cells in the sub-G1 fraction, mitochondrial membrane potential reduction, accumulation of cleaved caspase-3 and phosphatidylserine externalization. An increase in CD11b and CD15 differentiation markers with attenuated ROS generation was also observed in Cafestol-treated HL60 cells. These results were similar to those obtained following exposure of the same cell line to cytarabine (Ara-C), an antileukemic drug. Cafestol and Ara-C reduced the clonogenic potential of HL60 cells by 100%, but Cafestol spared murine colony forming unit- granulocyte/macrophage (CFU-GM), which retained their clonogenicity. The co-treatment of Cafestol and Ara-C reduced HL60 cell viability compared with both drugs administered alone. In conclusion, despite the distinct molecular mechanisms involved in the activity of Cafestol and Ara-C, a similar cytotoxicity towards leukemia cells was observed, which suggests a need for prophylactic-therapeutic pre-clinical studies regarding the anticancer properties of Cafestol. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines

PubMed Central

Yu, Vicky; Rahimy, Mehran; Korrapati, Avinaash; Xuan, Yinan; Zou, Angela E.; Krishnan, Aswini R.; Tsui, Tzuhan; Aguilera, Joseph A.; Advani, Sunil; Crotty Alexander, Laura E.; Brumund, Kevin T.; Wang-Rodriguez, Jessica

2016-01-01

Objectives Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. Materials and Methods HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 hours to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. Results E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. Conclusion E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed. PMID:26547127

Survival and characteristics of murine leukaemic and normal stem cells after hyperthermia: a murine model for human bone marrow purging.

PubMed

Gidáli, J; Szamosvölgyi, S; Fehér, I; Kovács, P

1990-01-01

The effect of hyperthermia in vitro on the survival and leukaemogenic effectiveness of WEHI 3-B cells and on the survival and transplantation efficiency of bone marrow cells was compared in a murine model system. Normal murine clonogenic haemopoietic cells (day 9 CFU-S and CFU-GM) proved to be significantly less sensitive to 42.5 degrees C hyperthermia (Do values: 54.3 and 41.1 min, respectively) than leukaemic clonogenic cells (CFU-L) derived from suspension culture or from bone marrow of leukaemic mice (Do: 17.8 min). Exposure for 120 min to 42.5 degrees C reduced the surviving fraction of CFU-L to 0.002 and that of CFU-S to 0.2. If comparable graft sizes were transplanted from normal or heat exposed bone marrow, 60-day survival of supralethally irradiated mice was similar. Surviving WEHI 3-B cells were capable of inducing leukaemia in vivo. The two log difference in the surviving fraction of CFU-L and CFU-S after 120 min exposure to 42.5 degrees C suggests that hyperthermia ex vivo may be a suitable purging method for autologous bone marrow transplantation.

PSI-Center Final Progress Report

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

Jarboe, Thomas R.; Shumlak, Uri; Sovinec, Carl

This is the Final Progress Report of the Plasma Science and Innovation Center (PSI-Center) covering March 2014 through February 2017. The Center has accomplished a great deal during this period. The PSI-Center is organized into four groups: Edge and Dynamic Neutrals; Transport and Kinetic Effects; Equilibrium, Stability, and Kinetic Effects in 3D Topologies; and Interface for Validation. Each group has made good progress and the results from each group are given in detail.

Altered Redox Status Accompanies Progression to Metastatic Human Bladder Cancer

PubMed Central

Hempel, Nadine; Ye, Hanqing; Abessia, Bryan; Mian, Badar; Melendez, J. Andres

2009-01-01

The role of reactive oxygen species (ROS) in bladder cancer progression remains an unexplored field. Expression levels of enzymes regulating ROS levels are often altered in cancer. Search of publicly available micro-array data reveals that expression of mitochondrial manganese superoxide dismutase (Sod2), responsible for the conversion of superoxide (O2-.) to hydrogen peroxide (H2O2), is consistently increased in high grade and advanced stage bladder tumors. Here we aim to identify the role of Sod2 expression and ROS in bladder cancer. Using an in vitro human bladder tumor model we monitored the redox state of both non-metastatic (253J) and highly metastatic (253J B-V) bladder tumor cell lines. 253J B-V cells displayed significantly higher Sod2 protein and activity levels compared to their parental 253J cell line. The increase in Sod2 expression was accompanied by a significant decrease in catalase activity, resulting in a net increase in H2O2 production in the 253J B-V line. Expression of pro-metastatic and –angiogenic factors, matrix metalloproteinase 9 (MMP-9) and vascular endothelial derived growth factor (VEGF), respectively, were similarly upregulated in the metastatic line. Expression of both MMP-9 and VEGF were shown to be H2O2-dependent, as removal of H2O2 by overexpression of catalase attenuated their expression. Similarly, expression of catalase effectively reduced the clonogenic activity of 253J B-V cells. These findings indicate that metastatic bladder cancer cells display an altered antioxidant expression profile, resulting in a net increase in ROS production, which leads to the induction of redox-sensitive pro-tumorigenic and pro-metastatic genes such as VEGF and MMP-9. PMID:18930813

Purging of the neuroblastoma stem cell compartment and tumor regression on exposure to hypoxia or cytotoxic treatment.

PubMed

Marzi, Ilaria; D'Amico, Massimo; Biagiotti, Tiziana; Giunti, Serena; Carbone, Maria Vittoria; Fredducci, David; Wanke, Enzo; Olivotto, Massimo

2007-03-15

We worked out an experimental protocol able to purge the stem cell compartment of the SH-SY5Y neuroblastoma clone. This protocol was based on the prolonged treatment of the wild-type cell population with either hypoxia or the antiblastic etoposide. Cell fate was monitored by immunocytochemical and electrophysiologic (patch-clamp) techniques. Both treatments produced the progressive disappearance of neuronal type (N) cells (which constitute the bulk of the tumor), leaving space for a special category of epithelial-like substrate-adherent cells (S(0)). The latter represent a minimal cell component of the untreated population and are endowed with immunocytochemical markers (p75, c-kit, and CD133) and the electrophysiologic "nude" profile, typical of the neural crest stem cells. S(0) cells displayed a highly clonogenic potency and a substantial plasticity, generating both the N component and an alternative subpopulation terminally committed to the fibromuscular lineage. Unlike the N component, this lineage was highly insensitive to the apoptotic activity of hypoxia and etoposide and developed only when the neuronal option was abolished. Under these conditions, the fibromuscular progeny of S(0) expanded and progressed up to the exhaustion of the staminal compartment and to the extinction of the tumor. When combined, hypoxia and etoposide cooperated in abolishing the N cell generation and promoting the conversion of the tumor described. This synergy might mirror a natural condition in the ischemic areas occurring in cancer. These results have relevant implications for the understanding of the documented tendency of neuroblastomas to regress from a malignant to a benign phenotype, either spontaneously or on antiblastic treatment.

Differential cellular responses to prolonged LDR-IR in MLH1-proficient and MLH1-deficient colorectal cancer HCT116 cells.

PubMed

Yan, Tao; Seo, Yuji; Kinsella, Timothy J

2009-11-15

MLH1 is a key DNA mismatch repair (MMR) protein involved in maintaining genomic stability by participating in the repair of endogenous and exogenous mispairs in the daughter strands during S phase. Exogenous mispairs can result following treatment with several classes of chemotherapeutic drugs, as well as with ionizing radiation. In this study, we investigated the role of the MLH1 protein in determining the cellular and molecular responses to prolonged low-dose rate ionizing radiation (LDR-IR), which is similar to the clinical use of cancer brachytherapy. An isogenic pair of MMR(+) (MLH1(+)) and MMR(-) (MLH1(-)) human colorectal cancer HCT116 cells was exposed to prolonged LDR-IR (1.3-17 cGy/h x 24-96 h). The clonogenic survival and gene mutation rates were examined. Cell cycle distribution was analyzed with flow cytometry. Changes in selected DNA damage repair proteins, DNA damage response proteins, and cell death marker proteins were examined with Western blotting. MLH1(+) HCT116 cells showed greater radiosensitivity with enhanced expression of apoptotic and autophagic markers, a reduced HPRT gene mutation rate, and more pronounced cell cycle alterations (increased late-S population and a G(2)/M arrest) following LDR-IR compared with MLH1(-) HCT116 cells. Importantly, a progressive increase in MLH1 protein levels was found in MLH1(+) cells during prolonged LDR-IR, which was temporally correlated with a progressive decrease in Rad51 protein (involved in homologous recombination) levels. MLH1 status significantly affects cellular responses to prolonged LDR-IR. MLH1 may enhance cell radiosensitivity to prolonged LDR-IR through inhibition of homologous recombination (through inhibition of Rad51).

Identification of novel posttranscriptional targets of the BCR/ABL oncoprotein by ribonomics: requirement of E2F3 for BCR/ABL leukemogenesis

PubMed Central

Eiring, Anna M.; Neviani, Paolo; Santhanam, Ramasamy; Oaks, Joshua J.; Chang, Ji Suk; Notari, Mario; Willis, William; Gambacorti-Passerini, Carlo; Volinia, Stefano; Marcucci, Guido; Caligiuri, Michael A.; Leone, Gustavo W.

2008-01-01

Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are aberrantly regulated at transcriptional or posttranslational levels by the constitutive kinase activity of the BCR/ABL oncoprotein. As a result, altered expression/function of RBPs leads to increased resistance to apoptotic stimuli, enhanced survival, growth advantage, and differentiation arrest of CD34+ progenitors from patients in CML blast crisis. Here, we identify the mRNAs bound to the hnRNP-A1, hnRNP-E2, hnRNP-K, and La/SSB RBPs in BCR/ABLtransformed myeloid cells. Interestingly, we found that the mRNA encoding the transcription factor E2F3 associates to hnRNP-A1 through a conserved binding site located in the E2F3 3′ untranslated region (UTR). E2F3 levels were up-regulated in CML-BCCD34+ in a BCR/ABL kinase– and hnRNP-A1 shuttling–dependent manner. Moreover, by using shRNA-mediated E2F3 knock-down and BCR/ABL-transduced lineage-negative bone marrow cells from E2F3+/+ and E2F3−/− mice, we show that E2F3 expression is important for BCR/ABL clonogenic activity and in vivo leukemogenic potential. Thus, the complexity of the mRNA/RBP network, together with the discovery of E2F3 as an hnRNP-A1–regulated factor, outlines the relevant role played by RBPs in posttranscriptional regulation of CML development and progression. PMID:17925491

46 CFR 172.195 - Survival conditions.

Code of Federal Regulations, 2014 CFR

2014-10-01

... assumed damage if it meets the following conditions in the final stage of flooding: (a) Final waterline... of an opening through which progressive flooding may take place, such as an air pipe, or an opening... least 3.94 inches (10 cm). (3) Each submerged opening must be weathertight. (d) Progressive flooding. If...

46 CFR 172.195 - Survival conditions.

Code of Federal Regulations, 2013 CFR

2013-10-01

... assumed damage if it meets the following conditions in the final stage of flooding: (a) Final waterline... of an opening through which progressive flooding may take place, such as an air pipe, or an opening... least 3.94 inches (10 cm). (3) Each submerged opening must be weathertight. (d) Progressive flooding. If...

46 CFR 172.195 - Survival conditions.

Code of Federal Regulations, 2012 CFR

2012-10-01

... assumed damage if it meets the following conditions in the final stage of flooding: (a) Final waterline... of an opening through which progressive flooding may take place, such as an air pipe, or an opening... least 3.94 inches (10 cm). (3) Each submerged opening must be weathertight. (d) Progressive flooding. If...

The small molecule SI113 synergizes with mitotic spindle poisons in arresting the growth of human glioblastoma multiforme

PubMed Central

Abbruzzese, Claudia; Catalogna, Giada; Gallo, Enzo; di Martino, Simona; Mileo, Anna M.; Carosi, Mariantonia; Dattilo, Vincenzo; Schenone, Silvia; Musumeci, Francesca; Lavia, Patrizia; Perrotti, Nicola; Amato, Rosario; Paggi, Marco G.

2017-01-01

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro, as well as inhibiting tumor growth in vivo. We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches. PMID:29340013

Correction of dog dystrophic epidermolysis bullosa by transplantation of genetically modified epidermal autografts.

PubMed

Gache, Yannick; Pin, Didier; Gagnoux-Palacios, Laurent; Carozzo, Claude; Meneguzzi, Guerrino

2011-10-01

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering condition caused by mutations in the gene coding for collagen type VII. Genetically engineered RDEB dog keratinocytes were used to generate autologous epidermal sheets subsequently grafted on two RDEB dogs carrying a homozygous missense mutation in the col7a1 gene and expressing baseline amounts of the aberrant protein. Transplanted cells regenerated a differentiated and vascularized auto-renewing epidermis progressively repopulated by dendritic cells and melanocytes. No adverse immune reaction was detected in either dog. In dog 1, the grafted epidermis firmly adhered to the dermis throughout the 24-month follow-up, which correlated with efficient transduction (100%) of highly clonogenic epithelial cells and sustained transgene expression. In dog 2, less efficient (65%) transduction of primary keratinocytes resulted in a loss of the transplanted epidermis and graft blistering 5 months after transplantation. These data provide the proof of principle for ex vivo gene therapy of RDEB patients with missense mutations in collagen type VII by engraftment of the reconstructed epidermis, and demonstrate that highly efficient transduction of epidermal stem cells is crucial for successful gene therapy of inherited skin diseases in which correction of the genetic defect confers no major selective advantage in cell culture.

The small molecule SI113 synergizes with mitotic spindle poisons in arresting the growth of human glioblastoma multiforme.

PubMed

Abbruzzese, Claudia; Catalogna, Giada; Gallo, Enzo; di Martino, Simona; Mileo, Anna M; Carosi, Mariantonia; Dattilo, Vincenzo; Schenone, Silvia; Musumeci, Francesca; Lavia, Patrizia; Perrotti, Nicola; Amato, Rosario; Paggi, Marco G

2017-12-19

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro , as well as inhibiting tumor growth in vivo . We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches.

Toward computer simulation of high-LET in vitro survival curves.

PubMed

Heuskin, A-C; Michiels, C; Lucas, S

2013-09-21

We developed a Monte Carlo based computer program called MCSC (Monte Carlo Survival Curve) able to predict the survival fraction of cells irradiated in vitro with a broad beam of high linear energy transfer particles. Three types of cell responses are studied: the usual high dose response, the bystander effect and the low-dose hypersensitivity (HRS). The program models the broad beam irradiation and double strand break distribution following Poisson statistics. The progression of cells through the cell cycle is taken into account while the repair takes place. Input parameters are experimentally determined for A549 lung carcinoma cells irradiated with 10 and 20 keV µm(-1) protons, 115 keV µm(-1) alpha particles and for EAhy926 endothelial cells exposed to 115 keV µm(-1) alpha particles. Results of simulations are presented and compared with experimental survival curves obtained for A549 and EAhy296 cells. Results are in good agreement with experimental data for both cell lines and all irradiation protocols. The benefits of MCSC are several: the gain of time that would have been spent performing time-consuming clonogenic assays, the capacity to estimate survival fraction of cell lines not forming colonies and possibly the evaluation of radiosensitivity parameters of given individuals.

Curcumin suppresses oncogenicity of human colon cancer cells by covalently modifying the cysteine 67 residue of SIRT1.

PubMed

Lee, Yeon-Hwa; Song, Na-Young; Suh, Jinyoung; Kim, Do-Hee; Kim, Wonki; Ann, Jihyae; Lee, Jeewoo; Baek, Jeong-Heum; Na, Hye-Kyung; Surh, Young-Joon

2018-05-25

SIRT1, an NAD + -dependent histone/protein deacetylase, has diverse physiological actions. Recent studies have demonstrated that SIRT1 is overexpressed in colorectal cancer, suggesting its oncogenic potential. However, the molecular mechanisms by which overexpressed SIRT1 induces the progression of colorectal cancer and its inhibition remain largely unknown. Curcumin (diferuloymethane), a major component of the spice turmeric derived from the plant Curcuma longa L., has been reported to exert chemopreventive and anti-carcinogenic effects on colon carcinogenesis. In the present study, we found that curcumin reduced the expression of SIRT1 protein without influencing its mRNA expression in human colon cancer cells, suggesting posttranslational regulation of SIRT1 by this phytochemical. Notably, ubiquitination and subsequent proteasomal degradation of SIRT1 were induced by curcumin treatment. Results of nano-LC-ESI-MS/MS revealed the direct binding of curcumin to cysteine 67 of SIRT1. In line with this result, the protein stability and clonogenicity of a mutant SIRT1 in which cysteine 67 was substituted by alanine were unaffected by curcumin. Taken together, these observations suggest that curcumin facilitates the proteasomal degradation of oncogenic SIRT1 through covalent modification of SIRT1 at the cysteine 67 residue. Copyright © 2018. Published by Elsevier B.V.

Three-dimensional culture and differentiation of human osteogenic cells in an injectable hydroxypropylmethylcellulose hydrogel.

PubMed

Trojani, Christophe; Weiss, Pierre; Michiels, Jean-François; Vinatier, Claire; Guicheux, Jérôme; Daculsi, Guy; Gaudray, Patrick; Carle, Georges F; Rochet, Nathalie

2005-09-01

The present work evaluates a newly developed silated hydroxypropylmethylcellulose (Si-HPMC)-based hydrogel as a scaffold for 3D culture of osteogenic cells. The pH variation at room temperature catalyzes the reticulation and self-hardening of the viscous polymer solution into a gelatine state. We designed reticulation time, final consistency and pH in order to obtain an easy handling matrice, suitable for in vitro culture and in vivo injection. Three human osteogenic cell lines and normal human osteogenic (HOST) cells were cultured in 3D inside this Si-HPMC hydrogel. We show here that osteosarcoma cells proliferate as clonogenic spheroids and that HOST colonies survive for at least 3 weeks. Mineralization assay and gene expression analysis of osteoblastic markers and cytokines, indicate that all the cells cultured in 3D into this hydrogel, exhibited a more mature differentiation status than cells cultured in monolayer on plastic. This study demonstrates that this Si-HPMC hydrogel is well suited to support osteoblastic survival, proliferation and differentiation when used as a new scaffold for 3D culture and represents also a potential basis for an innovative bone repair material.

MAPK pathway control of stem cell proliferation and differentiation in the embryonic pituitary provides insights into the pathogenesis of papillary craniopharyngioma

PubMed Central

Pozzi, Sara; Carreno, Gabriela; Manshaei, Saba; Panousopoulos, Leonidas; Gonzalez-Meljem, Jose Mario; Apps, John R.; Virasami, Alex; Thavaraj, Selvam; Gutteridge, Alice; Forshew, Tim; Marais, Richard; Brandner, Sebastian; Jacques, Thomas S.; Andoniadou, Cynthia L.

2017-01-01

Despite the importance of the RAS-RAF-MAPK pathway in normal physiology and disease of numerous organs, its role during pituitary development and tumourigenesis remains largely unknown. Here, we show that the over-activation of the MAPK pathway, through conditional expression of the gain-of-function alleles BrafV600E and KrasG12D in the developing mouse pituitary, results in severe hyperplasia and abnormal morphogenesis of the gland by the end of gestation. Cell-lineage commitment and terminal differentiation are disrupted, leading to a significant reduction in numbers of most of the hormone-producing cells before birth, with the exception of corticotrophs. Of note, Sox2+ stem cells and clonogenic potential are drastically increased in the mutant pituitaries. Finally, we reveal that papillary craniopharyngioma (PCP), a benign human pituitary tumour harbouring BRAF p.V600E also contains Sox2+ cells with sustained proliferative capacity and disrupted pituitary differentiation. Together, our data demonstrate a crucial function of the MAPK pathway in controlling the balance between proliferation and differentiation of Sox2+ cells and suggest that persistent proliferative capacity of Sox2+ cells may underlie the pathogenesis of PCP. PMID:28506993

Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers.

PubMed

De, Pradip; Carlson, Jennifer H; Wu, Hui; Marcus, Adam; Leyland-Jones, Brian; Dey, Nandini

2016-07-12

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronectin-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP.

Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers

PubMed Central

De, Pradip; Carlson, Jennifer H.; Wu, Hui; Marcus, Adam; Leyland-Jones, Brian; Dey, Nandini

2016-01-01

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronection-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP. PMID:27281609

The vascular disrupting agent ZD6126 shows increased antitumor efficacy and enhanced radiation response in large, advanced tumors

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

Siemann, Dietmar W.; Rojiani, Amyn M.

2005-07-01

Purpose: ZD6126 is a vascular-targeting agent that induces selective effects on the morphology of proliferating and immature endothelial cells by disrupting the tubulin cytoskeleton. The efficacy of ZD6126 was investigated in large vs. small tumors in a variety of animal models. Methods and Materials: Three rodent tumor models (KHT, SCCVII, RIF-1) and three human tumor xenografts (Caki-1, KSY-1, SKBR3) were used. Mice bearing leg tumors ranging in size from 0.1-2.0 g were injected intraperitoneally with a single 150 mg/kg dose of ZD6126. The response was assessed by morphologic and morphometric means as well as an in vivo to in vitromore » clonogenic cell survival assay. To examine the impact of tumor size on the extent of enhancement of radiation efficacy by ZD6126, KHT sarcomas of three different sizes were irradiated locally with a range of radiation doses, and cell survival was determined. Results: All rodent tumors and human tumor xenografts evaluated showed a strong correlation between increasing tumor size and treatment effect as determined by clonogenic cell survival. Detailed evaluation of KHT sarcomas treated with ZD6126 showed a reduction in patent tumor blood vessels that was {approx}20% in small (<0.3 g) vs. >90% in large (>1.0 g) tumors. Histologic assessment revealed that the extent of tumor necrosis after ZD6126 treatment, although minimal in small KHT sarcomas, became more extensive with increasing tumor size. Clonogenic cell survival after ZD6126 exposure showed a decrease in tumor surviving fraction from approximately 3 x 10{sup -1} to 1 x 10{sup -4} with increasing tumor size. When combined with radiotherapy, ZD6126 treatment resulted in little enhancement of the antitumor effect of radiation in small (<0.3 g) tumors but marked increases in cell kill in tumors larger than 1.0 g. Conclusions: Because bulky neoplastic disease is typically the most difficult to manage, the present findings provide further support for the continued development of vascular disrupting agents such as ZD6126 as a vascular-targeted approach to cancer therapy.« less

No adaptive response is induced by chronic low-dose radiation from Ra-226 in the CHSE/F fish embryonic cell line and the HaCaT human epithelial cell line

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

Shi, Xiaopei, E-mail: shix22@mcmaster.ca; Mothersi

Purpose: To determine whether chronic low-dose α-particle radiation from Ra-226 over multiple cell generations can lead to an adaptive response in CHSE/F fish embryonic cells or HaCaT human epithelial cells receiving subsequent acute high-dose γ-ray radiation. Methods: CHSE/F and HaCaT cells were exposed to very low doses of Ra-226 in medium for multiple generations prior to being challenged by a higher dose γ-ray radiation. The clonogenic assay was used to test the clonogenic survival of cells with or without being pretreated by radiation from Ra-226. Results: In general, pretreatment with chronic radiation has no significant influence on the reaction ofmore » cells to the subsequent challenge radiation. Compared to unprimed cells, the change in clonogenic survival of primed cells after receiving challenge radiation is mainly due to the influence of the chronic exposure, and there's little adaptive response induced. However at several dose points, pretreatment of CHSE/F fish cells with chronic radiation resulted in a radiosensitive response to a challenge dose of γ-ray radiation, and pretreatment of HaCaT cells resulted in no effect except for a slightly radioresistant response to the challenge radiation which was not significant. Conclusion: The results suggest that chronic low-dose radiation is not effective enough to induce adaptive response. There was a difference between human and fish cells and it may be important to consider results from multiple species before making conclusions about effects of chronic or low doses of radiation in the environment. The term “radiosensitive” or “adaptive” make no judgment about whether such responses are ultimately beneficial or harmful. - Highlights: • No obvious adaptive response is induced by chronic low-dose radiation from Ra-226. • Priming radiation from Ra-226 sensitized CHSE/F cells to the challenge radiation. • Linear model is inconsistent with current work using chronic low-dose radiation.« less

Radiation-induced biologic bystander effect elicited in vitro by targeted radiopharmaceuticals labeled with alpha-, beta-, and auger electron-emitting radionuclides.

PubMed

Boyd, Marie; Ross, Susan C; Dorrens, Jennifer; Fullerton, Natasha E; Tan, Ker Wei; Zalutsky, Michael R; Mairs, Robert J

2006-06-01

Recent studies have shown that indirect effects of ionizing radiation may contribute significantly to the effectiveness of radiotherapy by sterilizing malignant cells that are not directly hit by the radiation. However, there have been few investigations of the importance of indirect effects in targeted radionuclide treatment. Our purpose was to compare the induction of bystander effects by external beam gamma-radiation with those resultant from exposure to 3 radiohaloanalogs of metaiodobenzylguanidine (MIBG): (131)I-MIBG (low-linear-energy-transfer [LET] beta-emitter), (123)I-MIBG (potentially high-LET Auger electron emitter), and meta-(211)At-astatobenzylguanidine ((211)At-MABG) (high-LET alpha-emitter). Two human tumor cell lines-UVW (glioma) and EJ138 (transitional cell carcinoma of bladder)-were transfected with the noradrenaline transporter (NAT) gene to enable active uptake of MIBG. Medium from cells that accumulated the radiopharmaceuticals or were treated with external beam radiation was transferred to cells that had not been exposed to radioactivity, and clonogenic survival was determined in donor and recipient cultures. Over the dose range 0-9 Gy of external beam radiation of donor cells, 2 Gy caused 30%-40% clonogenic cell kill in recipient cultures. This potency was maintained but not increased by higher dosage. In contrast, no corresponding saturation of bystander cell kill was observed after treatment with a range of activity concentrations of (131)I-MIBG, which resulted in up to 97% death of donor cells. Cellular uptake of (123)I-MIBG and (211)At-MABG induced increasing recipient cell kill up to levels that resulted in direct kill of 35%-70% of clonogens. Thereafter, the administration of higher activity concentrations of these high-LET emitters was inversely related to the kill of recipient cells. Over the range of activity concentrations examined, neither direct nor indirect kill was observed in cultures of cells not expressing the NAT and, thus, incapable of active uptake of MIBG. Potent toxins are generated specifically by cells that concentrate radiohalogenated MIBG. These may be LET dependent and distinct from those elicited by conventional radiotherapy.

Guaranteed Time Observations Support for Goddard High Resolution Spectrograph (GHRS) on HST

NASA Technical Reports Server (NTRS)

Beaver, Edward

1998-01-01

We assemble this final grant report by combining our previously submitted progress reports with the last year's progress report. Section 2 is the progress report for the June 1, 1991 to Nov. 14, 1995 period. Section 4 is the progress report for the Nov. 14, 1996 to Dec. 31, 1996 period. Section 5 is the progress report for the Nov. 14 to Aug. 31, 1997 period. Section 6 is the new progress report for the Sept. 15, 1997 to Nov. 14, 1998 final period. Section 3 is a summary of our spare detector high voltage transient tests activity in 1992 in support of the renewed safe operation of the GHRS HST D1 detector. Note that we have left the format of each progress report the same as originally sent out. The slight differences in format presentation are thus intended.

Effects of neratinib and combination with irradiation and chemotherapy in head and neck cancer cells.

PubMed

Schneider, S; Thurnher, D; Kadletz, L; Seemann, R; Brunner, M; Kotowski, U; Schmid, R; Lill, C; Heiduschka, G

2016-11-01

Prognosis of patients with head and neck squamous cell carcinoma (HNSCC) is still poor. Novel therapeutic approaches are of great interest to improve the effects of radiochemotherapy. We evaluated the effects of tyrosine kinase inhibitor neratinib on HNSCC cell lines CAL27, SCC25 and FaDu as a single agent and in combination with irradiation and chemotherapy. Effects of neratinib were evaluated in HNSCC cell lines CAL27, SCC25 and FaDu. Effect on cell viability of neratinib and combination with cisplatin and irradiation was measured using CCK-8 assays and clonogenic assays. Western blot analysis was performed to distinguish the effect on epithelial growth factor receptor and HER2 expression. Apoptosis was evaluated by flow cytometry analysis. Growth inhibition was achieved in all cell lines, whereas combination of cisplatin and neratinib showed greater inhibition than each agent alone. Apoptosis was induced in all cell lines. Combination of neratinib with irradiation or cisplatin showed significantly increased apoptosis. In clonogenic assays, significant growth inhibition was observed in all investigated cell lines. Neratinib, as a single agent or in combination with chemo-irradiation, may be a promising treatment option for patients with head and neck cancer. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

DNA damage induction and/or repair as mammalian cell biomarker for the prediction of cellular radiation response

NASA Astrophysics Data System (ADS)

Baumstark-Khan, C.

DNA damage and its repair processes are key factors in cancer induction and also in the treatment of malignancies. Cancer prevention during extended space missions becomes a topic of great importance for space radiobiology. The knowledge of individual responsiveness would allow the protection strategy to be tailored optimally in each case. Radiobiological analysis of cultured cells derived from tissue explants from individuals has shown that measurement of the surviving fraction after 2 Gy (SF2) may be used to predict the individual responsiveness. However, clonogenic assays are timeconsuming, thus alternative assays for the determination of radiore-sponse are being sought. For that reason CHO cell strains having different repair capacities were used for examining whether DNA strand break repair is a suitable experimental design to allow predictive statements. Cellular survival (CFA assay) and DNA strand breaks (total DNA strand breaks: FADU technique; DSBs: non-denaturing elution) were determined in parallel immediately after irradiation as well as after a 24 hour recovery period according to dose. There were no correlations between the dose-response curves of the initial level of DNA strand breaks and parameters that describe clonogenic survival curves (SF2). A good correlation exists between intrinsic cellular radioresistance and the extent of residual DNA strand breaks.

Ionizing Radiation-Induced Responses in Human Cells with Differing TP53 Status

PubMed Central

Mirzayans, Razmik; Andrais, Bonnie; Scott, April; Wang, Ying W.; Murray, David

2013-01-01

Ionizing radiation triggers diverse responses in human cells encompassing apoptosis, necrosis, stress-induced premature senescence (SIPS), autophagy, and endopolyploidy (e.g., multinucleation). Most of these responses result in loss of colony-forming ability in the clonogenic survival assay. However, not all modes of so-called clonogenic cell “death” are necessarily advantageous for therapeutic outcome in cancer radiotherapy. For example, the crosstalk between SIPS and autophagy is considered to influence the capacity of the tumor cells to maintain a prolonged state of growth inhibition that unfortunately can be succeeded by tumor regrowth and disease recurrence. Likewise, endopolyploid giant cells are able to segregate into near diploid descendants that continue mitotic activities. Herein we review the current knowledge on the roles that the p53 and p21WAF1 tumor suppressors play in determining the fate of human fibroblasts (normal and Li-Fraumeni syndrome) and solid tumor-derived cells after exposure to ionizing radiation. In addition, we discuss the important role of WIP1, a p53-regulated oncogene, in the temporal regulation of the DNA damage response and its contribution to p53 dynamics post-irradiation. This article highlights the complexity of the DNA damage response and provides an impetus for rethinking the nature of cancer cell resistance to therapeutic agents. PMID:24232458

Cooperation of imipramine blue and tyrosine kinase blockade demonstrates activity against chronic myeloid leukemia

PubMed Central

Laidlaw, Kamilla M.E.; Berhan, Samuel; Liu, Suhu; Silvestri, Giovannino; Holyoake, Tessa L.; Frank, David A.; Aggarwal, Bharat; Bonner, Michael Y.; Perrotti, Danilo

2016-01-01

The use of tyrosine kinase inhibitors (TKI), including nilotinib, has revolutionized the treatment of chronic myeloid leukemia (CML). However current unmet clinical needs include combating activation of additional survival signaling pathways in persistent leukemia stem cells after long-term TKI therapy. A ubiquitous signaling alteration in cancer, including CML, is activation of reactive oxygen species (ROS) signaling, which may potentiate stem cell activity and mediate resistance to both conventional chemotherapy and targeted inhibitors. We have developed a novel nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, imipramine blue (IB) that targets ROS generation. ROS levels are known to be elevated in CML with respect to normal hematopoietic stem/progenitor cells and not corrected by TKI. We demonstrate that IB has additive benefit with nilotinib in inhibiting proliferation, viability, and clonogenic function of TKI-insensitive quiescent CD34+ CML chronic phase (CP) cells while normal CD34+ cells retained their clonogenic capacity in response to this combination therapy in vitro. Mechanistically, the pro-apoptotic activity of IB likely resides in part through its dual ability to block NF-κB and re-activate the tumor suppressor protein phosphatase 2A (PP2A). Combining BCR-ABL1 kinase inhibition with NADPH oxidase blockade may be beneficial in eradication of CML and worthy of further investigation. PMID:27438151

Damnacanthal, a noni anthraquinone, inhibits c-Met and is a potent antitumor compound against Hep G2 human hepatocellular carcinoma cells.

PubMed

García-Vilas, Javier A; Quesada, Ana R; Medina, Miguel A

2015-01-26

Damnacanthal, an anthraquinone present in noni plants, targets several tyrosine kinases and has antitumoral effects. This study aims at getting additional insight on the potential of damnacanthal as a natural antitumor compound. The direct effect of damnacanthal on c-Met was tested by in vitro activity assays. Additionally, Western blots of c-Met phosphorylation in human hepatocellular carcinoma Hep G2 cells were performed. The antitumor effects of damnacanthal were tested by using cell growth, soft agar clonogenic, migration and invasion assays. Their mechanisms were studied by Western blot, and cell cycle, apoptosis and zymographic assays. Results show that damnacanthal targets c-Met both in vitro and in cell culture. On the other hand, damnacanthal also decreases the phosphorylation levels of Akt and targets matrix metalloproteinase-2 secretion in Hep G2 cells. These molecular effects are accompanied by inhibition of the growth and clonogenic potential of Hep G2 hepatocellular carcinoma cells, as well as induction of Hep G2 apoptosis. Since c-Met has been identified as a new potential therapeutical target for personalized treatment of hepatocellular carcinoma, damnacanthal and noni extract supplements containing it could be potentially interesting for the treatment and/or chemoprevention of hepatocellular carcinoma through its inhibitory effects on the HGF/c-Met axis.

In vitro stemness characterization of radio-resistant clones isolated from a medulloblastoma cell line ONS-76

PubMed Central

Sun, Lue; Moritake, Takashi; Zheng, Yun-Wen; Suzuki, Kenshi; Gerelchuluun, Ariungerel; Hong, Zhengshan; Zenkoh, Junko; Taniguchi, Hideki; Tsuboi, Koji

2013-01-01

One-third of patients with medulloblastoma die due to recurrence after various treatments including radiotherapy. Although it has been postulated that cancer stem-like cells are radio-resistant and play an important role in tumor recurrence, the “stemness” of medulloblastoma cells surviving irradiation has not yet been elucidated. Using a medulloblastoma cell line ONS-76, cells that survived gamma irradiation were investigated on their “stemness” in vitro. From 10 500 cells, 20 radio-resistant clones were selected after gamma ray irradiation (5 Gy × two fractions) using the replica micro-well technique. These 20 resistant clones were screened for CD133 positivity by flow cytometry followed by side population assay, tumor sphere formation assay and clonogenic survival assay. Results revealed CD133 fractions were significantly elevated in three clones, which also exhibited significantly increased levels of tumor sphere formation ability and side population fraction. Clonogenic survival assay demonstrated that their radio-resistance was significantly higher than the parental ONS-76. This may support the hypothesis that a small number of cancer stem-like cells (CSCs) are the main culprits in local recurrence after radiotherapy, and disruption of the resistance mechanism of these CSCs is a critical future issue in improving the outcome of patients with medulloblastoma. PMID:22951319

Upregulation of mitochondrial NAD+ levels impairs the clonogenicity of SSEA1+ glioblastoma tumor-initiating cells.

PubMed

Son, Myung Jin; Ryu, Jae-Sung; Kim, Jae Yun; Kwon, Youjeong; Chung, Kyung-Sook; Mun, Seon Ju; Cho, Yee Sook

2017-06-09

Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD + ) metabolism. However, the functional role of NAD + metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD + levels affect the characteristics of glioma-driven SSEA1 + TICs, including clonogenic growth potential. An increase in the mitochondrial NAD + levels by the overexpression of the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly suppressed the sphere-forming ability and induced differentiation of TICs, suggesting a loss of the characteristics of TICs. In addition, increased SIRT3 activity and reduced lactate production, which are mainly observed in healthy and young cells, appeared following NNT-overexpressed TICs. Moreover, in vivo tumorigenic potential was substantially abolished by NNT overexpression. Conversely, the short interfering RNA-mediated knockdown of NNT facilitated the maintenance of TIC characteristics, as evidenced by the increased numbers of large tumor spheres and in vivo tumorigenic potential. Our results demonstrated that targeting the maintenance of healthy mitochondria with increased mitochondrial NAD + levels and SIRT3 activity could be a promising strategy for abolishing the development of TICs as a new therapeutic approach to treating aging-associated tumors.

Cellular and Molecular Characterization of Human Cardiac Stem Cells Reveals Key Features Essential for Their Function and Safety

PubMed Central

Vahdat, Sadaf; Mousavi, Seyed Ahmad; Omrani, Gholamreza; Gholampour, Maziar; Sotoodehnejadnematalahi, Fattah; Ghazizadeh, Zaniar; Gharechahi, Javad

2015-01-01

Cell therapy of heart diseases is emerging as one of the most promising known treatments in recent years. Transplantation of cardiac stem cells (CSCs) may be one of the best strategies to cure adult or pediatric heart diseases. As these patient-derived stem cells need to be isolated from small heart biopsies, it is important to select the best isolation method and CSC subpopulation with the best cardiogenic functionality. We employed three different protocols including c-KIT+ cell sorting, clonogenic expansion, and explants culture to isolate c-KIT+ cells, clonogenic expansion-derived cells (CEDCs), and cardiosphere-derived cells (CDCs), respectively. Evaluation of isolated CSC characteristics in vitro and after rat myocardial infarction (MI) model transplantation revealed that although c-KIT+ and CDCs had higher MI regenerative potential, CEDCs had more commitment into cardiomyocytes and needed lower passages that were essential to reach a definite cell count. Furthermore, genome-wide expression analysis showed that subsequent passages caused changes in characteristics of cells, downregulation of cell cycle-related genes, and upregulation of differentiation and carcinogenic genes, which might lead to senescence, commitment, and possible tumorigenicity of the cells. Because of different properties of CSC subpopulations, we suggest that appropriate CSCs subpopulation should be chosen based on their experimental or clinical use. PMID:25867933

Mechanisms of ozone toxicity in cultured cells. I. Reduced clonogenic ability of polyunsaturated fatty acid-supplemented fibroblasts. Effect of vitamin E

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

Konings, A.W.

1986-01-01

The direct action of ozone on viability and survival of normal and modified mouse lung fibroblasts has been studied. By cell manipulation of fibroblasts in culture, the content of polyunsaturated fatty acids (PUFA) in the phospholipids was increased from about 6% to about 40%. The cellular content of alpha-tocopherol (alpha-T) (vitamin E) could be drastically enhanced. Vitamin E supplementation to the cell did not influence the PUFA manipulation. Normal, PUFA, and PUFA(alpha-T) fibroblasts were exposed to ozone by bubbling 10 ppm through the cell suspensions for different periods of time (0-6 h). No significant effects of the ozone exposure couldmore » be established when normal fibroblasts were used. The PUFA fibroblasts, however, were very vulnerable to ozone toxicity, both in terms of dye uptake (Trypan blue) and cell death (clonogenic ability). When alpha-tocopherol was present in the cell (200 ng/10(6) cells), a clear protection against ozone toxicity was found. It is concluded that ozone toxicity might be higher under conditions of a relative high amount of polyunsaturated fatty acids in the membrane phospholipids of the cell and a low cellular antioxidant capacity. Cellular membranes are probably an important target for ozone-induced cell death.« less

Highly Tumorigenic Diffuse Large B Cell Lymphoma Cells Are Produced by Coculture with Stromal Cells.

PubMed

Lin, Zhiguang; Chen, Bobin; Wu, Ting; Xu, Xiaoping

2018-05-23

Diffuse large B cell lymphoma (DLBCL) is heterogeneous. We aimed to explore how tumor microenvironment promotes lymphoma cell aggressiveness and heterogeneity. We created a coculture system using human DLBCL cells and mouse bone marrow stromal cells. Proliferative capacity, drug resistance, clonogenicity, and tumorigenicity were compared in lymphoma cells from the coculture system and lymphoma cells cultured alone. Expression of Notch signaling associated genes was evaluated using real-time reverse transcriptase PCR and Western blot. Lymphoma cells in the coculture system differentiated into a suspended cell group and an adherent cell group. They acquired a stronger proliferative capacity and drug resistance than lymphoma cells cultured alone, and differences existed between the adherent cell and suspended cell groups. The suspended cell group acquired the most powerful clonogenic and tumorigenic potential. However, Notch3 was exclusively expressed in the adherent lymphoma cell group and the use of N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, an inhibitor of Notch pathway, could abolish the emergence of highly aggressive lymphoma cells. Highly tumorigenic lymphoma cells could be generated by coculture with stromal cells, and it was dependent on Notch3 expression in the adjacent lymphoma cells through interaction with stromal cells. © 2018 S. Karger AG, Basel.

Synergistic Integration of Mesenchymal Stem Cells and Hydrostatic Pressure in the Expansion and Maintenance of Human Hematopoietic/Progenitor Cells

PubMed Central

2018-01-01

Ex vivo expansion of hematopoietic stem/progenitor cell (HSPC) has been investigated to improve the clinical outcome of HSPC transplantation. However, ex vivo expansion of HSPCs still faces a major obstacle in that HPSCs tend to differentiate when proliferating. Here, we cocultured HSPCs with mesenchymal stem cells (MSCs) and divided the HSPCs into two fractions according to whether they came into adherent to MSCs or not. Additionally, we used hydrostatic pressure (HP) to mimic the physical conditions in vivo. Even nonadherent cells expanded to yield a significantly larger number of total nucleated cells (TNCs), adherent cells maintained the HSPC phenotype (CD34+, CD34+CD38−, and CD133+CD38−) to a greater extent than nonadherent cells and had superior clonogenic potential. Moreover, applying HP significantly increased the number of TNCs, the frequency of the immature HSPC phenotype, and the clonogenic potential. Furthermore, the genetic markers for the HSPC niche were significantly increased under HP. Our data suggest that the nonadherent fraction is the predominant site of HSPC expansion, whereas the adherent fraction seems to mimic the HSPC niche for immature cells. Moreover, HP has a synergistic effect on expansion and functional maintenance. This first study utilizing HP has a potential of designing clinically applicable expansion systems. PMID:29681947

Marrow stromal cells from patients affected by MPS I differentially support haematopoietic progenitor cell development.

PubMed

Baxter, M A; Wynn, R F; Schyma, L; Holmes, D K; Wraith, J E; Fairbairn, L J; Bellantuono, I

2005-01-01

Bone marrow transplantation is the therapy of choice in patients affected by MPS I (Hurler syndrome), but a high incidence of rejection limits the success of this treatment. The deficiency of alpha-L-iduronidase (EC 1.2.3.76), one of the enzymes responsible for the degradation of glycosaminoglycans, results in accumulation of heparan and dermatan sulphate in these patients. Heparan sulphate and dermatan sulphate are known to be important components of the bone marrow microenvironment and critical for haematopoietic cell development. In this study we compared the ability of marrow stromal cells from MPS I patients and healthy donors to support normal haematopoiesis in Dexter-type long term culture. We found an inverse stroma/supernatant ratio in the number of clonogenic progenitors, particularly the colony-forming unit granulocyte-machrophage in MPS I cultures when compared to normal controls. No alteration in the adhesion of haematopoietic cells to the stroma of MPS I patients was found, suggesting that the altered distribution in the number of clonogenic progenitors is probably the result of an accelerated process of differentiation and maturation. The use of alpha-L-iduronidase gene-corrected marrow stromal cells re-established normal haematopoiesis in culture, suggesting that correction of the bone marrow microenvironment with competent enzyme prior to transplantation might help establishment of donor haematopoiesis.

Flow Cytometry Techniques in Radiation Biology

DTIC Science & Technology

1988-06-01

Henidtopoietic stem cells SUMMARY Hematopoietic stem cells ( HSC ) are present in the marrow at a concentration of approximately 2-3 HSC per 1000 nucleated marrow...cells. In the past, only clonogenic assays requiring 8-13 days and ten irradiated recipient rodents were available for assaying HSC . Because of the...importance of HSC in the postirradiation syndrome, we have developed a new rapid method based on flow cytometry not only to assay but also to purify and

Hypoxia Enhances the Antiglioma Cytotoxicity of B10, a Glycosylated Derivative of Betulinic Acid

PubMed Central

Thiepold, Anna-Luisa; Harter, Patrick N.; Reichert, Sebastian; Kögel, Donat; Paschke, Reinhard; Mittelbronn, Michel; Weller, Michael; Steinbach, Joachim P.; Fulda, Simone; Bähr, Oliver

2014-01-01

B10 is a glycosylated derivative of betulinic acid with promising activity against glioma cells. Lysosomal cell death pathways appear to be essential for its cytotoxicity. We investigated the influence of hypoxia, nutrient deprivation and current standard therapies on B10 cytotoxicity. The human glioma cell lines LN-308 and LNT-229 were exposed to B10 alone or together with irradiation, temozolomide, nutrient deprivation or hypoxia. Cell growth and viability were evaluated by crystal violet staining, clonogenicity assays, propidium iodide uptake and LDH release assays. Cell death was examined using an inhibitor of lysosomal acidification (bafilomycin A1), a cathepsin inhibitor (CA074-Me) and a short-hairpin RNA targeting cathepsin B. Hypoxia substantially enhanced B10-induced cell death. This effect was sensitive to bafilomycin A1 and thus dependent on hypoxia-induced lysosomal acidification. Cathepsin B appeared to mediate cell death because either the inhibitor CA074-Me or cathepsin B gene silencing rescued glioma cells from B10 toxicity under hypoxia. B10 is a novel antitumor agent with substantially enhanced cytotoxicity under hypoxia conferred by increased lysosomal cell death pathway activation. Given the importance of hypoxia for therapy resistance, malignant progression, and as a result of antiangiogenic therapies, B10 might be a promising strategy for hypoxic tumors like malignant glioma. PMID:24743710

Protein Kinase D1 attenuates tumorigenesis in colon cancer by modulating β-catenin/T cell factor activity

PubMed Central

Sundram, Vasudha; Ganju, Aditya; Hughes, Joshua E.; Khan, Sheema; Chauhan, Subhash C.; Jaggi, Meena

2014-01-01

Over 80% of colon cancer development and progression is a result of the dysregulation of β-catenin signaling pathway. Herein, for the first time, we demonstrate that a serine-threonine kinase, Protein Kinase D1 (PKD1), modulates the functions of β-catenin to suppress colon cancer growth. Analysis of normal and colon cancer tissues reveals downregulation of PKD1 expression in advanced stages of colon cancer and its co-localization with β-catenin in the colon crypts. This PKD1 downregulation corresponds with the aberrant expression and nuclear localization of β-catenin. In-vitro investigation of the PKD1-β-catenin interaction in colon cancer cells reveal that PKD1 overexpression suppresses cell proliferation and clonogenic potential and enhances cell-cell aggregation. We demonstrate that PKD1 directly interacts with β-catenin and attenuates β-catenin transcriptional activity by decreasing nuclear β-catenin levels. Additionally, we show that inhibition of nuclear β-catenin transcriptional activity is predominantly influenced by nucleus targeted PKD1. This subcellular modulation of β-catenin results in enhanced membrane localization of β-catenin and thereby increases cell-cell adhesion. Studies in a xenograft mouse model indicate that PKD1 overexpression delayed tumor appearance, enhanced necrosis and lowered tumor hypoxia. Overall, our results demonstrate a putative tumor-suppressor function of PKD1 in colon tumorigenesis via modulation of β-catenin functions in cells. PMID:25149539

MRI surveillance of cancer cell fate in a brain metastasis model after early radiotherapy.

PubMed

Murrell, Donna H; Zarghami, Niloufar; Jensen, Michael D; Dickson, Fiona; Chambers, Ann F; Wong, Eugene; Foster, Paula J

2017-10-01

Incidence of brain metastasis attributed to breast cancer is increasing and prognosis is poor. It is thought that disseminated dormant cancer cells persist in metastatic organs and may evade treatments, thereby facilitating a mechanism for recurrence. Radiotherapy is used to treat brain metastases clinically, but assessment has been limited to macroscopic tumor volumes detectable by clinical imaging. Here, we use cellular MRI to understand the concurrent responses of metastases and nonproliferative or slowly cycling cancer cells to radiotherapy. MRI cell tracking was used to investigate the impact of early cranial irradiation on the fate of individual iron-labeled cancer cells and outgrowth of breast cancer brain metastases in the human MDA-MB-231-BR-HER2 cell model. Early whole-brain radiotherapy significantly reduced the outgrowth of metastases from individual disseminated cancer cells in treated animals compared to controls. However, the numbers of nonproliferative iron-retaining cancer cells in the brain were not significantly different. Radiotherapy, when given early in cancer progression, is effective in preventing the outgrowth of solitary cancer cells to brain metastases. Future studies of the nonproliferative cancer cells' clonogenic potentials are warranted, given that their persistent presence suggests that they may have evaded treatment. Magn Reson Med 78:1506-1512, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Imaging of Tumor Characteristics and Molecular Pathways With PET: Developments Over the Last Decade Toward Personalized Cancer Therapy.

PubMed

Marcu, Loredana Gabriela; Moghaddasi, Leyla; Bezak, Eva

2018-05-04

Improvements in personalized therapy are made possible by the advances in molecular biology that led to developments in molecular imaging, allowing highly specific in vivo imaging of biological processes. Positron emission tomography (PET) is the most specific and sensitive imaging technique for in vivo molecular targets and pathways, offering quantification and evaluation of functional properties of the targeted anatomy. This work is an integrative research review that summarizes and evaluates the accumulated current status of knowledge of recent advances in PET imaging for cancer diagnosis and treatment, concentrating on novel radiotracers and evaluating their advantages and disadvantages in cancer characterization. Medline search was conducted, limited to English publications from 2007 onward. Identified manuscripts were evaluated for most recent developments in PET imaging of cancer hypoxia, angiogenesis, proliferation, and clonogenic cancer stem cells (CSC). There is an expansion observed from purely metabolic-based PET imaging toward antibody-based PET to achieve more information on cancer characteristics to identify hypoxia, proangiogenic factors, CSC, and others. 64 Cu-ATSM, for example, can be used both as a hypoxia and a CSC marker. Progress in the field of functional imaging will possibly lead to more specific tumor targeting and personalized treatment, increasing tumor control and improving quality of life. Copyright © 2018 Elsevier Inc. All rights reserved.

DNA methylation enzyme inhibitor RG108 suppresses the radioresistance of esophageal cancer

PubMed Central

Ou, Yao; Zhang, Quan; Tang, Yiting; Lu, Zhonghua; Lu, Xujing; Zhou, Xifa; Liu, Changmin

2018-01-01

Esophageal cancer (EC) is the eighth most common highly aggressive cancer worldwide. The purpose of this study was to investigate the effect of the DNA methyltransferase inhibitor RG108 on the radiosensitivity of EC cells. MTT and clonogenic assays were performed to assess the effect of RG108 on the proliferation and radiosensitivity of Eca-109 and TE-1 human EC cells. The cell cycle progression and alterations in apoptosis were analyzed by flow cytometry. For the in vivo analysis, the Eca-109 cells were inoculated into nude mice to establish tumors. Tissues from xenografts were obtained to detect changes to microvessels and tumor growth by immunohistochemistry (IHC). RNA-seq was used to identify differentially expressed genes. We found that RG108 increased the radiosensitivity of EC cells. Apoptosis and G2/M-phase arrest were induced by X-ray irradiation and were significantly enhanced by RG108. In addition, growth of tumor xenografts from the Eca-109 cells was significantly inhibited by irradiation in combination with RG108. The RNA-seq analysis revealed that, compared with radiation alone, X-ray irradiation in combination with RG108 altered the expression of 121 genes in multiple pathways, including the TGF-β signaling pathway and the Epstein-Barr virus infection pathway. In conclusion, RG108 induced radiosensitivity in EC cells both in vitro and in vivo. PMID:29328411

Hypofractionated Radiotherapy Is Superior to Conventional Fractionation in an Orthotopic Model of Anaplastic Thyroid Cancer.

PubMed

Oweida, Ayman; Phan, Andy; Vancourt, Benjamin; Robin, Tyler; Hararah, Mohammad K; Bhatia, Shilpa; Milner, Dallin; Lennon, Shelby; Pike, Laura; Raben, David; Haugen, Bryan; Pozdeyev, Nikita; Schweppe, Rebecca; Karam, Sana D

2018-06-01

Anaplastic thyroid cancer (ATC) is an aggressive and highly lethal disease with poor outcomes and resistance to therapy. Despite multimodality treatment, including radiation therapy and chemotherapy, response rates remain <15%, with a median time to progression of less than three months. Recent advances in radiotherapy (RT) delivery and gene-expression profiling may help guide patient selection for personalized therapy. The purpose of this study was to characterize the response to radiation in a panel of ATC cell lines and to test alternative RT fractionation schedules for overcoming radioresistance. The cellular response to radiation was characterized based on clonogenic assays. Radiation response was correlated with microarray gene-expression data. Hypofractionated and conventional RT was tested in an orthotopic ATC tumor model, and tumor growth was assayed locally and distantly with in vivo and ex vivo bioluminescence imaging. A spectrum of radiosensitivities was observed in ATC cell lines. Radioresistant cell lines had higher levels of CXCR4 compared to radiosensitive cell lines. Compared to conventionally fractionated RT, hypofractionated RT resulted in significantly improved tumor growth delay, decreased regional and distant metastases, and improved overall survival. The findings demonstrate the heterogeneity of response to radiation in ATC tumors and the superiority of hypofractionated RT in improving local control, metastatic spread, and survival in preclinical models. These data support the design of clinical trials targeting radioresistant pathways in combination with hypofractionated RT.

Preservation of Differentiation and Clonogenic Potential of Human Hematopoietic Stem and Progenitor Cells during Lyophilization and Ambient Storage

PubMed Central

Buchanan, Sandhya S.; Pyatt, David W.; Carpenter, John F.

2010-01-01

Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC) and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450±230 CFU-GM, 430±140 BFU-E, and 50±40 CFU-GEMM per 50 µL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25°C in the dark. Cells reconstituted immediately after lyophilization produced 580±90 CFU-GM (∼40%, relative to unprocessed controls p<0.0001), 170±70 BFU-E (∼40%, p<0.0001), and 41±22 CFU-GEMM (∼82%, p = 0.4171), and cells reconstituted after 28 days at room temperature produced 513±170 CFU-GM (∼35%, relative to unprocessed controls, p<0.0001), 112±68 BFU-E (∼26%, p<0.0001), and 36±17 CFU-GEMM (∼82%, p = 0.2164) These studies are the first to document high level retention of CFU-GEMM following lyophilization and storage for 4 weeks at 25°C. This type of flexible storage stability would potentially permit the ability to ship and store HPC without the need for refrigeration. PMID:20824143

Preservation of differentiation and clonogenic potential of human hematopoietic stem and progenitor cells during lyophilization and ambient storage.

PubMed

Buchanan, Sandhya S; Pyatt, David W; Carpenter, John F

2010-09-01

Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC) and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450+/-230 CFU-GM, 430+/-140 BFU-E, and 50+/-40 CFU-GEMM per 50 microL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25 degrees C in the dark. Cells reconstituted immediately after lyophilization produced 580+/-90 CFU-GM ( approximately 40%, relative to unprocessed controls p<0.0001), 170+/-70 BFU-E (approximately 40%, p<0.0001), and 41+/-22 CFU-GEMM (approximately 82%, p = 0.4171), and cells reconstituted after 28 days at room temperature produced 513+/-170 CFU-GM (approximately 35%, relative to unprocessed controls, p<0.0001), 112+/-68 BFU-E (approximately 26%, p<0.0001), and 36+/-17 CFU-GEMM ( approximately 82%, p = 0.2164) These studies are the first to document high level retention of CFU-GEMM following lyophilization and storage for 4 weeks at 25 degrees C. This type of flexible storage stability would potentially permit the ability to ship and store HPC without the need for refrigeration.

Cytotoxic sesquiterpene lactones from the leaves of Vernonia guineensis Benth. (Asteraceae)

PubMed Central

Toyang, Ngeh J.; Wabo, Hippolyte K.; Ateh, Eugene N.; Davis, Harry; Tane, Pierre; Sondengam, Luc B.; Bryant, Joseph; Verpoorte, Rob

2015-01-01

Ethnopharmacological relevance Vernonia guineensis Benth. (Asteraceae) preparations are used in folk medicine in Cameroon to treat a number of ailments, including prostate cancer and malaria, and is used as an anthelmintic, adaptogen and antidote. The aim of this study was to continue the validation of the activity of Vernonia guineensis Benth. extracts and isolated molecules against cancer cell lines following the previous isolation of an anti-prostate cancer sugar ester from the root extract. Materials and methods Acetone extracts of Vernonia guineensis Benth. leaves were tested for activity against 10 cancer cell lines (Breast—MDA-MB-231, Breast—MCF-7, Colon—HCT-116, Leukemia—HL-60, Lung—A549, Melanoma—A375, Ovarian—OVCAR3, Pancreas—Mia-paca, Prostate—PC-3 and Prostate—DU-145). The acetone extract was subjected to bioactivity guided fractionation. Anti-proliferation and clonogenic activity of the isolated compounds were tested. The WST-1 assay was used for the anti-proliferation activity, while the standard clonogenic test was used to determine the clonogenic activity. Results The acetone extract of Vernonia guineensis Benth. demonstrated in vitro activity ranging from IC50 4–26 mg/mL against the 10 cell lines. Activity guided fractionation of this extract yielded two sesquiterpene lactones, isolated for the first time from the genus Vernonia. The compounds were characterized using spectroscopic experiments, including a combination of 1D and 2D NMR data. Vernopicrin (1) and Vernomelitensin (2) demonstrated in vitro activity against human cancer cell lines with IC50 ranging from 0.35–2.04 μM (P < 0.05) and 0.13–1.5 μM (P < 0.05), respectively, between the most and least sensitive cell lines for each compound. Vernopicrin was most active against the human melanoma (A375) cell line and least active against the lung cancer (A549) cell line, while Vernomelitensin was also most active against the human melanoma (A375) cell line and least active against the breast cancer (MCF-7) cell line. Both compounds also demonstrated anticlonogenic activity. Conclusion The cytotoxicity demonstrated by the crude extract and isolated sesquiterpenes against cancer cell lines highlights the medicinal potential of V. guineensis. The selective anti-proliferation and dose dependent anticlonogenic activities suggest that the identified sesquiterpenes could be potential antitumor agents.. PMID:23376285

MiR-224 expression increases radiation sensitivity of glioblastoma cells

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

Upraity, Shailendra; Kazi, Sadaf; Padul, Vijay

Highlights: • MiR-224 expression in established glioblastoma cell lines and sporadic tumor tissues is low. • Exogenous miR-224 expression was found to increase radiation sensitivity of glioblastoma cells. • MiR-224 expression brought about 55–60% reduction in API5 expression levels. • Transfection with API5 siRNA increased radiation sensitivity of glioblastoma cells. • Low miR-224 and high API5 expression correlated with worse survival of GBM patients. - Abstract: Glioblastoma (GBM) is the most common and highly aggressive primary malignant brain tumor. The intrinsic resistance of this brain tumor limits the efficacy of administered treatment like radiation therapy. In the present study, effectmore » of miR-224 expression on growth characteristics of established GBM cell lines was analyzed. MiR-224 expression in the cell lines as well as in primary GBM tumor tissues was found to be low. Exogenous transient expression of miR-224 using either synthetic mimics or stable inducible expression using doxycycline inducible lentiviral vector carrying miR-224 gene, was found to bring about 30–55% reduction in clonogenic potential of U87 MG cells. MiR-224 expression reduced clonogenic potential of U87 MG cells by 85–90% on irradiation at a dose of 6 Gy, a dose that brought about 50% reduction in clonogenic potential in the absence of miR-224 expression. MiR-224 expression in glioblastoma cells resulted in 55–65% reduction in the expression levels of API5 gene, a known target of miR-224. Further, siRNA mediated down-regulation of API5 was also found to have radiation sensitizing effect on glioblastoma cell lines. Analysis of the Cancer Genome Atlas data showed lower miR-224 expression levels in male GBM patients to correlate with poorer survival. Higher expression levels of miR-224 target API5 also showed significant correlation with poorer survival of GBM patients. Up-regulation of miR-224 or down-regulation of its target API5 in combination with radiation therapy, therefore appear as promising options for the treatment of glioblastoma, which is refractory to the existing treatment strategies.« less

Anti-tumor activity of olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, in cultured endometrial carcinoma cells

PubMed Central

2014-01-01

Background PTEN inactivation is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in nucleus. Poly (ADP-ribose) polymerase (PARP) plays key roles in the repair of DNA single-strand breaks, and a PARP inhibitor induces synthetic lethality in cancer cells with HR deficiency. We examined the anti-tumor activity of olaparib, a PARP inhibitor, and its correlation between the sensitivity and status of PTEN in endometrial cancer cell lines. Methods The response to olaparib was evaluated using a clonogenic assay with SF50 values (concentration to inhibit cell survival to 50%) in 16 endometrial cancer cell lines. The effects of PTEN on the sensitivity to olaparib and ionizing radiation (IR) exposure were compared between parental HEC-6 (PTEN-null) and HEC-6 PTEN + (stably expressing wild-type PTEN) cells by clonogenic assay, foci formation of RAD51 and γH2AX, and induction of cleaved PARP. The effects of siRNA to PTEN were analyzed in cells with wild-type PTEN. Results The SF50 values were 100 nM or less in four (25%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in four (25%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 746 ± 838 nM; Wild-type [n = 4]: 215 ± 85 nM, p = 0.26 by Student’s t test). RAD51 expression was observed broadly and was not associated with PTEN status in the 16 cell lines. The number of colonies in the clonogenic assay, the foci formation of RAD51 and γH2AX, and the induction of apoptosis were not affected by PTEN introduction in the HEC-6 PTEN + cells. The expression level of nuclear PTEN was not elevated within 24 h following IR in the HEC-6-PTEN + cells. In addition, knocking down PTEN by siRNA did not alter the sensitivity to olaparib in 2 cell lines with wild-type PTEN. Conclusions Our results suggest that olaparib, a PARP inhibitor, is effective on certain endometrial cancer cell lines. Inactivation of PTEN might not affect the DNA repair function. Predictive biomarkers are warranted to utilize olaparib in endometrial cancer. PMID:24625059

Progression of trunk imbalance in adolescent idiopathic scoliosis with a thoracolumbar/lumbar curve: is it predictable at the initial visit?

PubMed

Hwang, Chang Ju; Lee, Choon Sung; Lee, Dong-Ho; Cho, Jae Hwan

2017-11-01

OBJECTIVE Progression of trunk imbalance is an important finding during follow-up of patients with adolescent idiopathic scoliosis (AIS). Nevertheless, no factors that predict progression of trunk imbalance have been identified. The purpose of this study was to identify parameters that predict progression of trunk imbalance in cases of AIS with a structural thoracolumbar/lumbar (TL/L) curve. METHODS This study included 105 patients with AIS and a structural TL/L curve who were followed up at an outpatient clinic. Patients with trunk imbalance (trunk shift ≥ 20 mm) at the initial visit were excluded. All patients were followed up for more than 2 years. Patients were divided into the following groups according to progression of trunk imbalance: 1) Group P, trunk shift ≥ 20 mm at the final visit and degree of progression ≥ 10 mm; and 2) Group NP, trunk shift < 20 mm at the final visit or degree of progression < 10 mm. Radiological parameters included Cobb angle, upper end vertebrae and lower end vertebrae (LEV), LEV tilt, disc wedge angle between LEV and LEV+1, trunk shift, apical vertebral translation, and apical vertebral rotation (AVR). Each parameter was compared between groups. Radiological parameters were assessed at every visit using whole-spine standing anteroposterior radiographs. RESULTS Among the 105 patients examined, 13 showed trunk imbalance with progression ≥ 10 mm at the final visit (Group P). Multivariate logistic regression analysis identified a lower Risser grade (p = 0.002) and a greater initial AVR (p = 0.020) as predictors of progressive trunk imbalance. A change in LEV tilt during follow-up was associated with trunk imbalance (p = 0.001). CONCLUSIONS Risser grade and AVR measured at the initial visit may predict progression of trunk imbalance. Surgeons should consider the risk of progressive trunk imbalance if patients show skeletal immaturity and a greater AVR at the initial visit.

Final Technical Progress Report: Development of Low-Cost Suspension Heliostat; December 7, 2011 - December 6, 2012

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

Bender, W.

2013-01-01

Final technical progress report of SunShot Incubator Solaflect Energy. The project succeeded in demonstrating that the Solaflect Suspension Heliostat design is viable for large-scale CSP installations. Canting accuracy is acceptable and is continually improving as Solaflect improves its understanding of this design. Cost reduction initiatives were successful, and there are still many opportunities for further development and further cost reduction.

MAPK pathway control of stem cell proliferation and differentiation in the embryonic pituitary provides insights into the pathogenesis of papillary craniopharyngioma.

PubMed

Haston, Scott; Pozzi, Sara; Carreno, Gabriela; Manshaei, Saba; Panousopoulos, Leonidas; Gonzalez-Meljem, Jose Mario; Apps, John R; Virasami, Alex; Thavaraj, Selvam; Gutteridge, Alice; Forshew, Tim; Marais, Richard; Brandner, Sebastian; Jacques, Thomas S; Andoniadou, Cynthia L; Martinez-Barbera, Juan Pedro

2017-06-15

Despite the importance of the RAS-RAF-MAPK pathway in normal physiology and disease of numerous organs, its role during pituitary development and tumourigenesis remains largely unknown. Here, we show that the over-activation of the MAPK pathway, through conditional expression of the gain-of-function alleles BrafV600E and KrasG12D in the developing mouse pituitary, results in severe hyperplasia and abnormal morphogenesis of the gland by the end of gestation. Cell-lineage commitment and terminal differentiation are disrupted, leading to a significant reduction in numbers of most of the hormone-producing cells before birth, with the exception of corticotrophs. Of note, Sox2 + stem cells and clonogenic potential are drastically increased in the mutant pituitaries. Finally, we reveal that papillary craniopharyngioma (PCP), a benign human pituitary tumour harbouring BRAF p.V600E also contains Sox2 + cells with sustained proliferative capacity and disrupted pituitary differentiation. Together, our data demonstrate a crucial function of the MAPK pathway in controlling the balance between proliferation and differentiation of Sox2 + cells and suggest that persistent proliferative capacity of Sox2 + cells may underlie the pathogenesis of PCP. © 2017. Published by The Company of Biologists Ltd.

SCL/TAL1-mediated transcriptional network enhances megakaryocytic specification of human embryonic stem cells.

PubMed

Toscano, Miguel G; Navarro-Montero, Oscar; Ayllon, Veronica; Ramos-Mejia, Veronica; Guerrero-Carreno, Xiomara; Bueno, Clara; Romero, Tamara; Lamolda, Mar; Cobo, Marien; Martin, Francisco; Menendez, Pablo; Real, Pedro J

2015-01-01

Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34(+) progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs.

Effective targeting of chronic myeloid leukemia initiating activity with the combination of arsenic trioxide and interferon alpha.

PubMed

El Eit, Rabab M; Iskandarani, Ahmad N; Saliba, Jessica L; Jabbour, Mark N; Mahfouz, Rami A; Bitar, Nizar M A; Ayoubi, Hanadi R El; Zaatari, Ghazi S; Mahon, Francois-Xavier; De Thé, Hugues B; Bazarbachi, Ali A; Nasr, Rihab R

2014-02-15

Imatinib is the standard of care in chronic meloid leukemia (CML) therapy. However, imatinib is not curative since most patients who discontinue therapy relapse indicating that leukemia initiating cells (LIC) are resistant. Interferon alpha (IFN) induces hematologic and cytogenetic remissions and interestingly, improved outcome was reported with the combination of interferon and imatinib. Arsenic trioxide was suggested to decrease CML LIC. We investigated the effects of arsenic and IFN on human CML cell lines or primary cells and the bone marrow retroviral transduction/transplantation murine CML model. In vitro, the combination of arsenic and IFN inhibited proliferation and activated apoptosis. Importantly, arsenic and IFN synergistically reduced the clonogenic activity of primary bone marrow cells derived from CML patients. Finally, in vivo, combined interferon and arsenic treatment, but not single agents, prolonged the survival of primary CML mice. Importantly, the combination severely impaired engraftment into untreated secondary recipients, with some recipients never developing the disease, demonstrating a dramatic decrease in CML LIC activity. Arsenic/IFN effect on CML LIC activity was significantly superior to that of imatinib. These results support further exploration of this combination, alone or with imatinib aiming at achieving CML eradication rather than long-term disease control. © 2013 UICC.

Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells.

PubMed

Urueña, Claudia; Cifuentes, Claudia; Castañeda, Diana; Arango, Amparo; Kaur, Punit; Asea, Alexzander; Fiorentino, Susana

2008-11-18

There is ethnopharmacological evidence that Petiveria alliacea can have antitumor activity; however, the mechanism of its cytotoxic activity is not well understood. We assessed multiple in vitro biological activities of an ethyl acetate soluble plant fraction over several tumor cell lines. Tumor cell lines were evaluated using the following tests: trypan blue exclusion test, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], flow cytometry, cytoskeleton organization analysis, cell cycle, mitochondria membrane depolarization, clonogenicity test, DNA fragmentation test and differential protein expression by HPLC-Chip/MS analysis. F4 fraction characterization was made by HPLC-MS. Petiveria alliacea fraction characterized by de-replication was found to alter actin cytoskeleton organization, induce G2 cell cycle arrest and cause apoptotic cell death in a mitochondria independent way. In addition, we found down regulation of cytoskeleton, chaperone, signal transduction proteins, and proteins involved in metabolic pathways. Finally up regulation of proteins involved in translation and intracellular degradation was also observed. The results of this study indicate that Petiveria alliacea exerts multiple biological activities in vitro consistent with cytotoxicity. Further studies in animal models are needed but Petiveria alliacea appears to be a good candidate to be used as an antitumor agent.

Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells

PubMed Central

Urueña, Claudia; Cifuentes, Claudia; Castañeda, Diana; Arango, Amparo; Kaur, Punit; Asea, Alexzander; Fiorentino, Susana

2008-01-01

Background There is ethnopharmacological evidence that Petiveria alliacea can have antitumor activity; however, the mechanism of its cytotoxic activity is not well understood. We assessed multiple in vitro biological activities of an ethyl acetate soluble plant fraction over several tumor cell lines. Methods Tumor cell lines were evaluated using the following tests: trypan blue exclusion test, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], flow cytometry, cytoskeleton organization analysis, cell cycle, mitochondria membrane depolarization, clonogenicity test, DNA fragmentation test and differential protein expression by HPLC-Chip/MS analysis. F4 fraction characterization was made by HPLC-MS. Results Petiveria alliacea fraction characterized by de-replication was found to alter actin cytoskeleton organization, induce G2 cell cycle arrest and cause apoptotic cell death in a mitochondria independent way. In addition, we found down regulation of cytoskeleton, chaperone, signal transduction proteins, and proteins involved in metabolic pathways. Finally up regulation of proteins involved in translation and intracellular degradation was also observed. Conclusion The results of this study indicate that Petiveria alliacea exerts multiple biological activities in vitro consistent with cytotoxicity. Further studies in animal models are needed but Petiveria alliacea appears to be a good candidate to be used as an antitumor agent. PMID:19017389

Fibronectin promotes differentiation of neural crest progenitors endowed with smooth muscle cell potential

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

Costa-Silva, Bruno; Programa de Pos-graduacao em Neurociencias, Centro de Ciencias Biologicas, Universidade Federal de Santa Catarina, Campus Universitario - Trindade, 88040-900, Florianopolis, S.C.; Coelho da Costa, Meline

The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effectmore » was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.« less

Extracellular matrix and α5β1 integrin signaling control the maintenance of bone formation capacity by human adipose-derived stromal cells

PubMed Central

Di Maggio, Nunzia; Martella, Elisa; Frismantiene, Agne; Resink, Therese J.; Schreiner, Simone; Lucarelli, Enrico; Jaquiery, Claude; Schaefer, Dirk J.; Martin, Ivan; Scherberich, Arnaud

2017-01-01

Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, adipose-derived stromal/stem cells (ASC), even after minimal monolayer expansion, display poor osteogenic capacity in vivo. We investigated whether ASC bone-forming capacity may be maintained by culture within a self-produced extracellular matrix (ECM) that recapitulates the native environment. SVF cells expanded without passaging up to 28 days (Unpass-ASC) deposited a fibronectin-rich extracellular matrix and displayed greater clonogenicity and differentiation potential in vitro compared to ASC expanded only for 6 days (P0-ASC) or for 28 days with regular passaging (Pass-ASC). When implanted subcutaneously, Unpass-ASC produced bone tissue similarly to SVF cells, in contrast to P0- and Pass-ASC, which mainly formed fibrous tissue. Interestingly, clonogenic progenitors from native SVF and Unpass-ASC expressed low levels of the fibronectin receptor α5 integrin (CD49e), which was instead upregulated in P0- and Pass-ASC. Mechanistically, induced activation of α5β1 integrin in Unpass-ASC led to a significant loss of bone formation in vivo. This study shows that ECM and regulation of α5β1-integrin signaling preserve ASC progenitor properties, including bone tissue-forming capacity, during in vitro expansion. PMID:28290502

Mutational analysis of P-glycoprotein: suppression of caspase activation in the absence of ATP-dependent drug efflux.

PubMed

Tainton, K M; Smyth, M J; Jackson, J T; Tanner, J E; Cerruti, L; Jane, S M; Darcy, P K; Johnstone, R W

2004-09-01

P-glycoprotein (P-gp) can induce multidrug resistance (MDR) through the ATP-dependent efflux of chemotherapeutic agents. We have previously shown that P-gp can inhibit nondrug apoptotic stimuli by suppressing the activation of caspases. To determine if this additional activity is functionally linked to ATP hydrolysis, we expressed wild-type and ATPase-mutant P-gp and showed that cells expressing mutant P-gp could not efflux chemotherapeutic drugs but remained relatively resistant to apoptosis. CEM lymphoma cells expressing mutant P-gp treated with vincristine showed a decrease in the fraction of cells with apoptotic morphology, cytochrome c release from the mitochondria and suppression of caspase activation, yet still accumulated in mitosis and showed a loss of clonogenic potential. The loss of clonogenicity in vincristine-treated cells expressing mutant P-gp was associated with accumulation of cells in mitosis and the presence of multinucleated cells consistent with mitotic catastrophe. The antiapoptotic effect of mutant P-gp was not affected by antibodies that inhibit the efflux function of the protein. These data are consistent with a dual activity model for P-gp-induced MDR involving both ATPase-dependent drug efflux and ATPase-independent inhibition of apoptosis. The structure-function analyses described herein provide novel insight into the mechanisms of action of P-gp in mediating MDR.

Molecular effects of the phosphatidylinositol-3-kinase inhibitor NVP-BKM120 on T and B-cell acute lymphoblastic leukaemia.

PubMed

Pereira, João Kleber Novais; Machado-Neto, João Agostinho; Lopes, Matheus Rodrigues; Morini, Beatriz Corey; Traina, Fabiola; Costa, Fernando Ferreira; Saad, Sara Teresinha Olalla; Favaro, Patricia

2015-09-01

Constitutive activation of the PI3K pathway in T cell acute lymphoblastic leukaemia (T-ALL) has been reported and in a mouse model, PI3K activation, together with MYC, cooperates in Burkitt lymphoma (BL) pathogenesis. We investigated the effects of NVP-BKM120, a potent pan-class I PI3K inhibitor, in lymphoblastic leukaemia cell lines. Effects of NVP-BKM120 on cell viability, clonogenicity, apoptosis, cell cycle, cell signalling and autophagy were assessed in vitro on T-ALL (Jurkat and MOLT-4) and BL (Daudi and NAMALWA) cell lines. NVP-BKM120 treatment decreased cell viability and clonogenic growth in all tested cells. Moreover, the drug arrested cell cycling in association with a decrease in Cyclin B1 protein levels, and increased apoptosis. Immunoblotting analysis of cells treated with the drug revealed decreased phosphorylation, in a dose-dependent manner, of AKT, mTOR, P70S6K and 4EBP1, with stable total protein levels. Additionally, we observed a dose-dependent decrease in BAD phosphorylation, in association with augmented BAX:BCL2 ratio. Quantification of autophagy showed a dose-dependent increase in acidic vesicular organelles in all cells tested. In summary, our present study establishes that NVP-BKM120 presents an effective antitumour activity against T-ALL and BL cell lines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Density-dependent induction of apoptosis by transforming growth factor-beta 1 in a human ovarian carcinoma cell line.

PubMed

Mathieu, C; Jozan, S; Mazars, P; Côme, M G; Moisand, A; Valette, A

1995-01-01

Transforming growth factor-beta 1 inhibited proliferation of a human ovarian carcinoma cell line (NIH-OVCAR-3). The inhibition of NIH-OVCAR-3 cell proliferation was accompanied by a decrease in clonogenic potential, evidenced by the reduced ability of TGF-beta 1-treated NIH-OVCAR-3 cells to form colonies on a plastic substratum. This rapid decrease of clonogenic potential, which was detected 6 h after addition of TGF-beta 1 was dose-dependent (IC50 = 4 pM). Fluorescence microscopy of DAPI-stained cells supported by electron-microscopic examination showed that TGF-beta 1 induced chromatin condensation and nuclear fragmentation. In addition, oligonucleosomal-sized fragments were detected in the TGF-beta 1-treated cells. These features indicated that TGF-beta 1 induced NIH-OVCAR-3 cell death by an apoptosis-like mechanism. This TGF-beta 1 apoptotic effect was subject to modulation by cell density. It was observed that an increase in cell density (up to 20 x 10(3) cells/cm2) protected NIH-OVCAR-3 cells against apoptosis induced by TGF-beta 1. Conditioned medium from high-density cultures of NIH-OVCAR-3 cells did not inhibit apoptosis induced by TGF-beta 1 on NIH-OVCAR-3 cells cultured at low density, suggesting that the protective effect of cell density was not related to the cell secretion of a soluble survival factor.

Osteopontin and splice variant expression level in human malignant glioma: radiobiologic effects and prognosis after radiotherapy.

PubMed

Güttler, Antje; Giebler, Maria; Cuno, Peter; Wichmann, Henri; Keßler, Jacqueline; Ostheimer, Christian; Söling, Ariane; Strauss, Christian; Illert, Jörg; Kappler, Matthias; Vordermark, Dirk; Bache, Matthias

2013-09-01

We investigated the role of the hypoxia-associated secreted glycoprotein osteopontin (OPN) in the response of malignant glioma to radiotherapy by characterizing OPN and its splice variants in vitro and in patient material. The effect of siRNA knockdown of OPN splice variants on cellular and radiobiologic behavior was analyzed in U251MG cells using OpnS siRNA (inhibition of all OPN splice variants) and OpnAC siRNA (knockdown only of OPNa and OPNc). OPN and splice variant mRNA levels were quantified in archival material of 41 glioblastoma tumor samples. Plasma OPN was prospectively measured in 33 malignant glioma patients. Inhibition of OPNa and OPNc (OpnAC) reduced clonogenic survival in U251MG cells but did not affect proliferation, migration or apoptosis. Knockdown of all OPN splice variants (OpnS) resulted in an even stronger inhibition of clonogenic survival, while cell proliferation and migration were reduced and rate of apoptosis was increased. Additional irradiation had additive effects with both siRNAs. Plasma OPN increased continuously in malignant glioma patients and was associated with poor survival. OPNb is partially able to compensate the effects of OPNa and OPNc knockdown in U251MG cells. High OPN plasma levels at the end of radiotherapy are associated with poor survival. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Obatoclax, a Pan-BCL-2 Inhibitor, Targets Cyclin D1 for Degradation to Induce Antiproliferation in Human Colorectal Carcinoma Cells.

PubMed

Or, Chi-Hung R; Chang, Yachu; Lin, Wei-Cheng; Lee, Wee-Chyan; Su, Hong-Lin; Cheung, Muk-Wing; Huang, Chang-Po; Ho, Cheesang; Chang, Chia-Che

2016-12-27

Colorectal cancer is the third most common cancer worldwide. Aberrant overexpression of antiapoptotic BCL-2 (B-cell lymphoma 2) family proteins is closely linked to tumorigenesis and poor prognosis in colorectal cancer. Obatoclax is an inhibitor targeting all antiapoptotic BCL-2 proteins. A previous study has described the antiproliferative action of obatoclax in one human colorectal cancer cell line without elucidating the underlying mechanisms. We herein reported that, in a panel of human colorectal cancer cell lines, obatoclax inhibits cell proliferation, suppresses clonogenicity, and induces G₁-phase cell cycle arrest, along with cyclin D1 downregulation. Notably, ectopic cyclin D1 overexpression abrogated clonogenicity suppression but also G₁-phase arrest elicited by obatoclax. Mechanistically, pre-treatment with the proteasome inhibitor MG-132 restored cyclin D1 levels in all obatoclax-treated cell lines. Cycloheximide chase analyses further revealed an evident reduction in the half-life of cyclin D1 protein by obatoclax, confirming that obatoclax downregulates cyclin D1 through induction of cyclin D1 proteasomal degradation. Lastly, threonine 286 phosphorylation of cyclin D1, which is essential for initiating cyclin D1 proteasomal degradation, was induced by obatoclax in one cell line but not others. Collectively, we reveal a novel anticancer mechanism of obatoclax by validating that obatoclax targets cyclin D1 for proteasomal degradation to downregulate cyclin D1 for inducing antiproliferation.

Spatial and temporal characterization of endometrial mesenchymal stem-like cells activity during the menstrual cycle

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

Shan, Xu; Chan, Rachel W.S., E-mail: rwschan@hku.hk; Centre of Reproduction, Development of Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR

The human endometrium is a highly dynamic tissue with the ability to cyclically regenerate during the reproductive life. Endometrial mesenchymal stem-like cells (eMSCs) located throughout the endometrium have shown to functionally contribute to endometrial regeneration. In this study we examine whether the menstrual cycle stage and the location in the endometrial bilayer (superficial and deep portions of the endometrium) has an effect on stem cell activities of eMSCs (CD140b{sup +}CD146{sup +} cells). Here we show the percentage and clonogenic ability of eMSCs were constant in the various stages of the menstrual cycle (menstrual, proliferative and secretory). However, eMSCs from themore » menstrual endometrium underwent significantly more rounds of self-renewal and enabled a greater total cell output than those from the secretory phase. Significantly more eMSCs were detected in the deeper portion of the endometrium compared to the superficial layer but their clonogenic and self-renewal activities remained similar. Our findings suggest that eMSCs are activated in the menstrual phase for the cyclical regeneration of the endometrium. - Highlights: • The percentages of endometrial mesenchymal-like stem cells (eMSCs) were constant across the menstrual cycle. • Menstruation eMSCs display superior self-renewal and long-term proliferative activities. • More eMSCs reside in the deeper portion of the endometrium than the superficial layer.« less

Nelfinavir induces radiation sensitization in pituitary adenoma cells

PubMed Central

Zeng, Jing; See, Alfred P.; Aziz, Khaled; Thiyagarajan, Saravanan; Salih, Tarek; Gajula, Rajendra P.; Armour, Michael; Phallen, Jillian; Terezakis, Stephanie; Kleinberg, Lawrence; Redmond, Kristen; Hales, Russell K.; Salvatori, Roberto; Quinones-Hinojosa, Alfredo; Tran, Phuoc T.; Lim, Michael

2017-01-01

Pituitary adenomas with local invasion and high secretory activity remain a therapeutic challenge. The HIV protease inhibitor nelfinavir is a radiosensitizer in multiple tumor models. We tested nelfinavir as a radiosensitizer in pituitary adenoma cells in vitro and in vivo. We examined the effect of nelfinavir with radiation on in vitro cell viability, clonogenic survival, apoptosis, prolactin secretion, cell cycle distribution and the PI3K-AKT-mTOR pathway. We evaluated tumor growth delay and confirmed nelfinavir’s effect on the PI3K-AKT-mTOR pathway in a hind-flank model. Nelfinavir sensitized pituitary adenoma cells to ionizing radiation as shown by viability assays and clonogenic assay with an enhancement ratio of 1.2 (p < 0.05). There is increased apoptotic cell death, as determined by annexin-V expression and cleaved caspase-3 levels. Nelfinavir does not affect prolactin secretion or cell cycle distribution. In vivo, untreated tumors reached 4-fold volume in 12 d, 17 d with nelfinavir treatment, 27 d with radiation 6 Gy, and 41 d with nelfinavir plus radiation (one-way ANOVA p < 0.001). Decreased phospho-S6 on protein gel blotting in vitro and immunohistochemistry in vivo demonstrated nelfinavir inhibition of the PI3K-AKT-mTOR pathway. Our data suggests a promising combination therapy with nelfinavir plus radiation in pituitary adenomas, which should be investigated in clinical studies. PMID:21811091

Nelfinavir induces radiation sensitization in pituitary adenoma cells.

PubMed

Zeng, Jing; See, Alfred P; Aziz, Khaled; Thiyagarajan, Saravanan; Salih, Tarek; Gajula, Rajendra P; Armour, Michael; Phallen, Jillian; Terezakis, Stephanie; Kleinberg, Lawrence; Redmond, Kristen; Hales, Russell K; Salvatori, Roberto; Quinones-Hinojosa, Alfredo; Tran, Phuoc T; Lim, Michael

2011-10-01

Pituitary adenomas with local invasion and high secretory activity remain a therapeutic challenge. The HIV protease inhibitor nelfinavir is a radiosensitizer in multiple tumor models. We tested nelfinavir as a radiosensitizer in pituitary adenoma cells in vitro and in vivo. We examined the effect of nelfinavir with radiation on in vitro cell viability, clonogenic survival, apoptosis, prolactin secretion, cell cycle distribution, and the PI3K-AKT-mTOR pathway. We evaluated tumor growth delay and confirmed nelfinavir's effect on the PI3K-AKT-mTOR pathway in a hind-flank model. Nelfinavir sensitized pituitary adenoma cells to ionizing radiation as shown by viability assays and clonogenic assay with an enhancement ratio of 1.2 (p < 0.05). There is increased apoptotic cell death, as determined by annexin-V expression and cleaved caspase-3 levels. Nelfinavir does not affect prolactin secretion or cell cycle distribution. In vivo, untreated tumors reached 4-fold volume in 12 days, 17 days with nelfinavir treatment, 27 days with radiation 6 Gy, and 41 days with nelfinavir plus radiation (one-way ANOVA p < 0.001). Decreased phospho-S6 on Western blotting in vitro and immunohistochemistry in vivo demonstrated nelfinavir inhibition of the PI3K-AKT-mTOR pathway. Our data suggests a promising combination therapy with nelfinavir plus radiation in pituitary adenomas, which should be investigated in clinical studies.

Upregulation of mitochondrial NAD+ levels impairs the clonogenicity of SSEA1+ glioblastoma tumor-initiating cells

PubMed Central

Son, Myung Jin; Ryu, Jae-Sung; Kim, Jae Yun; Kwon, Youjeong; Chung, Kyung-Sook; Mun, Seon Ju; Cho, Yee Sook

2017-01-01

Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD+) metabolism. However, the functional role of NAD+ metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD+ levels affect the characteristics of glioma-driven SSEA1+ TICs, including clonogenic growth potential. An increase in the mitochondrial NAD+ levels by the overexpression of the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly suppressed the sphere-forming ability and induced differentiation of TICs, suggesting a loss of the characteristics of TICs. In addition, increased SIRT3 activity and reduced lactate production, which are mainly observed in healthy and young cells, appeared following NNT-overexpressed TICs. Moreover, in vivo tumorigenic potential was substantially abolished by NNT overexpression. Conversely, the short interfering RNA-mediated knockdown of NNT facilitated the maintenance of TIC characteristics, as evidenced by the increased numbers of large tumor spheres and in vivo tumorigenic potential. Our results demonstrated that targeting the maintenance of healthy mitochondria with increased mitochondrial NAD+ levels and SIRT3 activity could be a promising strategy for abolishing the development of TICs as a new therapeutic approach to treating aging-associated tumors. PMID:28604662

Antitumor activity of curcumin is involved in down-regulation of YAP/TAZ expression in pancreatic cancer cells.

PubMed

Zhou, Xiuxia; Su, Jingna; Feng, Shaoyan; Wang, Lixia; Yin, Xuyuan; Yan, Jingzhe; Wang, Zhiwei

2016-11-29

Pancreatic cancer (PC) is one of the most aggressive human malignancies worldwide and is the fourth leading cause of cancer-related deaths. Curcumin (diferuloylmethane) is a polyphenol derived from the Curcuma longa plant. Certain studies have demonstrated that curcumin exerts its anti-tumor function in a variety of human cancers including PC, via targeting multiple therapeutically important cancer signaling pathways. However, the detailed molecular mechanisms are not fully understood. Two transcriptional co-activators, YAP (Yes-associated protein) and its close paralog TAZ (transcriptional coactivator with PDZ-binding motif) exert oncogenic activities in various cancers. Therefore, in this study we aimed to determine the molecular basis of curcumin-induced cell proliferation inhibition in PC cells. First, we detected the anti-tumor effects of curcumin on PC cell lines using CTG assay, Flow cytometry, clonogenic assay, wound healing assay and Transwell invasion assay. We found that curcumin significantly suppressed cell growth, weakened clonogenic potential, inhibited migration and invasion, and induced apoptosis and cell cycle arrest in PC cells. We further measured that overexpression of YAP enhanced cell proliferation and abrogated the cytotoxic effects of curcumin on PC cells. Moreover, we found that curcumin markedly down-regulated YAP and TAZ expression and subsequently suppressed Notch-1 expression. Collectively, these findings suggest that pharmacological inhibition of YAP and TAZ activity may be a promising anticancer strategy for the treatment of PC patients.

Sensitization of thermotolerant SCK cells to hyperthermia and freezing with reduction of intracellular pH: implications for cryosurgery.

PubMed

Burgher, Abram H; Swanlund, David J; Griffin, Robert J; Song, Chang W; Bischof, John C; Roberts, Kenneth P

2003-03-01

During cryosurgery, cells frozen slowly at the outer part of the ice ball undergo severe dehydration and are subject to solute effects injury, which may be caused in part by protein denaturation. This study was undertaken to determine whether heat shock proteins (HSPs), the molecular chaperones that stabilize proteins against denaturation, have a protective effect on cells during slow freezing. In addition, we aimed to determine whether acidic conditions, similar to those found in many solid tumors, would effect this protection. SCK cells were frozen at 5 degrees C/min to -10 degrees C or -20 degrees C before or after induction of thermotolerance, and at neutral or low pH conditions. Lethal damage was determined by clonogenics. Clonogenic survival was decreased by 50% in thermotolerant cells frozen to -10 degrees C after culture in acidic conditions (pH 6.6) compared with non-thermotolerant cells cultured at neutral pH. Induction of thermotolerance alone or low pH alone did not significantly sensitize SCK cells to freezing. All treatment groups were equally susceptible to killing when frozen to -20 degrees C. Our results show that induction of thermal tolerance does not protect SCK cells against subsequent freezing injury and that a low pH environment actually sensitizes these cells to freeze injury. Copyright 2003 Wiley-Liss, Inc.

Enhancement of Radiation Response in Osteosarcoma and Rhabomyosarcoma Cell Lines by Histone Deacetylase Inhibition

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

Blattmann, Claudia, E-mail: claudia.blattmann@med.uni-heidelberg.d; Oertel, Susanne; Ehemann, Volker

2010-09-01

Purpose: Histone deacetylase inhibitors (HDACIs) can enhance the sensitivity of cells to photon radiation treatment (XRT) by altering numerous molecular pathways. We investigated the effect of pan-HDACIs such as suberoylanilide hydroxamic acid (SAHA) on radiation response in two osteosarcoma (OS) and two rhabdomyosarcoma (RMS) cell lines. Methods and Materials: Clonogenic survival, cell cycle analysis, and apoptosis were examined in OS (KHOS-24OS, SAOS2) and RMS (A-204, RD) cell lines treated with HDACI and HDACI plus XRT, respectively. Protein expression was investigated via immunoblot analysis, and cell cycle analysis and measurement of apoptosis were performed using flow cytometry. Results: SAHA induced anmore » inhibition of cell proliferation and clonogenic survival in OS and RMS cell lines and led to a significant radiosensitization of all tumor cell lines. Other HDACI such as M344 and valproate showed similar effects as investigated in one OS cell line. Furthermore, SAHA significantly increased radiation-induced apoptosis in the OS cell lines, whereas in the RMS cell lines radiation-induced apoptosis was insignificant with and without SAHA. In all investigated sarcoma cell lines, SAHA attenuated radiation-induced DNA repair protein expression (Rad51, Ku80). Conclusion: Our results show that HDACIs enhance radiation action in OS and RMS cell lines. Inhibition of DNA repair, as well as increased apoptosis induction after exposure to HDACIs, can be mechanisms of radiosensitization by HDACIs.« less

A gallotannin-rich fraction from Caesalpinia spinosa (Molina) Kuntze displays cytotoxic activity and raises sensitivity to doxorubicin in a leukemia cell line

PubMed Central

2012-01-01

Background Enhancement of tumor cell sensitivity may help facilitate a reduction in drug dosage using conventional chemotherapies. Consequently, it is worthwhile to search for adjuvants with the potential of increasing chemotherapeutic drug effectiveness and improving patient quality of life. Natural products are a very good source of such adjuvants. Methods The biological activity of a fraction enriched in hydrolysable polyphenols (P2Et) obtained from Caesalpinia spinosa was evaluated using the hematopoietic cell line K562. This fraction was tested alone or in combination with the conventional chemotherapeutic drugs doxorubicin, vincristine, etoposide, camptothecin and taxol. The parameters evaluated were mitochondrial depolarization, caspase 3 activation, chromatin condensation and clonogenic activity. Results We found that the P2Et fraction induced mitochondrial depolarization, activated caspase 3, induced chromatin condensation and decreased the clonogenic capacity of the K562 cell line. When the P2Et fraction was used in combination with chemotherapeutic drugs at sub-lethal concentrations, a fourfold reduction in doxorubicin inhibitory concentration 50 (IC50) was seen in the K562 cell line. This finding suggested that P2Et fraction activity is specific for the molecular target of doxorubicin. Conclusions Our results suggest that a natural fraction extracted from Caesalpinia spinosa in combination with conventional chemotherapy in combination with natural products on leukemia cells may increase therapeutic effectiveness in relation to leukemia. PMID:22490328

Prediction of cellular radiosensitivity from DNA damage induced by gamma-rays and carbon ion irradiation in canine tumor cells.

PubMed

Wada, Seiichi; Van Khoa, Tran; Kobayashi, Yasuhiko; Funayama, Tomoo; Ogihara, Kikumi; Ueno, Shunji; Ito, Nobuhiko

2005-11-01

Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after gamma-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with gamma-rays. The initial and residual (4 hr) DNA damage due to gamma-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.

Evaluation of the cytotoxic and genotoxic effects of benchmark multi-walled carbon nanotubes in relation to their physicochemical properties.

PubMed

Louro, Henriqueta; Pinhão, Mariana; Santos, Joana; Tavares, Ana; Vital, Nádia; Silva, Maria João

2016-11-16

To contribute with scientific evidence to the grouping strategy for the safety assessment of multi-walled carbon nanotubes (MWCNTs), this work describes the investigation of the cytotoxic and genotoxic effects of four benchmark MWCNTs in relation to their physicochemical characteristics, using two types of human respiratory cells. The cytotoxic effects were analysed using the clonogenic assay and replication index determination. A 48h-exposure of cells revealed that NM-401 was the only cytotoxic MWCNT in both cell lines, but after 8-days exposure, the clonogenic assay in A549 cells showed cytotoxic effects for all the tested MWCNTs. Correlation analysis suggested an association between the MWCNTs size in cell culture medium and cytotoxicity. No induction of DNA damage was observed after any MWCNTs in any cell line by the comet assay, while the micronucleus assay revealed that both NM-401 and NM-402 were genotoxic in A549 cells. NM-401 and NM-402 are the two longest MWCNTs analyzed in this work, suggesting that length may be determinant for genotoxicity. No induction of micronuclei was observed in BBEAS-2Beas-2B cell line and the different effect in both cell lines is explained in view of the size-distribution of MWCNTs in the cell culture medium, rather than cell's specificities. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Antitumor activity of curcumin is involved in down-regulation of YAP/TAZ expression in pancreatic cancer cells

PubMed Central

Wang, Lixia; Yin, Xuyuan; Yan, Jingzhe; Wang, Zhiwei

2016-01-01

Pancreatic cancer (PC) is one of the most aggressive human malignancies worldwide and is the fourth leading cause of cancer-related deaths. Curcumin (diferuloylmethane) is a polyphenol derived from the Curcuma longa plant. Certain studies have demonstrated that curcumin exerts its anti-tumor function in a variety of human cancers including PC, via targeting multiple therapeutically important cancer signaling pathways. However, the detailed molecular mechanisms are not fully understood. Two transcriptional co-activators, YAP (Yes-associated protein) and its close paralog TAZ (transcriptional coactivator with PDZ-binding motif) exert oncogenic activities in various cancers. Therefore, in this study we aimed to determine the molecular basis of curcumin-induced cell proliferation inhibition in PC cells. First, we detected the anti-tumor effects of curcumin on PC cell lines using CTG assay, Flow cytometry, clonogenic assay, wound healing assay and Transwell invasion assay. We found that curcumin significantly suppressed cell growth, weakened clonogenic potential, inhibited migration and invasion, and induced apoptosis and cell cycle arrest in PC cells. We further measured that overexpression of YAP enhanced cell proliferation and abrogated the cytotoxic effects of curcumin on PC cells. Moreover, we found that curcumin markedly down-regulated YAP and TAZ expression and subsequently suppressed Notch-1 expression. Collectively, these findings suggest that pharmacological inhibition of YAP and TAZ activity may be a promising anticancer strategy for the treatment of PC patients. PMID:27738325

Differential Radiosensitizing Effect of Valproic Acid in Differentiation Versus Self-Renewal Promoting Culture Conditions

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

Debeb, Bisrat G.; Xu Wei; Mok, Henry

2010-03-01

Purpose: It has been shown that valproic acid (VA) enhances the proliferation and self-renewal of normal hematopoietic stem cells and that breast cancer stem/progenitor cells can be resistant to radiation. From these data, we hypothesized that VA would fail to radiosensitize breast cancer stem/progenitor cells grown to three-dimensional (3D) mammospheres. Methods and Materials: We used the MCF7 breast cancer cell line grown under stem cell-promoting culture conditions (3D mammosphere) and standard nonstem cell monolayer culture conditions (two-dimensional) to examine the effect of pretreatment with VA on radiation sensitivity in clonogenic survival assays and on the expression of embryonic stem cellmore » transcription factors. Results: 3D-cultured MCF-7 cells expressed higher levels of Oct4, Nanog, and Sox2. The 3D passage enriched self-renewal and increased radioresistance in the 3D mammosphere formation assays. VA radiosensitized adherent cells but radioprotected 3D cells in single-fraction clonogenic assays. Moreover, fractionated radiation sensitized VA-treated adherent MCF7 cells but did not have a significant effect on VA-treated single cells grown to mammospheres. Conclusion: We have concluded that VA might preferentially radiosensitize differentiated cells compared with those expressing stem cell surrogates and that stem cell-promoting culture is a useful tool for in vitro evaluation of novel cancer therapeutic agents and radiosensitizers.« less

7 CFR 3402.23 - Documentation of progress on funded projects.

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2011-01-01

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2014-01-01

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2012-01-01

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L- and D-lactate enhance DNA repair and modulate the resistance of cervical carcinoma cells to anticancer drugs via histone deacetylase inhibition and hydroxycarboxylic acid receptor 1 activation.

PubMed

Wagner, Waldemar; Ciszewski, Wojciech M; Kania, Katarzyna D

2015-07-25

The consideration of lactate as an active metabolite is a newly emerging and attractive concept. Recently, lactate has been reported to regulate gene transcription via the inhibition of histone deacetylases (HDACs) and survival of cancer cells via hydroxycarboxylic acid receptor 1 (HCAR1). This study examined the role of L- and D-lactate in the DNA damage response in cervical cancer cells. Three cervical cancer cell lines were examined: HeLa, Ca Ski and C33A. The inhibitory activity of lactate on HDACs was analysed using Western blot and biochemical methods. The lactate-mediated stimulation of DNA repair and cellular resistance to neocarzinostatin, doxorubicin and cisplatin were studied using γ-H2AX, comet and clonogenic assays. HCAR1 and DNA repair gene expression was quantified by real-time PCR. DNA-PKcs activity and HCAR1 protein expression were evaluated via immunocytochemistry and Western blot, respectively. HCAR1 activation was investigated by measuring intracellular cAMP accumulation and Erk phosphorylation. HCAR1 expression was silenced using shRNA. L- and D-lactate inhibited HDACs, induced histone H3 and H4 hyperacetylation, and decreased chromatin compactness in HeLa cells. Treating cells with lactate increased LIG4, NBS1, and APTX expression by nearly 2-fold and enhanced DNA-PKcs activity. Based on γ-H2AX and comet assays, incubation of cells in lactate-containing medium increased the DNA repair rate. Furthermore, clonogenic assays demonstrated that lactate mediates cellular resistance to clinically used chemotherapeutics. Western blot and immunocytochemistry showed that all studied cell lines express HCAR1 on the cellular surface. Inhibiting HCAR1 function via pertussis toxin pretreatment partially abolished the effects of lactate on DNA repair. Down-regulating HCAR1 decreased the efficiency of DNA repair, abolished the cellular response to L-lactate and decreased the effect of D-lactate. Moreover, HCAR1 shRNA-expressing cells produced significantly lower mRNA levels of monocarboxylate transporter 4. Finally, the enhancement of DNA repair and cell survival by lactate was suppressed by pharmacologically inhibiting monocarboxylate transporters using the inhibitor α-cyano-4-hydroxycinnamic acid (α-CHCA). Our data indicate that L- and D-lactate present in the uterine cervix may participate in the modulation of cellular DNA damage repair processes and in the resistance of cervical carcinoma cells to anticancer therapy.

The differential susceptibilities of MCF-7 and MDA-MB-231 cells to the cytotoxic effects of curcumin are associated with the PI3K/Akt-SKP2-Cip/Kips pathway.

PubMed

Jia, Tao; Zhang, Li; Duan, Yale; Zhang, Min; Wang, Gang; Zhang, Jun; Zhao, Zheng

2014-01-01

The mechanism underlying the differential cytotoxicity of curcumin in various cancer types, however, remains largely unclear. The aims of this study is to examine the concentration- and time-related effects of curcumin on two different breast cancer cells, MCF-7 and MDA-MB-231, and investigated the functional changes induced by curcumin treatment, as well as their relationship to the PI3K/Akt-SKP2-Cip/Kips pathway. First, WST-1 and clonogenic assay were performed to determine the cytotoxicity of curcumin in MCF-7 and MDA-MB-231 cells. Then, the expression of CDK interacting protein/Kinase inhibitory protein (Cip/Kips) members (p27, p21 and p57) and S-phase kinase-associated protein-2 (SKP2) was investigated by QRT PCR and Western Blotting. Curcumin's effect on PI3K (phosphatidylinositol 3-kinase) /Akt and its substrates Foxo1 and Foxo3a were then studied by Western Blotting. Small interfering RNAs (siRNAs) targeting SKP2 was used to explore the relationship between SKP2 and Cip/Kips members. Finally, WST-1 assay was tested to explore the concomitant treatment with curcumin and the inhibition of PKB or SKP2 signaling on curcumin sensitivity in MCF-7 and MDA-MB-231 cells. We demonstrated MCF-7 and MDA-MB-231 cells exhibited differential responses to curcumin by WST-1 and clonogenic assay (MDA-MB-231 cells was sensitive, and MCF-7 cells was resistant), which were found to be related to the differential curcumin-mediated regulation of SKP2-Cip/Kips (p21 and p27 but not p57) signaling. The differential cellular responses were further linked to the converse effects of curcumin on PI3K/Akt and its substrates Foxo1 and Foxo3a. Importantly, PI3K inhibitor wortmannin could counteract both curcumin-induced phosphorylation of Akt and up-regulation of SKP2 in MCF-7 cells. Subsequent WST-1 assay demonstrated concomitant treatment with curcumin and wortmannin or SKP2 siRNA not only further augmented curcumin sensitivity in MDA-MB-231 cells but also overcame curcumin resistance in MCF-7 cells. Our study established PI3K/Akt-SKP2-Cip/Kips signaling pathway is involved in the mechanism of action of curcumin and revealed that the discrepant modulation of this pathway by curcumin is responsible for the differential susceptibilities of these two cell types to curcumin.

Creating a dashboard to track progress toward IOM recommendations for the future of nursing.

PubMed

Spetz, Joanne; Bates, Timothy; Chu, Lela; Lin, Jessica; Fishman, Nancy W; Melichar, Lori

2013-01-01

This article explains the process used to identify and develop a set of data used to track national progress toward the recommendations of the Institute of Medicine Committee for the Future of Nursing. The data are presented in a dashboard format to visually summarize information and quickly measure progress. The approach selected by the research team is outlined, the criteria for selecting candidate metrics are detailed, the process for seeking external guidance is described, and the final dashboard measures are presented. Finally, the methods for data collection for each metric are explicated, to guide states and local regions in the collection of their own data.

ERK1/2 signalling protects against apoptosis following endoplasmic reticulum stress but cannot provide long-term protection against BAX/BAK-independent cell death.

PubMed

Darling, Nicola J; Balmanno, Kathryn; Cook, Simon J

2017-01-01

Disruption of protein folding in the endoplasmic reticulum (ER) causes ER stress. Activation of the unfolded protein response (UPR) acts to restore protein homeostasis or, if ER stress is severe or persistent, drive apoptosis, which is thought to proceed through the cell intrinsic, mitochondrial pathway. Indeed, cells that lack the key executioner proteins BAX and BAK are protected from ER stress-induced apoptosis. Here we show that chronic ER stress causes the progressive inhibition of the extracellular signal-regulated kinase (ERK1/2) signalling pathway. This is causally related to ER stress since reactivation of ERK1/2 can protect cells from ER stress-induced apoptosis whilst ERK1/2 pathway inhibition sensitises cells to ER stress. Furthermore, cancer cell lines harbouring constitutively active BRAFV600E are addicted to ERK1/2 signalling for protection against ER stress-induced cell death. ERK1/2 signalling normally represses the pro-death proteins BIM, BMF and PUMA and it has been proposed that ER stress induces BIM-dependent cell death. We found no evidence that ER stress increased the expression of these proteins; furthermore, BIM was not required for ER stress-induced death. Rather, ER stress caused the PERK-dependent inhibition of cap-dependent mRNA translation and the progressive loss of pro-survival proteins including BCL2, BCLXL and MCL1. Despite these observations, neither ERK1/2 activation nor loss of BAX/BAK could confer long-term clonogenic survival to cells exposed to ER stress. Thus, ER stress induces cell death by at least two biochemically and genetically distinct pathways: a classical BAX/BAK-dependent apoptotic response that can be inhibited by ERK1/2 signalling and an alternative ERK1/2- and BAX/BAK-independent cell death pathway.

Lin28 induces resistance to anti-androgens via promotion of AR splice variant generation.

PubMed

Tummala, Ramakumar; Nadiminty, Nagalakshmi; Lou, Wei; Evans, Christopher P; Gao, Allen C

2016-04-01

Prostate cancer (PCa) is androgen-dependent initially and progresses to a castration-resistant state after androgen deprivation therapy. Treatment options for castration-resistant PCa include the potent second-generation anti-androgen enzalutamide or CYP17A1 inhibitor abiraterone. Recent clinical observations point to the development of resistance to these therapies which may be mediated by constitutively active alternative splice variants of the androgen receptor (AR). Sensitivity of LNCaP cells overexpressing Lin28 (LN-Lin28) to enzalutamide, abiraterone, or bicalutamide was compared to that of control LN-neo cells using cell growth assays, proliferation assays using MTT, anchorage-dependent clonogenic ability assays and soft agar assays. Ability of LN-Lin28 cells to maintain AR activation after treatment with enzalutamide, abiraterone, or bicalutamide was tested using immunofluorescence, Western blotting, ChIP assays, and qRT-PCR. Importance of Lin28 in enzalutamide resistance was assessed by the downregulation of Lin28 expression in C4-2B and 22Rv1 cells chronically treated with enzalutamide. Requirement for sustained AR signaling in LN-Lin28 cells was examined by the downregulation of either full length AR or AR-V7 using siRNA. We show that Lin28 promotes the development of resistance to currently used targeted therapeutics by enhancing the expression of AR splice variants such as AR-V7. PCa cells overexpressing Lin28 exhibit resistance to treatment with enzalutamide, abiraterone, or bicalutamide. Downregulation of Lin28 resensitizes enzalutamide-resistant PCa cells to enzalutamide treatment. We also show that the upregulation of splicing factors such as hnRNPA1 by Lin28 may mediate the enhanced generation of AR splice variants in Lin28-expressing cells. Our findings suggest that Lin28 plays a key role in the acquisition of resistance to AR-targeted therapies by PCa cells and establish the importance of Lin28 in PCa progression. © 2015 Wiley Periodicals, Inc.

Dual PI3K/mTOR inhibitors, GSK2126458 and PKI-587, suppress tumor progression and increase radiosensitivity in nasopharyngeal carcinoma.

PubMed

Liu, Tongxin; Sun, Quanquan; Li, Qi; Yang, Hua; Zhang, Yuqin; Wang, Rong; Lin, Xiaoshan; Xiao, Dong; Yuan, Yawei; Chen, Longhua; Wang, Wei

2015-02-01

Although combined chemoradiotherapy has provided considerable improvements for nasopharyngeal carcinoma (NPC), recurrence and metastasis are still frequent. The PI3K/Akt/mTOR pathway plays a critical role in tumor formation and tumor cell survival after radiation-induced DNA damage. In the present study, we evaluated whether inhibition of PI3K/mTOR by two novel dual inhibitors, GSK2126458 and PKI-587, could suppress tumor progression and sensitize NPC cells to radiation. Four NPC cell lines (CNE-1, CNE-2, 5-8F, and 6-10B) were used to analyze the effects of GSK216458 and PKI-587 on cell proliferation, migration, invasion, clonogenic survival, amount of residual γ-H2AX foci, cell cycle, and apoptosis after radiation. A 5-8F xenograft model was used to evaluate the in vivo effects of the two compounds in combination with ionizing radiation (IR). Both GSK216458 and PKI-587 effectively inhibited cell proliferation and motility in NPC cells and suppressed phosphorylation of Akt, mTOR, S6, and 4EBP1 proteins in a concentration- and time-dependent manner. Moreover, both compounds sensitized NPC cells to IR by increasing DNA damage, enhancing G2-M cell-cycle delay, and inducing apoptosis. In vivo, the combination of IR with GSK2126458 or PKI-587 significantly inhibited tumor growth. Antitumor effect was correlated with induction of apoptosis and suppression of the phosphorylation of mTOR, Akt, and 4EBP1. These new findings suggest the usefulness of PI3K/mTOR dual inhibition for antitumor and radiosensitizing. The combination of IR with a dual PI3K/mTOR inhibitor, GSK2126458 or PKI-587, might be a promising therapeutic strategy for NPC. ©2014 American Association for Cancer Research.

Hydroquinone-induced FOXP3-ADAM17-Lyn-Akt-p21 signaling axis promotes malignant progression of human leukemia U937 cells.

PubMed

Chen, Ying-Jung; Liu, Wen-Hsin; Chang, Long-Sen

2017-02-01

Hydroquinone (1,4-benzenediol; HQ), a major marrow metabolite of the leukemogen benzene, has been proven to evoke benzene-related hematological disorders and myelotoxicity in vitro and in vivo. The goal of the present study was to explore the role of FOXP3 in HQ-induced malignant progression of U937 human leukemia cells. U937 cells were treated with 5 μM HQ for 24 h, and the cells were re-suspended in serum-containing medium without HQ for 2 days. The same procedure was repeated three times, and the resulting U937/HQ cells were maintained in cultured medium containing 5 μM HQ. Proliferation and colony formation of U937/HQ cells were notably higher than those of U937 cells. Ten-eleven translocation methylcytosine dioxygenase-mediated demethylation of the Treg-specific demethylated region in FOXP3 gene resulted in higher FOXP3 expression in U937/HQ cells than in U937 cells. FOXP3-induced miR-183 expression reduced β-TrCP mRNA stability and suppressed β-TrCP-mediated Sp1 degradation, leading to up-regulation of Sp1 expression in U937/HQ cells. Sp1 up-regulation further increased ADAM17 and Lyn expression, and ADAM17 up-regulation stimulated Lyn activation in U937/HQ cells. Moreover, U937/HQ cells showed higher Lyn-mediated Akt activation and cytoplasmic p21 expression than U937 cells did. Abolishment of Akt activation decreased cytoplasmic p21 expression in U937/HQ cells. Suppression of FOXP3, ADAM17, and Lyn expression, as well as Akt inactivation, repressed proliferation and clonogenicity of U937/HQ cells. Together with the finding that cytoplasmic p21 shows anti-apoptotic and oncogenic activities in cancer cells, the present data suggest a role of FOXP3/ADAM17/Lyn/Akt/p21 signaling axis in HQ-induced hematological disorders.

Role of microRNA in Aggressive Prostate Cancer

DTIC Science & Technology

2015-09-01

j.issn.2223-4683.2013.08.01 Scan to your mobile device or view this article at: http://www.amepc.org/tau/article/view/2550/3635 229Translational Andrology...of Ezh2, accompanied with diminished clonogenic ability and sphere formation in PCa cells (45). Another let-7 target gene is High- mobility group AT...2013;32:4139-47. 89. Motoyama K, Inoue H, Nakamura Y, et al. Clinical significance of high mobility group A2 in human gastric cancer and its

Translational Control in Bone Marrow Failure

DTIC Science & Technology

2014-04-01

expressing the ATG2 ORF and the ATG3 ORF, we also evaluated the ATG> GTG mutation, which produces all 3 internal ORFs. (We did not test ORF4...expressing shortened forms of NE reduced clonogenic capacity (Fig. 4 of Tidwell et al.). When comparing the shorter isoforms of NE to each other, the GTG ...mutated from ATG to either ATA or GTG , shortened forms of NE were evident (Figure 2, c.3G.A, c.1A.G). When the highly conserved nucleotide in the Kozak

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Excellence in the Classroom

ERIC Educational Resources Information Center

Governor's Business Council (Texas), 2006

2006-01-01

Texas has made good progress in education since the school reform movement began in earnest 15 years ago. State test scores are up for all groups of students in all grades in all subjects. On the National Assessment of Educational Progress, Texas is close to the top in student performance in math, and has finally begun to make real progress in…

Enhanced radiosensitization of p53 mutant cells by oleamide.

PubMed

Lee, Yoon-Jin; Chung, Da Yeon; Lee, Su-Jae; Ja Jhon, Gil; Lee, Yun-Sil

2006-04-01

Effect of oleamide, an endogenous fatty-acid primary amide, on tumor cells exposed to ionizing radiation (IR) has never before been explored. NCI H460, human lung cancer cells, and human astrocytoma cell lines, U87 and U251, were used. The cytotoxicity of oleamide alone or in combination with IR was determined by clonogenic survival assay, and induction of apoptosis was estimated by FACS analysis. Protein expressions were confirmed by Western blotting, and immunofluorescence analysis of Bax by use of confocal microscopy was also performed. The combined effect of IR and oleamide to suppress tumor growth was studied by use of xenografts in the thighs of nude mice. Oleamide in combination with IR had a synergistic effect that decreased clonogenic survival of lung-carcinoma cell lines and also sensitized xenografts in nude mice. Enhanced induction of apoptosis of the cells by the combined treatment was mediated by loss of mitochondrial membrane potential, which resulted in the activation of caspase-8, caspase-9, and caspase-3 accompanied by cytochrome c release and Bid cleavage. The synergistic effects of the combined treatment were more enhanced in p53 mutant cells than in p53 wild-type cells. In p53 wild-type cells, both oleamide and radiation induced Bax translocation to mitochondria. On the other hand, in p53 mutant cells, radiation alone slightly induced Bax translocation to mitochondria, whereas oleamide induced a larger translocation. Oleamide may exhibit synergistic radiosensitization in p53 mutant cells through p53-independent Bax translocation to mitochondria.

Successful short-term cryopreservation of volume-reduced cord blood units in a cryogenic mechanical freezer: effects on cell recovery, viability, and clonogenic potential.

PubMed

Anagnostakis, Ioannis; Papassavas, Andreas C; Michalopoulos, Efstathios; Chatzistamatiou, Theofanis; Andriopoulou, Sofia; Tsakris, Athanassios; Stavropoulos-Giokas, Catherine

2014-01-01

Cord blood (CB) units are stored from weeks to years in liquid- or vapor-phase nitrogen until they are used for transplantation. We examined the effects of cryostorage in a mechanical freezer at -150°C on critical quality control variables of CB collections to investigate the possible use of mechanical freezers at -150°C as an alternative to storage in liquid- (or vapor-) phase nitrogen. A total of 105 CB units were thawed and washed at different time intervals (6, 12, 24, and 36 months). For every thawed CB unit, samples were removed and cell enumeration (total nucleated cells [TNCs], mononuclear cells [MNCs], CD34+, CD133+) was performed. In addition, viability was obtained with the use of flow cytometry, and recoveries were calculated. Also, total absolute colony-forming unit counts were performed and progenitor cell recoveries were studied by clonogenic assays. Significant differences (p < 0.05) were observed in certain variables (TNCs, MNC numbers, viability) when they were examined in relation with time intervals, while others (CD34+, CD133+) were relatively insensitive (p = NS) to the duration of time interval the CB units were kept in cryostorage condition. The data presented suggest that cryopreservation of CB units in a mechanical freezer at -150°C may represent an alternative cryostorage condition for CB cryopreservation. © 2013 American Association of Blood Banks.

Skeletal muscle-derived interstitial progenitor cells (PICs) display stem cell properties, being clonogenic, self-renewing, and multi-potent in vitro and in vivo.

PubMed

Cottle, Beverley J; Lewis, Fiona C; Shone, Victoria; Ellison-Hughes, Georgina M

2017-07-04

The development of cellular therapies to treat muscle wastage with disease or age is paramount. Resident muscle satellite cells are not currently regarded as a viable cell source due to their limited migration and growth capability ex vivo. This study investigated the potential of muscle-derived PW1 + /Pax7 - interstitial progenitor cells (PICs) as a source of tissue-specific stem/progenitor cells with stem cell properties and multipotency. Sca-1 + /PW1 + PICs were identified on tissue sections from hind limb muscle of 21-day-old mice, isolated by magnetic-activated cell sorting (MACS) technology and their phenotype and characteristics assessed over time in culture. Green fluorescent protein (GFP)-labelled PICs were used to determine multipotency in vivo in a tumour formation assay. Isolated PICs expressed markers of pluripotency (Oct3/4, Sox2, and Nanog), were clonogenic, and self-renewing with >60 population doublings, and a population doubling time of 15.8 ± 2.9 h. PICs demonstrated an ability to generate both striated and smooth muscle, whilst also displaying the potential to differentiate into cell types of the three germ layers both in vitro and in vivo. Moreover, PICs did not form tumours in vivo. These findings open new avenues for a variety of solid tissue engineering and regeneration approaches, utilising a single multipotent stem cell type isolated from an easily accessible source such as skeletal muscle.

Autophagy contributes to resistance of tumor cells to ionizing radiation.

PubMed

Chaachouay, Hassan; Ohneseit, Petra; Toulany, Mahmoud; Kehlbach, Rainer; Multhoff, Gabriele; Rodemann, H Peter

2011-06-01

Autophagy signaling is a novel important target to improve anticancer therapy. To study the role of autophagy on resistance of tumor cells to ionizing radiation (IR), breast cancer cell lines differing in their intrinsic radiosensitivity were used. Breast cancer cell lines MDA-MB-231 and HBL-100 were examined with respect to clonogenic cell survival and induction of autophagy after radiation exposure and pharmacological interference of the autophagic process. As marker for autophagy the appearance of LC3-I and LC3-II proteins was analyzed by SDS-PAGE and Western blotting. Formation of autophagic vacuoles was monitored by immunofluorescence staining of LC3. LC3-I and LC3-II formation differs markedly in radioresistant MDA-MB-231 versus radiosensitive HBL-100 cells. Western blot analyses of LC3-II/LC3-I ratio indicated marked induction of autophagy by IR in radioresistant MDA-MB-231 cells, but not in radiosensitive HBL-100 cells. Indirect immunofluorescence analysis of LC3-II positive vacuoles confirmed this differential effect. Pre-treatment with 3-methyladenine (3-MA) antagonized IR-induced autophagy. Likewise, pretreatment of radioresistant MDA-231 cells with autophagy inhibitors 3-MA or chloroquine (CQ) significantly reduced clonogenic survival of irradiated cells. Our data clearly indicate that radioresistant breast tumor cells show a strong post-irradiation induction of autophagy, which thus serves as a protective and pro-survival mechanism in radioresistance. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Relative biological effectiveness (RBE) of alpha radiation in cultured porcine aortic endothelial cells.

PubMed

Thomas, Patricia; Tracy, Bliss; Ping, Tilly; Baweja, Anar; Wickstrom, Mark; Sidhu, Narinder; Hiebert, Linda

2007-03-01

Northern peoples can receive elevated radiation doses (1- 10 mSv/y) from transfer of polonium-210 (210Po) through the lichen-caribou-human food chain. Ingested 210Po is primarily blood-borne and thus many of its short range alpha particles irradiate the endothelial cells lining the blood vessels. The relative biological effectiveness (RBE) of alpha particles vs. x-rays was examined in porcine aortic endothelial cells as a surrogate for understanding what might happen to human endothelial cells in northern populations consuming traditional foods. Cultured porcine aortic endothelial cells were exposed to x-ray and 210Po alpha particle radiation. Alpha irradiation was applied to the cell cultures internally via the culture medium and externally, using thin-bottomed culture dishes. The results given here are based on the external irradiation method, which was found to be more reliable. Dose-response curves were compared for four lethal endpoints (cell viability, live cell fraction, release of lactate dehydrogenase [LDH] and clonogenic survival) to determine the relative biological effectiveness (RBE) of alpha radiation. The alpha RBE for porcine cells varied from 1.6-21, depending on the endpoint: 21.2+/-4.5 for cell viability, 12.9+/-2.7 for decrease in live cell number, 5.3+/-0.4 for LDH release to the medium but only 1.6 +/-0.1 for clonogenic survival. The low RBE of 1.6 was due to x-ray hypersensitivity of endothelial cells at low doses.

Relative biological effectiveness (RBE) of 210Po alpha-particles versus X-rays on lethality in bovine endothelial cells.

PubMed

Thomas, P A; Tracy, B L; Ping, T; Wickstrom, M; Sidhu, N; Hiebert, L

2003-02-01

Alpha-radiation from polonium-210 ((210)Po) can elevate background radiation dose by an order of magnitude in people consuming large quantities of meat and seafood, particularly caribou and reindeer. Because up to 50% of the ingested (210)Po body burden is initially found in the blood, a primary target for the short range alpha-particles is the endothelial cells lining the blood vessels. This study examined the relative biological effectiveness (RBE) of (210)Po alpha-particles versus 250 kVp X-rays in producing injury to cultured bovine aortic endothelial cells. Radiation effects on cells were measured in four different ways: the percentage viable cells by trypan blue dye exclusion, the number of live cells, the lactate dehydrogenase (LDH) release to medium and the ability to form colonies (clonogenic survival). Comparison of dose-response curves yielded RBE values of 13.1+/-2.5 (SEM) for cell viability, 10.3+/-1.0 for live cell number and 11.1+/-3.0 for LDH activity. The RBE values for clonogenic survival were 14.0+/-1.0 based on the ratio of the initial slopes of the dose-response curves and 13.1, 9.9 and 7.7 for 50, 10 and 1% survival rate, respectively. At X-ray doses <0.25 Gy, a pronounced stimulatory effect on proliferation was noted. Exposure to (210)Po alpha-particles was seven to 14 times more effective than X-ray exposure in causing endothelial cell damage.

Targeting HSP70 and GRP78 in canine osteosarcoma cells in combination with doxorubicin chemotherapy.

PubMed

Asling, Jonathan; Morrison, Jodi; Mutsaers, Anthony J

2016-11-01

Heat shock proteins (HSPs) are molecular chaperones subdivided into several families based on their molecular weight. Due to their cytoprotective roles, these proteins may help protect cancer cells against chemotherapy-induced cell death. Investigation into the biologic activity of HSPs in a variety of cancers including primary bone tumors, such as osteosarcoma (OSA), is of great interest. Both human and canine OSA tumor samples have aberrant production of HSP70. This study assessed the response of canine OSA cells to inhibition of HSP70 and GRP78 by the ATP-mimetic VER-155008 and whether this treatment strategy could sensitize cells to doxorubicin chemotherapy. Single-agent VER-155008 treatment decreased cellular viability and clonogenic survival and increased apoptosis in canine OSA cell lines. However, combination schedules with doxorubicin after pretreatment with VER-155008 did not improve inhibition of cellular viability, apoptosis, or clonogenic survival. Treatment with VER-155008 prior to chemotherapy resulted in an upregulation of target proteins HSP70 and GRP78 in addition to the co-chaperone proteins Herp, C/EBP homologous transcription protein (CHOP), and BAG-1. The increased GRP78 was more cytoplasmic in location compared to untreated cells. Single-agent treatment also revealed a dose-dependent reduction in activated and total Akt. Based on these results, targeting GRP78 and HSP70 may have biologic activity in canine osteosarcoma. Further studies are required to determine if and how this strategy may impact the response of osteosarcoma cells to chemotherapy.

Upregulation of NAD(P)H:Quinone Oxidoreductase By Radiation Potentiates the Effect of Bioreductive β-Lapachone on Cancer Cells1

PubMed Central

Choi, Eun K; Terai, Kaoru; Ji, In-Mi; Kook, Yeon H; Park, Kyung H; Oh, Eun T; Griffin, Robert J; Lim, Byung U; Kim, Jin-Seok; Lee, Doo S; Boothman, David A; Loren, Melissa; Song, Chang W; Park, Heon Joo

2007-01-01

We found that β-lapachone (β-lap), a novel bioreductive drug, caused rapid apoptosis and clonogenic cell death in A549 human lung epithelial cancer cells in vitro in a dose-dependent manner. The clonogenic cell death caused by β-lap could be significantly inhibited by dicoumarol, an inhibitor of NAD(P)H:quinone oxido-reductase (NQO1), and also by siRNA for NQO1, demonstrating that NQO1-induced bioreduction of β-lap is an essential step in β-lap-induced cell death. Irradiation of A549 cells with 4 Gy caused a long-lasting upregulation of NQO1, thereby increasing NQO1-mediated β-lap-induced cell deaths. Although the direct cause of β-lap-induced apoptosis is not yet clear, β-lap treatment reduced the expression of p53 and NF-κB, whereas it increased cytochrome C release, caspase-3 activity, and γH2AX foci formation. Importantly, β-lap treatment immediately after irradiation enhanced radiation-induced cell death, indicating that β-lap sensitizes cancer cells to radiation, in addition to directly killing some of the cells. The growth of A549 tumors induced in immunocompromised mice could be markedly suppressed by local radiation therapy when followed by β-lap treatment. This is the first study to demonstrate that combined radiotherapy and β-lap treatment can have a significant effect on human tumor xenografts. PMID:17786182

Development of an XTT tetrazolium salt-based assay for detection of specific hyperthermia sensitizers in a high-flux screening programme.

PubMed

Lechpammer, S; Asea, A; Mallick, R; Zhong, R; Sherman, M Y; Calderwood, S K

2002-01-01

It is now possible to search for new drugs using high-throughput screening of chemical libraries accumulated over the past few years. To detect potential new hyperthermia sensitizers, we are screening for chemical inhibitors of thermotolerance. For the screening of a large chemical library, a rapid and simple assay based on the XTT-tetrazolium salt with the addition of intermediate electron acceptor, phenazine methosulphate (PMS) as a promoter, was developed. It was found that the sensitivity of the XTT/PMS assay is sufficient for assessing thermal cell killing and thermotolerance, although it was highly dependent on cell number and type. When the formazan assay system was challenged with the bioflavonoid drug quercetin (up to 25mm) and validated against the clonogenic cell survival assay, significant decreases in thermotolerant cell viability were observed, directly reflecting inhibition of thermotolerance. Although short-term assays can, in some instances, underestimate overall cell killing, the dose dependency of inhibition of thermotolerance by quercetin recorded in this study by clonogenic and XTT/PMS assays was similar. Application of the XTT/PMS assay in chemical library screening was highly effective in differentiating potential thermotolerance inhibitors from both chemicals with lack of efficacy and from toxic compounds. Taken together, these results show that the XTT/PMS assay, when carried out under careful conditions, is well suited for primary high-flux screen of many thousands of compounds, thus opening up new areas for discovery of hyperthermia sensitizers.

Spontaneous regression of curve in immature idiopathic scoliosis - does spinal column play a role to balance? An observation with literature review.

PubMed

Modi, Hitesh N; Suh, Seung-Woo; Yang, Jae-Hyuk; Hong, Jae-Young; Venkatesh, Kp; Muzaffar, Nasir

2010-11-04

Child with mild scoliosis is always a subject of interest for most orthopaedic surgeons regarding progression. Literature described Hueter-Volkmann theory regarding disc and vertebral wedging, and muscular imbalance for the progression of adolescent idiopathic scoliosis. However, many authors reported spontaneous resolution of curves also without any reason for that and the rate of resolution reported is almost 25%. Purpose of this study was to question the role of paraspinal muscle tuning/balancing mechanism, especially in patients with idiopathic scoliosis with early mild curve, for spontaneous regression or progression as well as changing pattern of curves. An observational study of serial radiograms in 169 idiopathic scoliosis children (with minimum follow-up one year) was carried. All children with Cobb angle < 25° and who were diagnosed for the first time were selected. As a sign of immaturity at the time of diagnosis, all children had Risser sign 0. No treatment was given to entire study group. Children were divided in three groups at final follow-up: Group A, B and C as children with regression, no change and progression of their curves, respectively. Additionally changes in the pattern of curve were also noted. Average age was 9.2 years at first visit and 10.11 years at final follow-up with an average follow-up of 21 months. 32.5% (55/169), 41.4% (70/169) and 26% (44/169) children exhibited regression, no change and progression in their curves, respectively. 46.1% of children (78/169) showed changing pattern of their curves during the follow-up visits before it settled down to final curve. Comparing final fate of curve with side of curve and number of curves it did not show any relationship (p > 0.05) in our study population. Possible reason for changing patterns could be better explained by the tuning/balancing mechanism of spinal column that makes an effort to balance the spine and result into spontaneous regression or prevent further progression of curve. If this which we called as "tuning/balancing mechanism" fails, curve will ultimately progress.

Gemcitabine treatment causes resistance and malignancy of pancreatic cancer stem-like cells via induction of lncRNA HOTAIR

PubMed Central

Wang, Li; Dong, Ping; Wang, Weiguo; Huang, Mingquan; Tian, Bole

2017-01-01

Gemcitabine is the first-line chemotherapeutic agent for advanced adenocarcinoma of the pancreas, despite the high risk of chemoresistance as a major disadvantage. In the past few years, significant advances have been made in the field of pancreatic cancer stem-like cells (CSCs) and their critical roles in drug resistance, invasion and metastasis, which are tightly regulated by long non-coding RNAs (lncRNAs). The present study demonstrated that HOX antisense intergenic RNA (HOTAIR) is not different between the pancreatic cancer cell line PANC-1 and its enriched CSC sub-population. However, after gemcitabine treatment, the expression levels of HOTAIR in CSCs were induced, but not in PANC-1 cells. HOTAIR induced by gemcitabine failed to cause chemoresistance, but promoted the clonogenicity, proliferation and migration of the cells. By introducing HOTAIR using lentivirus, chemoresistance was induced and the self-renewal capacity, proliferation and migration were significantly promoted. By contrast, HOTAIR knockdown in PANC-1 CSCs treated with or without gemcitabine decreased the cell proliferation, altered the cell cycle progression and induced apoptosis, demonstrating its critical roles in regulating the malignant character of PANC-1 CSCs. In conclusion, the present study demonstrated that HOTAIR may be induced by gemcitabine and acts as a tumor promoter by inhibiting the chemosensitivity, and promoting the self-renewal capacity, proliferation and migration of PANC-1 CSCs, which supports its potential application as a novel therapeutic approach for pancreatic cancer. PMID:29201179

Synergistic effect of Ebselen and gamma radiation on breast cancer cells.

PubMed

Thabet, Noura M; Moustafa, Enas M

2017-08-01

To explore the synergistic effect of a seleno-organic compound Ebselen (Ebs) and/or γ-radiation to exert antitumor effects on human breast cancer (MCF-7) cell line in vitro. Ebs cytotoxicity at various concentrations (10, 25, 50 and 75 μg), cell proliferation and clonogenic assay of Ebs and/or γ-radiation (at 1, 3 and 6 Gy), expression of p-IκBα and NF-κB, inflammatory cytokines levels (TNF-α, IL-2, INF-γ, IL-10 and TGF-β), apoptotic factors (Caspase-3, Granzyme-B and TRAIL) and angiogenic factor (VEGF) were investigated. The results showed that the effective dosage of this combination was observed at 25 μg/ml of Ebs with γ-radiation at 6 Gy. Data displayed a significant reduction in NF-κB mRNA along with an elevation in granzyme-B mRNA and TRAIL mRNA expression. Furthermore, protein expression of caspase-3 was elevated, whereas p-IκBα and p-NF-κB(p65) protein expression was reduced significantly. Also, a significant decline in TNF-α, IL-2, INF-γ, TGF-β with a significant increase in IL-10 levels were revealed. Meanwhile, a significant decrease in VEGF level and proliferation capacity were observed. We conclude that a combination of Ebs with radiotherapy has a major antitumor efficiency in inducing apoptosis and inhibiting cancer cell progression, due to the synergistic effect in regulating gene and protein expression, and in a modulating response of pro-and anti-inflammatory cytokines.

The developing cancer stem-cell model: clinical challenges and opportunities.

PubMed

Vermeulen, Louis; de Sousa e Melo, Felipe; Richel, Dick J; Medema, Jan Paul

2012-02-01

During the past decade, a stem-cell-like subset of cancer cells has been identified in many malignancies. These cells, referred to as cancer stem cells (CSCs), are of particular interest because they are believed to be the clonogenic core of the tumour and therefore represent the cell population that drives growth and progression. Many efforts have been made to design therapies that specifically target the CSC population, since this was predicted to be the crucial population to eliminate. However, recent insights have complicated the initial elegant model, by showing a dominant role for the tumour microenvironment in determining CSC characteristics within a malignancy. This is particularly important since dedifferentiation of non-tumorigenic tumour cells towards CSCs can occur, and therefore the CSC population in a neoplasm is expected to vary over time. Moreover, evidence suggests that not all tumours are driven by rare CSCs, but might instead contain a large population of tumorigenic cells. Even though these results suggest that specific targeting of the CSC population might not be a useful therapeutic strategy, research into the hierarchical cellular organisation of malignancies has provided many important new insights in the biology of tumours. In this Personal View, we highlight how the CSC concept is developing and influences our thinking on future treatment for solid tumours, and recommend ways to design clinical trials to assess drugs that target malignant disease in a rational fashion. Copyright © 2012 Elsevier Ltd. All rights reserved.

Extra-virgin olive oil phenols block cell cycle progression and modulate chemotherapeutic toxicity in bladder cancer cells

PubMed Central

Coccia, Andrea; Mosca, Luciana; Puca, Rosa; Mangino, Giorgio; Rossi, Alessandro; Lendaro, Eugenio

2016-01-01

Epidemiological data indicate that the daily consumption of extra-virgin olive oil (EVOO), a common dietary habit of the Mediterranean area, lowers the incidence of certain types of cancer, in particular bladder neoplasm. The aim of the present study was to evaluate the antiproliferative activity of polyphenols extracted from EVOO on bladder cancer (BCa), and to clarify the biological mechanisms that trigger cell death. Furthermore, we also evaluated the ability of low doses of extra-virgin olive oil extract (EVOOE) to modulate the in vitro activity of paclitaxel or mitomycin, two antineoplastic drugs used in the management of different types of cancer. Our results showed that EVOOE significantly inhibited the proliferation and clonogenic ability of T24 and 5637 BCa cells in a dose-dependent manner. Furthermore, cell cycle analysis after EVOOE treatment showed a marked growth arrest prior to mitosis in the G2/M phase for both cell lines, with the subsequent induction of apoptosis only in the T24 cells. Notably, simultaneous treatment of mitomycin C and EVOOE reduced the drug cytotoxicity due to inhibition of ROS production. Conversely, the co-treatment of T24 cells with paclitaxel and the polyphenol extract strongly increased the apoptotic cell death at each tested concentration compared to paclitaxel alone. Our results support the epidemiological evidence indicating that olive oil consumption exerts health benefits and may represent a starting point for the development of new anticancer strategies. PMID:27748855

Mannose phosphate isomerase regulates fibroblast growth factor receptor family signaling and glioma radiosensitivity.

PubMed

Cazet, Aurélie; Charest, Jonathan; Bennett, Daniel C; Sambrooks, Cecilia Lopez; Contessa, Joseph N

2014-01-01

Asparagine-linked glycosylation is an endoplasmic reticulum co- and post-translational modification that enables the transit and function of receptor tyrosine kinase (RTK) glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI), an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization.

Early LC3 lipidation induced by d-limonene does not rely on mTOR inhibition, ERK activation and ROS production and it is associated with reduced clonogenic capacity of SH-SY5Y neuroblastoma cells.

PubMed

Berliocchi, Laura; Chiappini, Carlotta; Adornetto, Annagrazia; Gentile, Debora; Cerri, Silvia; Russo, Rossella; Bagetta, Giacinto; Corasaniti, Maria Tiziana

2018-02-01

d-Limonene is a natural monoterpene abundant in Citrus essential oils. It is endowed with several biological activities, including inhibition of carcinogenesis and promotion of tumour regression. Recently, d-limonene has been shown to modulate autophagic markers in vitro at concentrations found in vivo, in clinical pharmacokinetic studies. Autophagy is an intracellular catabolic process serving as both an adaptive metabolic response and a quality control mechanism. Because autophagy defects have been linked to a wide range of human pathologies, including neurodegeneration and cancer, there is a need for new pharmacological tools to control deregulated autophagy. To better understand the effects of d-limonene on autophagy, to identify the molecular mechanisms through which this monoterpene rapidly triggers LC3 lipidation and to evaluate the role for autophagy in long-term effects of d-limonene. Human SH-SY5Y neuroblastoma, HepG2 hepatocellular carcinoma and MCF7 breast cancer cells were used. Endogenous LC3-II levels were evaluated by western blotting. Autophagic flux assay was performed using bafilomycin A1 and chloroquine. Intracellular distribution of LC3 protein was studied by confocal microscopy analysis of LC3B-GFP transduced cells. Expression of lysosomal-membrane protein LAMP-1 was assessed by immunofluorescence analysis. Phosphorylated levels of downstream substrates of mTOR kinase (p70S6 kinase, 4E-BP1, and ULK1) and ERK were analyzed by western blotting. Production of reactive oxygen species (ROS) was assessed by live confocal microscopy of cells loaded with CellROX ® Green Reagent. Clonogenic assay was used to evaluate the ability of treated cells to proliferate and form colonies. LC3 lipidation promoted by d-limonene correlates with autophagosome formation and stimulation of basal autophagy. LC3 lipidation does not rely on inhibition of mTOR kinase, which instead appears to be transiently activated. In addition, d-limonene rapidly activates ERK and stimulates ROS generation, yet none of these events is implicated in lipidation of LC3, which was only partly reduced by chelation of intracellular calcium. The early LC3 lipidation induced by d-limonene is associated with inhibition of clonogenic capacity which is reverted by the autophagy inhibitor chloroquine. d-Limonene rapidly stimulates the autophagic flux in cultured cancer cells, which could be usefully exploited for therapeutic purposes. Copyright © 2018 Elsevier GmbH. All rights reserved.

MiR-33a Decreases High-Density Lipoprotein-Induced Radiation Sensitivity in Breast Cancer

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

Wolfe, Adam R.; Bambhroliya, Arvind; Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, Houston, Texas

Purpose: We previously showed that high-density lipoprotein (HDL) radiosensitizes inflammatory breast cancer (IBC) cells in vitro and is associated with better local control after radiation therapy in IBC patients. The microRNA miR-33 family negatively regulates the adenosine triphosphate binding cassette transporter subfamily A member 1. We hypothesized that variations in miR-33a expression in IBC cancer cells versus non-IBC cells would correlate with radiation sensitivity following exposure to HDL in vitro. Methods and Materials: MiR-33a expression was analyzed by reverse transcriptase–polymerase chain reaction in 4 cell lines representing common clinical breast cancer subtypes. Overexpression and knockdown of miR-33a was demonstrated via transfection of anmore » miR-33a mimic or an anti-miR-33a construct in high- and low-expressing miR-33a cell lines. Clonogenic survival in vitro in these cells was quantified at baseline and following HDL treatment. MiR-33a expression on distant relapse-free survival (DRFS) of 210 cases downloaded from the Oxford breast cancer dataset was determined. Results: Expression levels of miR-33a were lower in IBC cell lines and IBC tumor samples than in non-IBC cell lines and normal breast tissue. Cholesterol concentrations in the cell membranes were higher in IBC cells than in non-IBC cells. Clonogenic survival following 24 hours of HDL treatment was decreased in response to irradiation in the low-miR-33a–expressing cell lines SUM149 and KPL4, but survival following HDL treatment decreased in the high-miR-33a–expressing cell lines MDA-MB-231 and SUM159. In the high-miR-33a–expressing cell lines, anti-miR-33a transfection decreased radiation resistance in clonogenic assays. Conversely, in the low-miR-33a–expressing cell lines, the miR-33a mimic reversed the HDL-induced radiation sensitization. Breast cancer patients in the top quartile based on miR-33a expression had markedly lower rates of DRFS than the bottom quartile (P=.0228, log-rank test). For breast cancer patients treated with radiation, high miR-33a expression predicted worse overall survival (P=.06). Conclusions: Our results reveal miR-33a negatively regulates HDL-induced radiation sensitivity in breast cancer.« less

40 CFR 60.2575 - What are my requirements for meeting increments of progress and achieving final compliance?

Code of Federal Regulations, 2012 CFR

2012-07-01

... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) STANDARDS OF PERFORMANCE FOR NEW STATIONARY... Units Model Rule-Increments of Progress § 60.2575 What are my requirements for meeting increments of...

40 CFR 60.2575 - What are my requirements for meeting increments of progress and achieving final compliance?

Code of Federal Regulations, 2014 CFR

2014-07-01

... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) STANDARDS OF PERFORMANCE FOR NEW STATIONARY... Units Model Rule-Increments of Progress § 60.2575 What are my requirements for meeting increments of...

40 CFR 60.2575 - What are my requirements for meeting increments of progress and achieving final compliance?

Code of Federal Regulations, 2013 CFR

2013-07-01

... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) STANDARDS OF PERFORMANCE FOR NEW STATIONARY... Units Model Rule-Increments of Progress § 60.2575 What are my requirements for meeting increments of...

FINAL Progress Report DOE Grant DE-FG02-04ER15587

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

Mullins, Charles Buddie

Catalysis Program - Viviane Schwartz Program Manager This Final Report discusses several archival journal articles that have been published that present and discuss the results that were discovered through this DOE grant.

78 FR 60226 - Amendments To Implement Certain Provisions of the Moving Ahead for Progress in the 21st Century...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-10-01

... Moving Ahead for Progress in the 21st Century Act (MAP-21) AGENCY: Federal Motor Carrier Safety...) adopts, as final, certain regulations required by the Moving Ahead for Progress in the 21st Century... required by MAP-21. Benefits and Costs The rule provisions considered both individually and in the...

Making Progress: The Use of Multiple Progress Reports to Enhance Advertising Students' Media Plan Term Projects

ERIC Educational Resources Information Center

Kritz, Gary H.; Lozada, Hector R.; Long, Mary M.

2007-01-01

Since the AACSB mandates that students demonstrate effective oral and written communication skills, it is imperative that business professors do what is necessary to improve such skills. The authors investigate whether the use of using multiple progress reports in an Advertising class project improves the final product. The data results show that…

78 FR 65705 - Request for Comments on the Annual Progress Report on the Outer Continental Shelf (OCS) Oil and...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-01

... (Five Year Program). The Annual Progress Report is available for review at: www.boem.gov/Five-Year-Program-Annual-Progress-Report/ . Information on the Five Year Program is available online at http://www... final on August 27, 2012, after the required 60-day congressional review period. Section 18(e) of the...

The hitchhikers guide to cancer stem cell theory: markers, pathways and therapy.

PubMed

Fábián, Ákos; Vereb, György; Szöllősi, János

2013-01-01

Cancer stem cell (CSC) biology is a rapidly developing field within cancer research. CSCs are postulated to be a unique cell population exclusively capable of infinite self renewal, multilineage differentiation and with ability to evade conventional cytotoxic cancer therapy. These traits distinguish CSCs from their more differentiated counterparts, which possess only limited or no potential for self renewal and tumor initiation. Therefore, CSCs would be the driving motor of malignant growth and therapy resistance. Accordingly, successful cancer treatment would need to eliminate this highly potent group of cells, since even small residual numbers would suffice to recapitulate the disease after therapy. Putative CSCs has been identified in a broad range of human malignancies and several cell surface markers have been associated with their stem cell phenotype. Despite all efforts, a pure CSC population has not been isolated and often in vitro clonogenic and in vivo tumorigenic potential is found in several cell populations with occasionally contradictory surface marker signatures. Here, we give a brief overview of recent advances in CSC theory, including the signaling pathways in CSCs that also appear crucial for stem cells homeostasis in normal tissues. We discuss evidence for the interaction of CSCs with the stromal tumor environment. Finally, we review the emerging potentially effective CSC-targeted treatment strategies and their future role in therapy. Copyright © 2012 International Society for Advancement of Cytometry.

Tech Prep II: Implementation Final Report.

ERIC Educational Resources Information Center

Brown, Jane A.

This document contains the final progress report on a tech prep implementation project and the Work Force Challenge 2000 Report developed during the project. The final report lists these major accomplishments: approximately 1,500 educators in grades K-12 were provided information concerning future global issues in the work force and the effects in…

40 CFR 60.1585 - What are my requirements for meeting increments of progress and achieving final compliance?

Code of Federal Regulations, 2010 CFR

2010-07-01

... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) STANDARDS OF PERFORMANCE FOR NEW STATIONARY... or Before August 30, 1999 Model Rule-Increments of Progress § 60.1585 What are my requirements for...

RECENT PROGRESS IN GLYCOCHEMISTRY AND GREEN CHEMISTRY. (R826123)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Computational Methods for Nonlinear Dynamic Problems in Solid and Structural Mechanics: Progress in the Theory and Modeling of Friction and in the Control of Dynamical Systems with Frictional Forces

DTIC Science & Technology

1989-03-31

present several numerical studies designed to reveal the effect that some of the governing parameters have on the behavior of the system and, whenever...Friction and in the Control of Dynamical Systems with Frictional Forces FINAL TECHNICAL REPORT March 31, 1989 _ -- I -.7: .-.- - : AFOSR Contract F49620...SOLID AND STRUCTURAL MECHANICS: Progress in the Theory and Modeling of Friction and in the Control of Dynamical Systems with Frictional Forces I I * FINAL

The use of Aotus Trivirgatus and Macaca Mulatta as Tools for Studies on Prevention and Therapy of Infections with Plasmodium Falciparum and Plasmodium Vivax

DTIC Science & Technology

1976-08-13

INFECTIONS WITH PLASMODIUM FALCIPARUM AND PLASMODIUM VIVAX (U) FINAL PROGRESS REPORT ( PROJECT 2284-XXIX) For the Period I May 1975 to 30 April...IT» IOC mit settiM I’jtf Section ^ I» ’■■■■• BisTtmunM/MWUiiun cooa DiJÜ iWBU. UK/» FINAL PROGRESS REPORT ( PROJECT 2284-XXIX) S...quinolinemethanols pyridinemethanols I ’As in previous years, the activities of this Project were focused on development of: (a) agents fully effective

A Novel siRNA-Based Approach to Study Mechanisms of Resistance/Action of a New Drug in Treatment of Breast Cancer. Addendum

DTIC Science & Technology

2006-08-01

electroporation, were tested in the MCF-7 breast cancer cell line. The cell line was then treated with a lethal dose of ET-743 and cytarabine , however no...drugs with known mechanisms of resistance, methotrexate (MTX) and cytarabine , using a clonogenic assay and MCF-7 breast cancer cells. 3. To employ...Aim 2. To test the ability of the generated siRNA library by using two drugs with known mechanisms of resistance, methotrexate (MTX) and cytarabine

Sensitization of Prostate Cancer Cells to Androgen Deprivation and Radiation via Manipulation of the MDM2 Pathway

DTIC Science & Technology

2005-04-01

cell number apoptosis, and clonogenic assays of LNCaP- MST. Months 1-6. c. Time course experiments of AS effects on AD, RT, and AD+RT in LNCaP and LNCaP...to AS- MDM2, and have not found much of an effect . More recently, we >" 0" have initiated the measurement of SmRNA expression using the Oligo Pollack...AL, Joon DL, Meistrich M, Hachem P, Pollack A. Effect of sequencing androgen deprivation and radiation on prostate cancer growth. Int J Radiat Oncol

40 CFR 60.1630 - How do I comply with the increment of progress for achieving final compliance?

Code of Federal Regulations, 2010 CFR

2010-07-01

... PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) STANDARDS OF PERFORMANCE FOR NEW STATIONARY SOURCES... Before August 30, 1999 Model Rule-Increments of Progress § 60.1630 How do I comply with the increment of...

75 FR 6012 - National Assessment of Educational Progress (NAEP) in Reading

Federal Register 2010, 2011, 2012, 2013, 2014

2010-02-05

... comment for evaluating and finalizing achievement levels definitions for the National Assessment of... comment and recommendations for improvements to the achievement levels definitions for the National Assessment of Educational Progress (NAEP) in reading. These achievement levels definitions describe the...

29 CFR 97.50 - Closeout.

Code of Federal Regulations, 2010 CFR

2010-07-01

... limited to: (1) Final performance or progress report. (2) Financial Status Report (SF 269) or Outlay Report and Request for Reimbursement for Construction Programs (SF-271) (as applicable.) (3) Final request for payment (SF-270) (if applicable). (4) Invention disclosure (if applicable). (5) Federally...

36 CFR 1207.50 - Closeout.

Code of Federal Regulations, 2010 CFR

2010-07-01

... limited to: (1) Final performance or progress report. (2) Financial Status Report (SF 269) or Outlay Report and Request for Reimbursement for Construction Programs (SF-271) (as applicable). (3) Final request for payment (SF-270) (if applicable). (4) Invention disclosure (if applicable). (5) Federally...

DNA double strand breaks induced by the indirect effect of radiation are more efficiently repaired by non-homologous end joining compared to homologous recombination repair.

PubMed

Bajinskis, Ainars; Natarajan, Adayapalam T; Erixon, Klaus; Harms-Ringdahl, Mats

2013-08-30

The aim of this study was to investigate the relative involvement of three major DNA repair pathways, i.e., non-homologous end joining (NHEJ), homologous recombination (HRR) and base excision (BER) in repair of DNA lesions of different complexity induced by low- or high-LET radiation with emphasis on the contribution of the indirect effect of radiation for these radiation qualities. A panel of DNA repair-deficient CHO cell lines was irradiated by (137)Cs γ-rays or radon progeny α-particles. Irradiation was also performed in the presence of 2M DMSO to reduce the indirect effect of radiation and the complexity of the DNA damage formed. Clonogenic survival and micronucleus assays were used to estimate efficiencies of the different repair pathways for DNA damages produced by direct and indirect effects. Removal of the indirect effect of low-LET radiation by DMSO increased clonogenic survival and decreased MN formation for all cell lines investigated. A direct contribution of the indirect effect of radiation to DNA base damage was suggested by the significant protection by DMSO seen for the BER deficient cell line. Lesions formed by the indirect effect are more readily repaired by the NHEJ pathway than by HRR after irradiation with γ-rays or α-particles as evaluated by cell survival and the yields of MN. The results obtained with BER- and NHEJ-deficient cells suggest that the indirect effect of radiation contributes significantly to the formation of repair substrates for these pathways. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of Selective Checkpoint Kinase 1 Inhibition on Cytarabine Cytotoxicity in Acute Myelogenous Leukemia Cells in Vitro

PubMed Central

Schenk, Erin L.; Koh, Brian D.; Flatten, Karen S.; Peterson, Kevin L.; Parry, David; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Karnitz, Larry M.; Kaufmann, Scott H.

2012-01-01

Purpose Previous studies have demonstrated that the replication checkpoint, which involves the kinases ATR and Chk1, contributes to cytarabine resistance in cell lines. In the present study, we examined whether this checkpoint is activated in clinical AML during cytarabine infusion in vivo and then assessed the impact of combining cytarabine with the recently described Chk1 inhibitor SCH 900776 in vitro. Experimental design AML marrow aspirates harvested before and during cytarabine infusion were examined by immunoblotting. Human AML lines treated with cytarabine in the absence or presence of SCH 900776 were assayed for checkpoint activation by immunoblotting, nucleotide incorporation into DNA and flow cytometry. Long-term effects in AML lines, clinical AML isolates, and normal myeloid progenitors were assayed using clonogenic assays. Results Immunoblotting demonstrated increased Chk1 phosphorylation, a marker of checkpoint activation, in over half of Chk1-containing AMLs after 48 h of cytarabine infusion. In human AML lines, SCH 900776 not only disrupted cytarabine-induced Chk1 activation and S phase arrest, but also markedly increased cytarabine-induced apoptosis. Clonogenic assays demonstrated that SCH 900776 enhanced the anti-proliferative effects of cytarabine in AML cell lines and clinical AML samples at concentrations that had negligible impact on normal myeloid progenitors. Conclusions These results not only provide evidence for cytarabine-induced S phase checkpoint activation in AML in the clinical setting, but also show that a selective Chk1 inhibitor can overcome the S phase checkpoint and enhance the cytotoxicity of cytarabine. Accordingly, further investigation of the cytarabine/SCH 900776 combination in AML appears warranted. PMID:22869869

Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

PubMed Central

Concannon, Caoimhin G.; Rehm, Markus; Kögel, Donat; Prehn, Jochen H. M.

2008-01-01

Background The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases. Methodology/Principal Findings To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations. Conclusions/Significance Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling. PMID:18665234

Synergistic Effect of Simvastatin Plus Radiation in Gastric Cancer and Colorectal Cancer: Implications of BIRC5 and Connective Tissue Growth Factor.

PubMed

Lim, Taekyu; Lee, Inkyoung; Kim, Jungmin; Kang, Won Ki

2015-10-01

We investigated the synergistic effect of simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor plus radiation therapy, on the proliferation and survival of gastric cancer (GC) and colorectal cancer (CRC) cells. We also studied several genes involved in the simvastatin/radiation-induced effects. Gastric cancer (AGS, SNU601, MKN1, and MKN28) and CRC (CoLo320, SW48, HT29, and HCT8) cell lines were treated with 0.2 μM simvastatin alone, or in combination with 0 to 4 Gy of radiation, and subjected to clonogenic survival and proliferation assays in vitro. To assess the molecular mechanism of the combination treatment, we performed microarray analysis, immunoblot assays, small interfering RNA knockdown experiments, and plasmid rescue assays. The antitumoral effects of simvastatin and radiation were evaluated in vivo using xenograft models. The combination therapy of simvastatin plus radiation inhibited basal clonogenic survival and proliferation of GC and CRC cells in vitro. Simvastatin suppressed the expression of BIRC5 and CTGF genes in these cancer cells. In vivo, the combined treatment with simvastatin and radiation significantly reduced the growth of xenograft tumors compared with treatment with radiation alone. We suggest that simvastatin has a synergistic effect with radiation on GC and CRC through the induction of apoptosis, which may be mediated by a simultaneous inhibition of BIRC5 and CTGF expression. A clinical trial of simvastatin in combination with radiation in patients with GC or CRC is warranted. Copyright © 2015 Elsevier Inc. All rights reserved.

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

La Russa, D

Purpose: The purpose of this project is to develop a robust method of parameter estimation for a Poisson-based TCP model using Bayesian inference. Methods: Bayesian inference was performed using the PyMC3 probabilistic programming framework written in Python. A Poisson-based TCP regression model that accounts for clonogen proliferation was fit to observed rates of local relapse as a function of equivalent dose in 2 Gy fractions for a population of 623 stage-I non-small-cell lung cancer patients. The Slice Markov Chain Monte Carlo sampling algorithm was used to sample the posterior distributions, and was initiated using the maximum of the posterior distributionsmore » found by optimization. The calculation of TCP with each sample step required integration over the free parameter α, which was performed using an adaptive 24-point Gauss-Legendre quadrature. Convergence was verified via inspection of the trace plot and posterior distribution for each of the fit parameters, as well as with comparisons of the most probable parameter values with their respective maximum likelihood estimates. Results: Posterior distributions for α, the standard deviation of α (σ), the average tumour cell-doubling time (Td), and the repopulation delay time (Tk), were generated assuming α/β = 10 Gy, and a fixed clonogen density of 10{sup 7} cm−{sup 3}. Posterior predictive plots generated from samples from these posterior distributions are in excellent agreement with the observed rates of local relapse used in the Bayesian inference. The most probable values of the model parameters also agree well with maximum likelihood estimates. Conclusion: A robust method of performing Bayesian inference of TCP data using a complex TCP model has been established.« less

The targeted histone deacetylase inhibitor tefinostat (CHR-2845) shows selective in vitro efficacy in monocytoid-lineage leukaemias

PubMed Central

Zabkiewicz, Joanna; Gilmour, Marie; Hills, Robert; Vyas, Pares; Bone, Elizabeth; Davidson, Alan; Burnett, Alan; Knapper, Steven

2016-01-01

Tefinostat (CHR-2845) is a novel monocyte/macrophage-targeted histone deacetylase (HDAC) inhibitor which is cleaved into its active acid by the intracellular esterase human carboxylesterase-1 (hCE-1). The in vitro efficacy of tefinostat was characterised in cell lines and in a cohort of 73 primary AML and CMML samples. Dose-dependent induction of apoptosis and significant growth inhibitory effects were seen in myelomonocytic (M4), monocytic/monoblastic (M5) and CMML samples in comparison to non-monocytoid AML sub-types (p = 0.007). Importantly, no growth inhibitory effects were seen in normal bone marrow CD34+ cells exposed to AML-toxic doses of tefinostat in clonogenic assays. Expression of hCE-1 was measured by intracellular flow cytometry and immunoblotting across the cohort, with highest levels seen in M5 AML patients. hCE-1 levels correlated with significantly increased tefinostat sensitivity (low EC50) as measured by growth inhibition assays (p = 0.001) and concomitant elevation of the mature monocytoid marker CD14+. Strong induction of intracellular histone protein acetylation was observed in tefinostat-responsive samples, as were high levels of the DNA damage sensor γ-H2A.X, highlighting potential biomarkers of patient responsiveness. Synergistic interaction between tefinostat and the current standard treatment cytarabine was demonstrated in dose response and clonogenic assays using simultaneous drug addition in primary samples (median Combination Index value = 0.51). These data provide a strong rationale for the further clinical evaluation of tefinostat in monocytoid-lineage haematological neoplasms including CMML and monocyte-lineage AMLs. PMID:26934551

Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery.

PubMed

Buckel, Lisa; Savariar, Elamprakash N; Crisp, Jessica L; Jones, Karra A; Hicks, Angel M; Scanderbeg, Daniel J; Nguyen, Quyen T; Sicklick, Jason K; Lowy, Andrew M; Tsien, Roger Y; Advani, Sunil J

2015-04-01

Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor-targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell-penetrating peptide targeting matrix metalloproteinases and RGD-binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low-passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule- and dose-dependent manner, correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double-strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with nontargeted free MMAE or tumor-targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor-targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell-penetrating peptides. ©2015 American Association for Cancer Research.

Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery

PubMed Central

Crisp, Jessica L.; Jones, Karra A.; Hicks, Angel M.; Scanderbeg, Daniel J.; Nguyen, Quyen T.; Sicklick, Jason K.; Lowy, Andrew M.; Tsien, Roger Y.; Advani, Sunil J.

2015-01-01

Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell penetrating peptide targeting matrix metalloproteinases and RGD binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule and dose dependent manner correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with non-targeted free MMAE or tumor targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell penetrating peptides. PMID:25681274

Functional activation of PPARγ in human upper aerodigestive cancer cell lines.

PubMed

Wright, Simon K; Wuertz, Beverly R; Harris, George; Abu Ghazallah, Raed; Miller, Wendy A; Gaffney, Patrick M; Ondrey, Frank G

2017-01-01

Upper aerodigestive cancer is an aggressive malignancy with relatively stagnant long-term survival rates over 20 yr. Recent studies have demonstrated that exploitation of PPARγ pathways may be a novel therapy for cancer and its prevention. We tested whether PPARγ is expressed and inducible in aerodigestive carcinoma cells and whether it is present in human upper aerodigestive tumors. Human oral cancer CA-9-22 and NA cell lines were treated with the PPAR activators eicosatetraynoic acid (ETYA), 15-deoxy-δ- 12,14-prostaglandin J2 (PG-J2), and the thiazolidinedione, ciglitazone, and evaluated for their ability to functionally activate PPARγ luciferase reporter gene constructs. Cellular proliferation and clonogenic potential after PPARγ ligand treatment were also evaluated. Aerodigestive cancer specimens and normal tissues were evaluated for PPARγ expression on gene expression profiling and immunoblotting. Functional activation of PPARγ reporter gene constructs and increases in PPARγ protein were confirmed in the nuclear compartment after PPARγ ligand treatment. Significant decreases in cell proliferation and clonogenic potential resulted from treatment. Lipid accumulation was induced by PPARγ activator treatment. 75% of tumor specimens and 100% of normal control tissues expressed PPARγ RNA, and PPARγ protein was confirmed in 66% of tumor specimens analyzed by immunoblotting. We conclude PPARγ can be functionally activated in upper aerodigestive cancer and that its activation downregulates several features of the neoplastic phenotype. PPARγ expression in human upper aerodigestive tract tumors and normal cells potentially legitimizes it as a novel intervention target in this disease. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Selective targeting of KRAS-Mutant cells by miR-126 through repression of multiple genes essential for the survival of KRAS-Mutant cells

PubMed Central

Hara, Toshifumi; Jones, Matthew F.; Subramanian, Murugan; Li, Xiao Ling; Ou, Oliver; Zhu, Yuelin; Yang, Yuan; Wakefield, Lalage M.; Hussain, S. Perwez; Gaedcke, Jochen; Ried, Thomas; Luo, Ji; Caplen, Natasha J.; Lal, Ashish

2014-01-01

MicroRNAs (miRNAs) regulate the expression of hundreds of genes. However, identifying the critical targets within a miRNA-regulated gene network is challenging. One approach is to identify miRNAs that exert a context-dependent effect, followed by expression profiling to determine how specific targets contribute to this selective effect. In this study, we performed miRNA mimic screens in isogenic KRAS-Wild-type (WT) and KRAS-Mutant colorectal cancer (CRC) cell lines to identify miRNAs selectively targeting KRAS-Mutant cells. One of the miRNAs we identified as a selective inhibitor of the survival of multiple KRAS-Mutant CRC lines was miR-126. In KRAS-Mutant cells, miR-126 over-expression increased the G1 compartment, inhibited clonogenicity and tumorigenicity, while exerting no effect on KRAS-WT cells. Unexpectedly, the miR-126-regulated transcriptome of KRAS-WT and KRAS-Mutant cells showed no significant differences. However, by analyzing the overlap between miR-126 targets with the synthetic lethal genes identified by RNAi in KRAS-Mutant cells, we identified and validated a subset of miR-126-regulated genes selectively required for the survival and clonogenicity of KRAS-Mutant cells. Our strategy therefore identified critical target genes within the miR-126-regulated gene network. We propose that the selective effect of miR-126 on KRAS-Mutant cells could be utilized for the development of targeted therapy for KRAS mutant tumors. PMID:25245095

Treprostinil indirectly regulates endothelial colony forming cell angiogenic properties by increasing VEGF-A produced by mesenchymal stem cells.

PubMed

Smadja, David M; Levy, Marilyne; Huang, Lan; Rossi, Elisa; Blandinières, Adeline; Israel-Biet, Dominique; Gaussem, Pascale; Bischoff, Joyce

2015-10-01

Pulmonary vasodilators and prostacyclin therapy in particular, have markedly improved the outcome of patients with pulmonary hypertension (PH). Endothelial dysfunction is a key feature of PH, and we previously reported that treprostinil therapy increases number and proliferative potential of endothelial colony forming cells (ECFC) isolated from PH patients' blood. In the present study, the objective was to determine how treprostinil contributes to the proangiogenic functions of ECFC. We examined the effect of treprostinil on ECFC obtained from cord blood in terms of colony numbers, proliferative and clonogenic properties in vitro, as well as in vivo vasculogenic properties. Surprisingly, treprostinil inhibited viability of cultured ECFC but did not modify their clonogenic properties or the endothelial differentiation potential from cord blood stem cells. Treprostinil treatment significantly increased the vessel-forming ability of ECFC combined with mesenchymal stem cells (MSC) in Matrigel implanted in nude mice. In vitro, ECFC proliferation was stimulated by conditioned media from treprostinil-pretreated MSC, and this effect was inhibited either by the use of VEGF-A blocking antibodies or siRNA VEGF-A in MSC. Silencing VEGF-A gene in MSC also blocked the pro-angiogenic effect of treprostinil in vivo. In conclusion, increased VEGF-A produced by MSC can account for the increased vessel formation observed during treprostinil treatment. The clinical relevance of these data was confirmed by the high level of VEGF-A detected in plasma from patients with paediatric PH who had been treated with treprostinil. Moreover, our results suggest that VEGF-A level in patients could be a surrogate biomarker of treprostinil efficacy.

Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro.

PubMed

Wang, Z-M; Kang, Y-H; Yang, X; Wang, J-F; Zhang, Q; Yang, B-X; Zhao, K-L; Xu, L-P; Yang, L-P; Ma, J-X; Huang, G-H; Cai, J; Sun, X-C

2016-01-01

To explore the radiosensitivity of andrographolide on esophageal cancer cell line ECA109. The inhibition effects of andrographolide were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Clonogenic survival assay was used to evaluate the effects of andrographolide on the radiosensitivity of esophageal cancer cells. Immunofluorescence was employed to examine Bax expression. The changes in cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of NF-κb/Cleaved-Caspase3/Bax/Bcl-2 was measured using Western blot analysis. DNA damage was detected via γ-H2AX foci counting. With a clear dose and time effects, andrographolide was found to inhibit the proliferation of esophageal cell line ECA109. The results of the clonogenic survival assay show that andrographolide could markedly enhance radiosensitivity (P < 0.05) with a sensitizing enhancement ratio of 1.28. Andrographolide caused a dose-dependent increase in Cleaved-Caspase3/Bax protein expression and a decrease in Bcl-2/NF-κb expression. Apoptosis in andrographolide-treated ECA-109 increased significantly compared with the apoptosis in the simple drug and radiation combined with drug groups (P < 0.001; P < 0.05). Moreover, compared with the independent radiation group, the andrographolide combined with radiation group increased the number of DNA double chain breaks. Andrographolide can increase the radiosensitivity of esophageal cell line ECA109. This result may be associated with the decrease in the NF-κb level and the induced apoptosis of esophageal cancer cells. © 2014 International Society for Diseases of the Esophagus.

Glucose starvation impairs DNA repair in tumour cells selectively by blocking histone acetylation.

PubMed

Ampferl, Rena; Rodemann, Hans Peter; Mayer, Claus; Höfling, Tobias Tim Alexander; Dittmann, Klaus

2018-03-01

Tumour cells are characterized by aerobic glycolysis and thus have high glucose consumption. Because repairing radiation-induced DNA damage is an energy-demanding process, we hypothesized that glucose starvation combined with radiotherapy could be an effective strategy to selectively target tumour cells. We glucose-starved tumour cells (A549, FaDu) in vitro and analysed their radiation-induced cell responses compared to normal fibroblasts (HSF7). Irradiation depleted intracellular ATP levels preferentially in cancer cells. Consequently, glucose starvation impaired DNA double-strand break (DSB) repair and radiosensitized confluent tumour cells but not normal fibroblasts. In proliferating tumour cells glucose starvation resulted in a reduction of proliferation, but failed to radiosensitize cells. Glucose supply was indispensable during the late DSB repair in confluent tumour cells starting approximately 13 h after irradiation, and glucose starvation inhibited radiation-induced histone acetylation, which is essential for chromatin relaxation. Sirtinol - an inhibitor of histone deacetylases - reverted the effects of glucose depletion on histone acetylation and DNA DSB repair in tumour cells. Furthermore, a glucose concentration of 2.8 mmol/L was sufficient to impair DSB repair in tumour cells and reduced their clonogenic survival under a fractionated irradiation regimen. In resting tumour cells, glucose starvation combined with irradiation resulted in the impairment of late DSB repair and the reduction of clonogenic survival, which was associated with disrupted radiation-induced histone acetylation. However, in normal cells, DNA repair and radiosensitivity were not affected by glucose depletion. Copyright © 2017 Elsevier B.V. All rights reserved.

Sarcosine influences apoptosis and growth of prostate cells via cell-type specific regulation of distinct sets of genes.

PubMed

Rodrigo, Miguel A Merlos; Strmiska, Vladislav; Horackova, Eva; Buchtelova, Hana; Michalek, Petr; Stiborova, Marie; Eckschlager, Tomas; Adam, Vojtech; Heger, Zbynek

2018-02-01

Sarcosine is a widely discussed oncometabolite of prostate cells. Although several reports described connections between sarcosine and various phenotypic changes of prostate cancer (PCa) cells, there is still a lack of insights on the complex phenomena of its effects on gene expression patterns, particularly in non-malignant and non-metastatic cells. To shed more light on this phenomenon, we performed parallel microarray profiling of RNA isolated from non-malignant (PNT1A), malignant (22Rv1), and metastatic (PC-3) prostate cell lines treated with sarcosine. Microarray results were experimentally verified using semi-quantitative-RT-PCR, clonogenic assay, through testing of the susceptibility of cells pre-incubated with sarcosine to anticancer agents with different modes of actions (inhibitors of topoisomerase II, DNA cross-linking agent, antimicrotubule agent and inhibitor of histone deacetylases) and by evaluation of activation of executioner caspases 3/7. We identified that irrespective of the cell type, sarcosine stimulates up-regulation of distinct sets of genes involved in cell cycle and mitosis, while down-regulates expression of genes driving apoptosis. Moreover, it was found that in all cell types, sarcosine had pronounced stimulatory effects on clonogenicity. Except of an inhibitor of histone deacetylase valproic acid, efficiency of all agents was significantly (P < 0.05) decreased in sarcosine pre-incubated cells. Our comparative study brings evidence that sarcosine affects not only metastatic PCa cells, but also their malignant and non-malignant counterparts and induces very similar changes in cells behavior, but via distinct cell-type specific targets. © 2017 Wiley Periodicals, Inc.

E-cigarette vapour is not inert and exposure can lead to cell damage.

PubMed

Holliday, Richard; Kist, Ralf; Bauld, Linda

2016-03-01

In vitro experiments were performed on normal epithelial cells as well as head and neck squamous cell carcinoma (HNSCC) cell lines. The widely available cell line HaCat, a spontaneously transformed immortal keratinocyte and the HNSCC cell lines HN30 and UMSCC10B were used. Cells were exposed to nicotine-containing and nicotine-free vapour extract from two popular e-cigarette brands for periods ranging from 48 hours to eight weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapour nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. In conclusion, our study strongly suggests that electronic cigarettes are not as safe as their marketing makes them appear to the public. Our in vitro experiments employing two brands of e-cigs show that at biologically relevant doses, vapourised e-cig liquids induce increased DNA strand breaks and cell death, and decreased clono- genic survival in both normal epithelial and HNSCC cell lines independently of nicotine content. Further research is needed to definitively determine the long-term effects of e-cig usage, as well as whether the DNA damage shown in our study as a result of e-cig exposure will lead to mutations that ultimately result in cancer.

78 FR 63875 - Progress Reports Rules Revision

Federal Register 2010, 2011, 2012, 2013, 2014

2013-10-25

... Reports Rules Revision AGENCY: Bureau of Prisons, Justice. ACTION: Final rule. SUMMARY: In this document, the Bureau of Prisons (Bureau) removes from regulations and/or modifies two types of progress reports: transfer reports and triennial reports. DATES: This rule is effective on November 25, 2013. FOR FURTHER...

Next Generation Attenuation Relationships for the Eastern United States (NGA-East)

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

Mahin, Stephen; Bozorgnia, Yousef

2016-04-11

This is a progress report to DOE for project Next Generation Attenuation for Central & Eastern US (NGA-East).This progress report consists of numerous monthly progress segments starting June 1, 2010 until December 31, 2015. Please note: the December 2015 progress report was issued in January 2016 due to the final university financial reporting at the end of this project. For each month, there is a technical progress list, and an update on the financial progress of the project. As you know, this project is jointly funded by the DOE, US Nuclear Regulatory Commission (NRC) and Electric Power Research Institute (EPRI).more » Thus, each segment includes financial progress for these three funding agencies.« less

Extreme Performance Scalable Operating Systems Final Progress Report (July 1, 2008 - October 31, 2011)

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

Malony, Allen D; Shende, Sameer

This is the final progress report for the FastOS (Phase 2) (FastOS-2) project with Argonne National Laboratory and the University of Oregon (UO). The project started at UO on July 1, 2008 and ran until April 30, 2010, at which time a six-month no-cost extension began. The FastOS-2 work at UO delivered excellent results in all research work areas: * scalable parallel monitoring * kernel-level performance measurement * parallel I/0 system measurement * large-scale and hybrid application performance measurement * onlne scalable performance data reduction and analysis * binary instrumentation

Partial Return Yoke for MICE Step IV and Final Step

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

Witte, Holger; Plate, Stephen; Berg, J.Scott

2015-06-01

This paper reports on the progress of the design and construction of a retro-fitted return yoke for the international Muon Ionization Cooling Experiment (MICE). MICE is a proof-of-principle experiment aiming to demonstrate ionization cooling experimentally. In earlier studies we outlined how a partial return yoke can be used to mitigate stray magnetic field in the experimental hall; we report on the progress of the construction of the partial return yoke for MICE Step IV. We also discuss an extension of the Partial Return Yoke for the final step of MICE; we show simulation results of the expected performance.

Partial return yoke for MICE step IV and final step

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

Witte, H.; Plate, S.; Berg, J. S.

2015-05-03

This paper reports on the progress of the design and construction of a retro-fitted return yoke for the international Muon Ionization Cooling Experiment (MICE). MICE is a proof-of-principle experiment aiming to demonstrate ionization cooling experimentally. In earlier studies we outlined how a partial return yoke can be used to mitigate stray magnetic field in the experimental hall; we report on the progress of the construction of the partial return yoke for MICE Step IV. We also discuss an extension of the Partial Return Yoke for the final step of MICE; we show simulation results of the expected performance.

7 CFR 3016.50 - Closeout.

Code of Federal Regulations, 2010 CFR

2010-01-01

... are not limited to: (1) Final performance or progress report. (2) Financial Status Report (SF 269) or Outlay Report and Request for Reimbursement for Construction Programs (SF-271) (as applicable.) (3) Final request for payment (SF-270) (if applicable). (4) Invention disclosure (if applicable). (5) Federally...

Evaluation of a Progressive Failure Analysis Methodology for Laminated Composite Structures

NASA Technical Reports Server (NTRS)

Sleight, David W.; Knight, Norman F., Jr.; Wang, John T.

1997-01-01

A progressive failure analysis methodology has been developed for predicting the nonlinear response and failure of laminated composite structures. The progressive failure analysis uses C plate and shell elements based on classical lamination theory to calculate the in-plane stresses. Several failure criteria, including the maximum strain criterion, Hashin's criterion, and Christensen's criterion, are used to predict the failure mechanisms. The progressive failure analysis model is implemented into a general purpose finite element code and can predict the damage and response of laminated composite structures from initial loading to final failure.

Final report : for the period of December 1999 through November 30, 2000 : Florida Transit Training Program (1999/2000) : Florida Technical Assistance Program (1999/2000)

DOT National Transportation Integrated Search

2000-01-01

The following progress report is intended to highlight the significant activities of the Florida Transit Training Program and Florida Technical Assistant Program. The following progress report is intended to highlight the significant activities of th...

Evaluation of the National Assessment of Educational Progress. Study Reports

ERIC Educational Resources Information Center

Buckendahl, Chad W.; Davis, Susan L.; Plake, Barbara S.; Sireci, Stephen G.; Hambleton, Ronald K.; Zenisky, April L.; Wells, Craig S.

2009-01-01

The "Evaluation of the National Assessment of Educational Progress: Study Reports" describes the special studies that comprised the design of the evaluation. In the Final Report, the authors presented a practical discussion of the evaluation studies to its primary, intended audience, namely policymakers. On this accompanying CD, readers…

10 CFR 600.341 - Monitoring and reporting program and financial performance.

Code of Federal Regulations, 2012 CFR

2012-01-01

... will be taken to address the deviations. (2) A final technical report if the award is for research and... dates for reports. At a minimum, requirements must include: (1) Periodic progress reports (at least... follows: (i) The program portions of the reports must address progress toward achieving program...

10 CFR 600.341 - Monitoring and reporting program and financial performance.

Code of Federal Regulations, 2014 CFR

2014-01-01

... will be taken to address the deviations. (2) A final technical report if the award is for research and... dates for reports. At a minimum, requirements must include: (1) Periodic progress reports (at least... follows: (i) The program portions of the reports must address progress toward achieving program...

10 CFR 600.341 - Monitoring and reporting program and financial performance.

Code of Federal Regulations, 2013 CFR

2013-01-01

... will be taken to address the deviations. (2) A final technical report if the award is for research and... dates for reports. At a minimum, requirements must include: (1) Periodic progress reports (at least... follows: (i) The program portions of the reports must address progress toward achieving program...

10 CFR 600.341 - Monitoring and reporting program and financial performance.

Code of Federal Regulations, 2011 CFR

2011-01-01

... will be taken to address the deviations. (2) A final technical report if the award is for research and... dates for reports. At a minimum, requirements must include: (1) Periodic progress reports (at least... follows: (i) The program portions of the reports must address progress toward achieving program...

Reduced posting and marking of flight progress strips for en route air traffic control : final report.

DOT National Transportation Integrated Search

2000-02-01

The new Display System Replacement (DSR) being implemented in air route traffic control centers (ARTCCs) will allow the data-side controller less room to post Flight Progress Strips (FPSs). We tested a new FPS marking and posting procedure designed t...

Minorities in Higher Education: A Pipeline Problem?

ERIC Educational Resources Information Center

Sethna, Beheruz N.

2011-01-01

This paper uses national data from the American Council on Education (ACE) to study the progress of different ethnic groups through the academic pipeline--stages studied include the Bachelor's, Master's, doctoral, levels, and then progress to the Assistant, Associate, and (full) Professor stages, to full-time administrators and finally to the CEO…

46 CFR 172.245 - Survival conditions.

Code of Federal Regulations, 2013 CFR

2013-10-01

... conditions. A vessel is presumed to survive assumed damage if it meets the following conditions in the final..., and trim must be below the lower edge of an opening through which progressive flooding may take place... inches (50 mm) when the vessel is in the equilibrium position. (e) Progressive flooding. In the design...

46 CFR 172.245 - Survival conditions.

Code of Federal Regulations, 2012 CFR

2012-10-01

... conditions. A vessel is presumed to survive assumed damage if it meets the following conditions in the final..., and trim must be below the lower edge of an opening through which progressive flooding may take place... inches (50 mm) when the vessel is in the equilibrium position. (e) Progressive flooding. In the design...

46 CFR 172.245 - Survival conditions.

Code of Federal Regulations, 2014 CFR

2014-10-01

... conditions. A vessel is presumed to survive assumed damage if it meets the following conditions in the final..., and trim must be below the lower edge of an opening through which progressive flooding may take place... inches (50 mm) when the vessel is in the equilibrium position. (e) Progressive flooding. In the design...

Progress in linear optics, non-linear optics and surface alignment of liquid crystals

NASA Astrophysics Data System (ADS)

Ong, H. L.; Meyer, R. B.; Hurd, A. J.; Karn, A. J.; Arakelian, S. M.; Shen, Y. R.; Sanda, P. N.; Dove, D. B.; Jansen, S. A.; Hoffmann, R.

We first discuss the progress in linear optics, in particular, the formulation and application of geometrical-optics approximation and its generalization. We then discuss the progress in non-linear optics, in particular, the enhancement of a first-order Freedericksz transition and intrinsic optical bistability in homeotropic and parallel oriented nematic liquid crystal cells. Finally, we discuss the liquid crystal alignment and surface effects on field-induced Freedericksz transition.

Progressive Fracture of Fiber Composite Build-Up Structures

NASA Technical Reports Server (NTRS)

Gotsis, Pascal K.; Chamis, C. C.; Minnetyan, Levon

1997-01-01

Damage progression and fracture of built-up composite structures is evaluated by using computational simulation. The objective is to examine the behavior and response of a stiffened composite (0/ +/- 45/90)(sub s6) laminate panel by simulating the damage initiation, growth, accumulation, progression and propagation to structural collapse. An integrated computer code, CODSTRAN, was augmented for the simulation of the progressive damage and fracture of built-up composite structures under mechanical loading. Results show that damage initiation and progression have significant effect on the structural response. Influence of the type of loading is investigated on the damage initiation, propagation and final fracture of the build-up composite panel.

Progressive Fracture of Fiber Composite Build-Up Structures

NASA Technical Reports Server (NTRS)

Minnetyan, Levon; Gotsis, Pascal K.; Chamis, C. C.

1997-01-01

Damage progression and fracture of built-up composite structures is evaluated by using computational simulation. The objective is to examine the behavior and response of a stiffened composite (0 +/-45/90)(sub s6) laminate panel by simulating the damage initiation, growth, accumulation, progression and propagation to structural collapse. An integrated computer code CODSTRAN was augmented for the simulation of the progressive damage and fracture of built-up composite structures under mechanical loading. Results show that damage initiation and progression to have significant effect on the structural response. Influence of the type of loading is investigated on the damage initiation, propagation and final fracture of the build-up composite panel.

Procedures for Handling Retaliation Complaints Under Section 31307 of the Moving Ahead for Progress in the 21st Century Act (MAP-21). Final rule.

PubMed

2016-12-14

On March 16, 2016, the Occupational Safety and Health Administration (OSHA) of the U.S. Department of Labor (Department) issued an interim final rule (IFR) that provided procedures for the Department's processing of complaints under the employee protection (retaliation or whistleblower) provisions of Section 31307 of the Moving Ahead for Progress in the 21st Century Act (MAP-21). The IFR established procedures and time frames for the handling of retaliation complaints under MAP-21, including procedures and time frames for employee complaints to OSHA, investigations by OSHA, appeals of OSHA determinations to an administrative law judge (ALJ) for a hearing de novo, hearings by ALJs, review of ALJ decisions by the Administrative Review Board (ARB) (acting on behalf of the Secretary of Labor) and judicial review of the Secretary's final decision. It also set forth the Department's interpretations of the MAP-21 whistleblower provisions on certain matters. This final rule adopts, without change, the IFR.

Isolation of hair follicle bulge stem cells from YFP-expressing reporter mice.

PubMed

Nakrieko, Kerry-Ann; Irvine, Timothy S; Dagnino, Lina

2013-01-01

In this article we provide a method to isolate hair follicle stem cells that have undergone targeted gene inactivation. The mice from which these cells are isolated are bred into a Rosa26-yellow fluorescent protein (YFP) reporter background, which results in YFP expression in the targeted stem cell population. These cells are isolated and purified by fluorescence-activated cell sorting, using epidermal stem cell-specific markers in conjunction with YFP fluorescence. The purified cells can be used for gene expression studies, clonogenic experiments, and biological assays, such as viability and capacity for directional migration.

Synthesis and Antiproliferative Activity of 2,5-bis(3′-Indolyl)pyrroles, Analogues of the Marine Alkaloid Nortopsentin

PubMed Central

Carbone, Anna; Parrino, Barbara; Barraja, Paola; Spanò, Virginia; Cirrincione, Girolamo; Diana, Patrizia; Maier, Armin; Kelter, Gerhard; Fiebig, Heinz-Herbert

2013-01-01

2,5-bis(3′-Indolyl)pyrroles, analogues of the marine alkaloid nortopsentin, were conveniently prepared through a three step procedure in good overall yields. Derivatives 1a and 1b exhibited concentration-dependent antitumor activity towards a panel of 42 human tumor cell lines with mean IC50 values of 1.54 μM and 0.67 μM, respectively. Investigating human tumor xenografts in an ex-vivo clonogenic assay revealed selective antitumor activity, whereas sensitive tumor models were scattered among various tumor histotypes. PMID:23455514

Investigating the anti-proliferative activity of styrylazanaphthalenes and azanaphthalenediones.

PubMed

Mrozek-Wilczkiewicz, Anna; Kalinowski, Danuta S; Musiol, Robert; Finster, Jacek; Szurko, Agnieszka; Serafin, Katarzyna; Knas, Magdalena; Kamalapuram, Sishir K; Kovacevic, Zaklina; Jampilek, Josef; Ratuszna, Alicja; Rzeszowska-Wolny, Joanna; Richardson, Des R; Polanski, Jaroslaw

2010-04-01

A group of styrylazanaphthalenes and azanaphthalenediones were synthesized and tested for their anti-proliferative activity. Most of the compounds were obtained with the use of microwave-assisted synthesis. The lipophilicity of the compounds was measured by RP-HPLC and their anti-proliferative activity was assayed against the human SK-N-MC neuroepithelioma and HCT116 human colon carcinoma cell lines. Active compounds were also tested in clonogenity and comet assays. Several quinazolinone and styrylquinazoline analogues were found to have markedly greater anti-proliferative activity than desferoxamine and cis-platin. Copyright 2010 Elsevier Ltd. All rights reserved.

No Evidence for a Low Linear Energy Transfer Adaptive Response in Irradiated RKO Cells

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

Sowa, Marianne B.; Goetz, Wilfried; Baulch, Janet E.

2011-01-06

It has become increasingly evident from reports in the literature that there are many confounding factors that are capable of modulating radiation induced non-targeted responses such as the bystander effect and the adaptive response. In this paper we examine recent data that suggest that the observation of non-targeted responses may not be universally observable for differing radiation qualities. We have conducted a study of the adaptive response following low LET exposures for human colon carcinoma cells and failed to observe adaption for the endpoints of clonogenic survival or micronucleus formation.

Genomic amplification of Fanconi anemia complementation group A (FancA) in head and neck squamous cell carcinoma (HNSCC): Cellular mechanisms of radioresistance and clinical relevance.

PubMed

Hess, Julia; Unger, Kristian; Orth, Michael; Schötz, Ulrike; Schüttrumpf, Lars; Zangen, Verena; Gimenez-Aznar, Igor; Michna, Agata; Schneider, Ludmila; Stamp, Ramona; Selmansberger, Martin; Braselmann, Herbert; Hieber, Ludwig; Drexler, Guido A; Kuger, Sebastian; Klein, Diana; Jendrossek, Verena; Friedl, Anna A; Belka, Claus; Zitzelsberger, Horst; Lauber, Kirsten

2017-02-01

Radio (chemo) therapy is a crucial treatment modality for head and neck squamous cell carcinoma (HNSCC), but relapse is frequent, and the underlying mechanisms remain largely elusive. Therefore, novel biomarkers are urgently needed. Previously, we identified gains on 16q23-24 to be associated with amplification of the Fanconi anemia A (FancA) gene and to correlate with reduced progression-free survival after radiotherapy. Here, we analyzed the effects of FancA on radiation sensitivity in vitro, characterized the underlying mechanisms, and evaluated their clinical relevance. Silencing of FancA expression in HNSCC cell lines with genomic gains on 16q23-24 resulted in significantly impaired clonogenic survival upon irradiation. Conversely, overexpression of FancA in immortalized keratinocytes conferred increased survival accompanied by improved DNA repair, reduced accumulation of chromosomal translocations, but no hyperactivation of the FA/BRCA-pathway. Downregulation of interferon signaling as identified by microarray analyses, enforced irradiation-induced senescence, and elevated production of the senescence-associated secretory phenotype (SASP) appeared to be candidate mechanisms contributing to FancA-mediated radioresistance. Data of the TCGA HNSCC cohort confirmed the association of gains on 16q24.3 with FancA overexpression and impaired overall survival. Importantly, transcriptomic alterations similar to those observed upon FancA overexpression in vitro strengthened the clinical relevance. Overall, FancA amplification and overexpression appear to be crucial for radiotherapeutic failure in HNSCC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Ligand stimulation of ErbB4 and a constitutively-active ErbB4 mutant result in different biological responses in human pancreatic tumor cell lines

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

Mill, Christopher P.; Auburn University Harrison School of Pharmacy, Auburn, AL 36849-5501; Gettinger, Kathleen L.

2011-02-15

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Indeed, it has been estimated that 37,000 Americans will die from this disease in 2010. Late diagnosis, chemoresistance, and radioresistance of these tumors are major reasons for poor patient outcome, spurring the search for pancreatic cancer early diagnostic and therapeutic targets. ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases (RTKs), a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. These RTKs play central roles in many human malignancies by regulating cell proliferation, survival, differentiation, invasiveness, motility,more » and apoptosis. In this report we demonstrate that human pancreatic tumor cell lines exhibit minimal ErbB4 expression; in contrast, these cell lines exhibit varied and in some cases abundant expression and basal tyrosine phosphorylation of EGFR, ErbB2, and ErbB3. Expression of a constitutively-dimerized and -active ErbB4 mutant inhibits clonogenic proliferation of CaPan-1, HPAC, MIA PaCa-2, and PANC-1 pancreatic tumor cell lines. In contrast, expression of wild-type ErbB4 in pancreatic tumor cell lines potentiates stimulation of anchorage-independent colony formation by the ErbB4 ligand Neuregulin 1{beta}. These results illustrate the multiple roles that ErbB4 may be playing in pancreatic tumorigenesis and tumor progression.« less

In vitro and in vivo antitumor effects of chloroquine on oral squamous cell carcinoma

PubMed Central

Jia, Lihua; Wang, Juan; Wu, Tong; Wu, Jinan; Ling, Junqi; Cheng, Bin

2017-01-01

Chloroquine, which is a widely used antimalarial drug, has been reported to exert anticancer activity in some tumor types; however, its potential effects on oral squamous cell carcinoma (OSCC) remain unclear. The present study aimed to explore the effects and possible underlying mechanisms of chloroquine against OSCC. MTT and clonogenic assays were conducted to evaluate the effects of chloroquine on the human OSCC cell lines SCC25 and CAL27. Cell cycle progression and apoptosis were detected using flow cytometry. Autophagy was monitored using microtubule-associated protein 1A/1B-light chain 3 as an autophagosomal marker. In order to determine the in vivo antitumor effects of chloroquine on OSCC, a CAL27 xenograft model was used. The results demonstrated that chloroquine markedly inhibited the proliferation and the colony-forming ability of both OSCC cell lines in a dose- and time-dependent manner in vitro. Chloroquine also disrupted the cell cycle, resulting in the cell cycle arrest of CAL27 and SCC25 cells at G0/G1 phase, via downregulation of cyclin D1. In addition, chloroquine inhibited autophagy, and induced autophagosome and autolysosome accumulation in the cytoplasm, thus interfering with degradation; however, OSCC apoptosis was barely affected by chloroquine. The results of the in vivo study demonstrated that chloroquine effectively inhibited OSCC tumor growth in the CAL27 xenograft model. In conclusion, the present study reported the in vitro and in vivo antitumor effects of chloroquine on OSCC, and the results indicated that chloroquine may be considered a potent therapeutic agent against human OSCC. PMID:28849182

Antitumor potential of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H-pyrazoles in human bladder cancer cells.

PubMed

Tessmann, Josiane Weber; Buss, Julieti; Begnini, Karine Rech; Berneira, Lucas Moraes; Paula, Favero Reisdorfer; de Pereira, Claudio Martin Pereira; Collares, Tiago; Seixas, Fabiana Kömmling

2017-10-01

Bladder cancer is a genitourinary malignant disease common worldwide. Current chemotherapy is often limited mainly due to toxicity and drug resistance. Thus, there is a continued need to discover new therapies. Recently evidences shows that pyrazoline derivatives are promising antitumor agents in many types of cancers, but there are no studies with bladder cancer. In order to find potent and novel chemotherapy drugs for bladder cancer, a series of pyrazoline derivatives 2a-2d were tested for their antitumor activity in two human bladder cancer cell lines 5647 and T24. The MTT assay showed that the compounds 1-thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazole (2a) and 1-thiocarbamoyl-5-(4-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole (2c) decrease the cell viability of 5637 cells. Molecular modeling indicated that these compounds had a good oral bioavailability and low toxicities. Clonogenic assay and flow cytometric analysis were used to assess colony formation, apoptosis induction and cell cycle distribution. Overall, our results suggest that pyrazoline 2a and 2c, with the substituents hydrogen and chlorine respectively, may decrease cell viability and colony formation of bladder cancer 5637 cell line by inhibition of cell cycle progression, and for pyrazoline 2a, by induction of apoptosis. As indicated by the physicochemical properties of these compounds, the steric factor influences the activity. Therefore, these pyrazoline derivatives can be considered promising anticancer agents for the treatment of bladder cancer. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Induction of caspase-dependent extrinsic apoptosis by apigenin through inhibition of signal transducer and activator of transcription 3 (STAT3) signalling in HER2-overexpressing BT-474 breast cancer cells.

PubMed

Seo, Hye-Sook; Jo, Jae Kyung; Ku, Jin Mo; Choi, Han-Seok; Choi, Youn Kyung; Woo, Jong-Kyu; Kim, Hyo In; Kang, Soo-Yeon; Lee, Kang Min; Nam, Koong Won; Park, Namkyu; Jang, Bo-Hyoung; Shin, Yong Cheol; Ko, Seong-Gyu

2015-10-23

Phytoestrogen intake is known to be beneficial to decrease breast cancer incidence and progression. But its molecular mechanisms of action are still unknown. The present study aimed to examine the effect of apigenin on proliferation and apoptosis in HER2-expressing breast cancer cells. In our experiments, apigenin inhibited the proliferation of BT-474 cells in a dose- and time-dependent manner. Apigenin also inhibited clonogenic survival (anchorage-dependent and -independent) of BT-474 cells in a dose-dependent manner. These growth inhibitions were accompanied with an increase in sub-G0/G1 apoptotic populations. Apigenin-induced extrinsic a caspase-dependent apoptosis up-regulating the levels of cleaved caspase-8 and cleaved caspase-3, and inducing the cleavage of poly (ADP-ribose) polymerase (PARP). Whereas, apigenin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease mitochondrial membrane potential without affecting the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX). Apigenin reduced the expression of phospho-JAK1, phospho-JAK2 and phospho-STAT3 and decreased signal transducer and activator of transcription 3 (STAT3) dependent luciferase reporter gene activity in BT-474 cells. Apigenin inhibited CoCl2-induced VEGF secretion and decreased the nuclear translocation of STAT3. Our study indicates that apigenin induces apoptosis through inhibition of STAT3 signalling and could serve as a useful compound to prevent or treat HER2-overexpressing breast cancer. © 2015 Authors.

Induction of caspase-dependent extrinsic apoptosis by apigenin through inhibition of signal transducer and activator of transcription 3 (STAT3) signalling in HER2-overexpressing BT-474 breast cancer cells

PubMed Central

Seo, Hye-Sook; Jo, Jae Kyung; Ku, Jin Mo; Choi, Han-Seok; Choi, Youn Kyung; Woo, Jong-Kyu; in Kim, Hyo; Kang, Soo-yeon; Lee, Kang min; Nam, Koong Won; Park, Namkyu; Jang, Bo-Hyoung; Shin, Yong Cheol; Ko, Seong-Gyu

2015-01-01

Phytoestrogen intake is known to be beneficial to decrease breast cancer incidence and progression. But its molecular mechanisms of action are still unknown. The present study aimed to examine the effect of apigenin on proliferation and apoptosis in HER2-expressing breast cancer cells. In our experiments, apigenin inhibited the proliferation of BT-474 cells in a dose- and time-dependent manner. Apigenin also inhibited clonogenic survival (anchorage-dependent and -independent) of BT-474 cells in a dose-dependent manner. These growth inhibitions were accompanied with an increase in sub-G0/G1 apoptotic populations. Apigenin-induced extrinsic a caspase-dependent apoptosis up-regulating the levels of cleaved caspase-8 and cleaved caspase-3, and inducing the cleavage of poly (ADP-ribose) polymerase (PARP). Whereas, apigenin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease mitochondrial membrane potential without affecting the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX). Apigenin reduced the expression of phospho-JAK1, phospho-JAK2 and phospho-STAT3 and decreased signal transducer and activator of transcription 3 (STAT3) dependent luciferase reporter gene activity in BT-474 cells. Apigenin inhibited CoCl2-induced VEGF secretion and decreased the nuclear translocation of STAT3. Our study indicates that apigenin induces apoptosis through inhibition of STAT3 signalling and could serve as a useful compound to prevent or treat HER2-overexpressing breast cancer. PMID:26500281

miR-320a regulates cell proliferation and apoptosis in multiple myeloma by targeting pre-B-cell leukemia transcription factor 3

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

Lu, Yinghao; Department of Hematology, Affiliated Hospital of Guizhou Medical University, The Hematopoietic Stem Cell Transplant Center of Guizhou Province, Blood Diseases Diagnosis and Treatment Center of Guizhou Province, Guiyang, 550004, Guizhou Province; Wu, Depei, E-mail: wudepei@medmail.com.cn

2016-05-13

Aberrant expression of microRNAs (miRNAs) is implicated in cancer development and progression. While miR-320a is reported to be deregulated in many malignancy types, its biological role in multiple myeloma (MM) remains unclear. Here, we observed reduced expression of miR-320a in MM samples and cell lines. Ectopic expression of miR-320a dramatically suppressed cell viability and clonogenicity and induced apoptosis in vitro. Mechanistic investigation led to the identification of Pre-B-cellleukemia transcription factor 3 (PBX3) as a novel and direct downstream target of miR-320a. Interestingly, reintroduction of PBX3 abrogated miR-320a-induced MM cell growth inhibition and apoptosis. In a mouse xenograft model, miR-320a overexpression inhibitedmore » tumorigenicity and promoted apoptosis. Our findings collectively indicate that miR-320a inhibits cell proliferation and induces apoptosis in MM cells by directly targeting PBX3, supporting its utility as a novel and potential therapeutic agent for miRNA-based MM therapy. -- Highlights: •Expression of miR-320a in MM cell induces apoptosis in vitro. •miR-320a represses PBX3 via targeting specific sequences in the 3′UTR region. •Exogenous expression of PBX3 reverses the effects of miR-320a in inhibiting MM cell growth and promoting apoptosis. •Overexpression of miR-320a inhibits tumor growth and increases apoptosis in vivo.« less

Preclinical and clinical efficacy of XPO1/CRM1 inhibition by the karyopherin inhibitor KPT-330 in Ph+ leukemias

PubMed Central

Walker, Christopher J.; Oaks, Joshua J.; Santhanam, Ramasamy; Neviani, Paolo; Harb, Jason G.; Ferenchak, Gregory; Ellis, Justin J.; Landesman, Yosef; Eisfeld, Ann-Kathrin; Gabrail, Nash Y.; Smith, Carrie L.; Caligiuri, Michael A.; Hokland, Peter; Roy, Denis Claude; Reid, Alistair; Milojkovic, Dragana; Goldman, John M.; Apperley, Jane; Garzon, Ramiro; Marcucci, Guido; Shacham, Sharon; Kauffman, Michael G.

2013-01-01

As tyrosine kinase inhibitors (TKIs) fail to induce long-term response in blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL), novel therapies targeting leukemia-dysregulated pathways are necessary. Exportin-1 (XPO1), also known as chromosome maintenance protein 1, regulates cell growth and differentiation by controlling the nucleocytoplasmic trafficking of proteins and RNAs, some of which are aberrantly modulated in BCR-ABL1+ leukemias. Using CD34+ progenitors from CML, B-ALL, and healthy individuals, we found that XPO1 expression was markedly increased, mostly in a TKI-sensitive manner, in CML-BC and Ph+ B-ALL. Notably, XPO1 was also elevated in Ph− B-ALL. Moreover, the clinically relevant XPO1 inhibitor KPT-330 strongly triggered apoptosis and impaired the clonogenic potential of leukemic, but not normal, CD34+ progenitors, and increased survival of BCR-ABL1+ mice, 50% of which remained alive and, mostly, became BCR-ABL1 negative. Moreover, KPT-330 compassionate use in a patient with TKI-resistant CML undergoing disease progression significantly reduced white blood cell count, blast cells, splenomegaly, lactate dehydrogenase levels, and bone pain. Mechanistically, KPT-330 altered the subcellular localization of leukemia-regulated factors including RNA-binding heterogeneous nuclear ribonucleoprotein A1 and the oncogene SET, thereby inducing reactivation of protein phosphatase 2A tumor suppressor and inhibition of BCR-ABL1 in CML-BC cells. Because XPO1 is important for leukemic cell survival, KPT-330 may represent an alternative therapy for TKI-refractory Ph+ leukemias. PMID:23970380

Reciprocal regulation of the cholinic phenotype and epithelial-mesenchymal transition in glioblastoma cells

PubMed Central

Koch, Katharina; Hartmann, Rudolf; Schröter, Friederike; Suwala, Abigail Kora; Maciaczyk, Donata; Krüger, Andrea Caroline; Willbold, Dieter; Kahlert, Ulf Dietrich; Maciaczyk, Jaroslaw

2016-01-01

Glioblastoma (GBM) is the most malignant brain tumor with very limited therapeutic options. Standard multimodal treatments, including surgical resection and combined radio-chemotherapy do not target the most aggressive subtype of glioma cells, brain tumor stem cells (BTSCs). BTSCs are thought to be responsible for tumor initiation, progression, and relapse. Furthermore, they have been associated with the expression of mesenchymal features as a result of epithelial-mesenchymal transition (EMT) thereby inducing tumor dissemination and chemo resistance. Using high resolution proton nuclear magnetic resonance spectroscopy (1H NMR) on GBM cell cultures we provide evidence that the expression of well-known EMT activators of the ZEB, TWIST and SNAI families and EMT target genes N-cadherin and VIMENTIN is associated with aberrant choline metabolism. The cholinic phenotype is characterized by high intracellular levels of phosphocholine and total choline derivatives and was associated with malignancy in various cancers. Both genetic and pharmacological inhibition of the cardinal choline metabolism regulator choline kinase alpha (CHKα) significantly reduces the cell viability, invasiveness, clonogenicity, and expression of EMT associated genes in GBM cells. Moreover, in some cell lines synergetic cytotoxic effects were observed when combining the standard of care chemotherapeutic temozolomide with the CHKα inhibitor V-11-0711. Taken together, specific inhibition of the enzymatic activity of CHKα is a powerful strategy to suppress EMT which opens the possibility to target chemo-resistant BTSCs through impairing their mesenchymal transdifferentiation. Moreover, the newly identified EMT-oncometabolic network may be helpful to monitor the invasive properties of glioblastomas and the success of anti-EMT therapy. PMID:27705917

[Technologies for hair reconstruction and their applicability for pharmaceutical research].

PubMed

Matsuzaki, Takashi

2008-01-01

Hair follicles are the organs that produce hair shafts. They periodically regenerate throughout the life of the organisms, which is called the hair cycle. To develop new drugs to treat hair disorders and diseases, reproducible and high throughput assays or screening methods have been required to estimate the efficacy of various factors on hair follicle function. Although organ culture of hair follicles is one of the useful ways to carry out such research, it is not suitable for manipulating the genes or cells present in hair follicles. Patch assay is a method used to reconstruct hair follicles from enzymatically dissociated skin cells and has many advantages compared to the conventional Chamber method. Using the Patch method, transferring genes into follicular cells becomes easier than ever before. Chimeric follicles could be produced with dissociated cells by modifying the combination of cells or by simply merging cells of different origins. These applications certainly help the progress of hair research. However, we recently found that some functions of dermal papillae and follicular epithelia change during the growing phase (anagen) of the hair cycle. Dermal papillae produce different factors in early anagen and mid anagen. The signals from dermal papillae in early anagen could produce hair bulbs with clonogenic epithelial precursors but not with dormant epithelial precursors. On the other hand, the signals from dermal papillae in mid anagen strongly promote hair formation with dormant epithelial precursors. Therefore, more attention should be given to the hair cycle stages when using organ culture of hair follicles and conducting reconstruction experiments with follicularly derived cells.

The epigenetically regulated miR-494 associates with stem-cell phenotype and induces sorafenib resistance in hepatocellular carcinoma.

PubMed

Pollutri, Daniela; Patrizi, Clarissa; Marinelli, Sara; Giovannini, Catia; Trombetta, Elena; Giannone, Ferdinando A; Baldassarre, Maurizio; Quarta, Santina; Vandewynckel, Y P; Vandierendonck, A; Van Vlierberghe, H; Porretti, Laura; Negrini, Massimo; Bolondi, Luigi; Gramantieri, Laura; Fornari, Francesca

2018-01-05

Hepatocellular carcinoma (HCC) represents the second cause of cancer-related mortality worldwide and is associated with poor prognosis, especially in patients not amenable for curative treatments. The multi-kinase inhibitor sorafenib represents the first-line treatment option for advanced HCC; nevertheless, its effectiveness is limited due to tumor heterogeneity as well as innate or acquired drug resistance, raising the need for new therapeutic strategies. MicroRNAs (miRNAs) involvement in treatment response as well as their safety and efficacy in preclinical models and clinical trials have been widely documented in the oncologic field, including HCC. Here, we identified miR-494 upregulation in a subgroup of human and rat HCCs with stem cell-like characteristics, as well as multiple epigenetic mechanisms involved in its aberrant expression in HCC cell lines and patients. Moreover, we identified p27, puma and pten among miR-494 targets, contributing to speed up cell cycle progression, enhance survival potential in stressful conditions and increase invasive and clonogenic capabilities. MiR-494 overexpression increased sorafenib resistance via mTOR pathway activation in HCC cell lines and, in line, high miR-494 levels associated with decreased sorafenib response in two HCC animal models. A sorafenib-combined anti-miR-494-based strategy revealed an enhanced anti-tumor potential with respect to sorafenib-only treatment in our HCC rat model. In conclusion, our findings suggested miR-494 as a possible therapeutic target as well as a candidate biomarker for patient stratification in advanced HCC.

Characterization of potent and selective iodonium-class inhibitors of NADPH oxidases.

PubMed

Lu, Jiamo; Risbood, Prabhakar; Kane, Charles T; Hossain, Md Tafazzal; Anderson, Larry; Hill, Kimberly; Monks, Anne; Wu, Yongzhong; Antony, Smitha; Juhasz, Agnes; Liu, Han; Jiang, Guojian; Harris, Erik; Roy, Krishnendu; Meitzler, Jennifer L; Konaté, Mariam; Doroshow, James H

2017-11-01

The NADPH oxidases (NOXs) play a recognized role in the development and progression of inflammation-associated disorders, as well as cancer. To date, several NOX inhibitors have been developed, through either high throughput screening or targeted disruption of NOX interaction partners, although only a few have reached clinical trials. To improve the efficacy and bioavailability of the iodonium class NOX inhibitor diphenylene iodonium (DPI), we synthesized 36 analogs of DPI, focusing on improved solubility and functionalization. The inhibitory activity of the analogs was interrogated through cell viability and clonogenic studies with a colon cancer cell line (HT-29) that depends on NOX for its proliferative potential. Lack of altered cellular respiration at relevant iodonium analog concentrations was also demonstrated. Additionally, inhibition of ROS generation was evaluated with a luminescence assay for superoxide, or by Amplex Red® assay for H 2 O 2 production, in cell models expressing specific NOX isoforms. DPI and four analogs (NSCs 740104, 751140, 734428, 737392) strongly inhibited HT-29 cell growth and ROS production with nanomolar potency in a concentration-dependent manner. NSC 737392 and 734428, which both feature nitro functional groups at the meta position, had >10-fold higher activity against ROS production by cells that overexpress dual oxidase 2 (DUOX2) than the other compounds examined (IC 50 ≈200-400nM). Based on these results, we synthesized and tested NSC 780521 with optimized potency against DUOX2. Iodonium analogs with anticancer activity, including the first generation of targeted agents with improved specificity against DUOX2, may provide a novel therapeutic approach to NOX-driven tumors. Published by Elsevier Inc.

Targeting CDH17 Suppresses Tumor Progression in Gastric Cancer by Downregulating Wnt/β-Catenin Signaling

PubMed Central

Ren, Chao; Zeng, Zhao-lei; Wu, Wen-jing; Luo, Hui-yan; Zhou, Zhi-wei; Xu, Rui-hua

2013-01-01

Purpose Gastric cancer remains one of the leading causes of cancer death worldwide. Patients usually present late with local invasion or metastasis, for which there are no effective therapies available. Following previous studies that identified the adhesion molecule Cadherin-17(CDH17) as a potential marker for gastric carcinoma, we performed proof-of-principle studies to develop rational therapeutic approaches targeting CDH17 for treating this disease. Methods Immunohistochemistry was used to study the expression of CDH17 in 156 gastric carcinomas, and the relationship between survival and CDH17 expression was studied by multivariate analyses. The effect of RNA interference–mediated knockdown of CDH17 on proliferation of gastric carcinoma cell lines was examined in vitro and in vivo, as well as the effects on downstream signaling by immunoblotting. Results CDH17 was consistently up-regulated in human gastric cancers, and overall survival in patients with CDH17 upregulation was poorer than in those without expression of this gene (5 yrs overall survival rate 29.0% vs. 45.0%, P<0.01). Functional assays demonstrated that CDH17 knockdown inhibited cell proliferation, adhesion, migration, invasion, clonogenicity and induce G0/G1 arrest. In mice, shRNA-mediated CDH17 knockdown markedly inhibits tumor growth; intratumoral injection of CDH17 shRNAs results in significant antitumor effects on transplanted tumor models. The antitumor mechanisms underlying CDH17 inhibition involve inactivation of Wnt/β-catenin signaling. Conclusion Our results identify CDH17 as a biomarker of gastric carcinoma and attractive therapeutic target for this aggressive malignancy. PMID:23554857

Support services relating to geothermal programs. Final report

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

Not Available

1981-08-01

Activities designed to assist in the assessment of processes through which geopressured methane production and geopressure-geothermal power can be increased are discussed. Progress is reported on the following: general support, hot-dry-rock review, R and D plan, and the Edna Delcambre final report. (MHR)

Uncertainty and Variability in Physiologically-Based Pharmacokinetic (PBPK) Models: Key Issues and Case Studies (Final Report)

EPA Science Inventory

EPA announced the availability of the final report, Uncertainty and Variability in Physiologically-Based Pharmacokinetic (PBPK) Models: Key Issues and Case Studies. This report summarizes some of the recent progress in characterizing uncertainty and variability in physi...

Differential control of growth, cell cycle progression, and expression of NF-{kappa}B in human breast cancer cells MCF-7, MCF-10A, and MDA-MB-231 by ponicidin and oridonin, diterpenoids from the chinese herb Rabdosia rubescens

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

Hsieh Tzechen; Brander Cancer Research Institute, New York Medical College, Hawthorne, NY 10532; Wijeratne, E. Kithsiri

2005-11-11

Ponicidin and oridonin are novel diterpenoids isolated from Rabdosia rubescens. We tested their effects in MCF-7 and MDA-MB-231 cells, as representing low and high invasive breast carcinoma, with normal MCF-10A cells. Clonogenicity and proliferation in MCF-7 cells were inhibited more significantly by ponicidin than oridonin, while the reverse was observed in MCF-10A cells. Ponicidin and oridonin induced S/G{sub 2}M arrest and G{sub 1}/S block in MCF-7 cells. In MCF-10A cells treated with either diterpenoid, induction of apoptosis was observed. Moreover, oridonin almost completely blocked MCF-10A progression from S to G{sub 2}/M phase; in contrast, ponicidin-treated MCF-10A cells showed no discernablemore » changes in cell cycle phase distribution. Neither diterpenoid affected growth of MDA-MB-231 cells, at the dose range effective for MCF-7 or MCF-10A cells. Ponicidin-treated MCF-7 cells expressed reduced levels of cyclin B1, cdc2, transcription factor E2F, and Rb including phosphorylation at S780. Less pronounced effects were found in cells treated with oridonin. Neither compound altered cyclin D1 and cdk4 in MCF-7 cells. In MCF-10A cells, oridonin was more active than ponicidin in inhibiting the expression of cyclin B1, cdc2, S780-phosphorylated Rb, and E2F. To further investigate induction of apoptosis in MCF-10A cells, we measured changes in NF-{kappa}B. Decreases in p65 or p50 forms of NF-{kappa}B and its upstream regulator I-{kappa}B were found in oridonin-treated MCF-10A and not MCF-7 cells. Taken together, these results provide a mechanistic framework for the cellular effects of ponicidin and oridonin in different stage breast cancer cells.« less

Hydroxychavicol, a betel leaf component, inhibits prostate cancer through ROS-driven DNA damage and apoptosis

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

Gundala, Sushma Reddy; Yang, Chunhua; Mukkavilli, Rao

Dietary phytochemicals are excellent ROS-modulating agents and have been shown to effectively enhance ROS levels beyond toxic threshold in cancer cells to ensure their selective killing while leaving normal cells unscathed. Here we demonstrate that hydroxychavicol (HC), extracted and purified from Piper betel leaves, significantly inhibits growth and proliferation via ROS generation in human prostate cancer, PC-3 cells. HC perturbed cell-cycle kinetics and progression, reduced clonogenicity and mediated cytotoxicity by ROS-induced DNA damage leading to activation of several pro-apoptotic molecules. In addition, HC treatment elicited a novel autophagic response as evidenced by the appearance of acidic vesicular organelles and increasedmore » expression of autophagic markers, LC3-IIb and beclin-1. Interestingly, quenching of ROS with tiron, an antioxidant, offered significant protection against HC-induced inhibition of cell growth and down regulation of caspase-3, suggesting the crucial role of ROS in mediating cell death. The collapse of mitochondrial transmembrane potential by HC further revealed the link between ROS generation and induction of caspase-mediated apoptosis in PC-3 cells. Our data showed remarkable inhibition of prostate tumor xenografts by ∼ 72% upon daily oral administration of 150 mg/kg bw HC by quantitative tumor volume measurements and non-invasive real-time bioluminescent imaging. HC was well-tolerated at this dosing level without any observable toxicity. This is the first report to demonstrate the anti-prostate cancer efficacy of HC in vitro and in vivo, which is perhaps attributable to its selective prooxidant activity to eliminate cancer cells thus providing compelling grounds for future preclinical studies to validate its potential usefulness for prostate cancer management. - Highlights: • HC perturbs cell-cycle progression by induction of reactive oxygen species (ROS). • HC mediated cytotoxicity by ROS-induced DNA damage leading to apoptosis. • HC induced ROS-mediated autophagic response. • It inhibited prostate tumor growth by ∼ 72% without any observable toxicity. • Its anticancer efficacy is likely due to its selective prooxidant activity.« less

Curcumin-mediated regulation of Notch1/hairy and enhancer of split-1/survivin: molecular targeting in cholangiocarcinoma.

PubMed

Koprowski, Steven; Sokolowski, Kevin; Kunnimalaiyaan, Selvi; Gamblin, T Clark; Kunnimalaiyaan, Muthusamy

2015-10-01

Cholangiocarcinoma (CCA) is highly malignant and characterized by poor prognosis with chemotherapeutic resistance. Therefore, continued development of novel, effective approaches are needed. Notch expression is markedly upregulated in CCA, but the utility of Notch1 inhibition is not defined. Based on recent findings, we hypothesized that curcumin, a polyphenolic phytochemical, suppresses CCA growth in vitro via inhibition of Notch1 signaling. Established CCA cell lines CCLP-1 and SG-231 were treated with varying concentrations of curcumin (0-20 μM). Viability was assessed through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and clonogenic assays. Evaluation of apoptosis was determined via Western analysis for apoptotic markers and Caspase-Glo 3/7 assay. Cell lysates were further analyzed via Western blotting for Notch1/HES-1/survivin pathway expression, cell cycle progression, and survival. Curcumin-treated CCA cells exhibited reduced viability compared with control treatment. Statistically significant reductions in cell viability were observed with curcumin treatment at concentrations of 7.5, 10, and 15 μM by approximately 10%, 48%, and 56% for CCLP-1 and 13%, 25%, and 50% for SG-231, respectively. On Western analysis, concentrations of ≥10 μM showed reductions in Notch1, HES-1, and survivin. Apoptosis was evidenced by an increase in expression of cleaved poly [ADP] ribose polymerase and an increase in caspase activity. Cyclin D1 (cell cycle progression) expression levels were also reduced with treatment. Curcumin effectively induces CCA (CCLP-1 and SG-231) growth suppression and apoptosis at relatively low treatment concentrations when compared with the previous research. A concomitant reduction of Notch1, HES-1, and survivin expression in CCA cell lines provides novel evidence for a potential antitumorigenic mechanism-of-action. To our knowledge, this is the first report showing reduction in HES-1 expression via protein analysis after treatment with curcumin. Such findings merit further investigation of curcumin-mediated inhibition of Notch signaling in CCA either alone or in combination with chemotherapeutic agents. Copyright © 2015 Elsevier Inc. All rights reserved.

Experimental and Theoretical Progress on the GEM Theory

NASA Astrophysics Data System (ADS)

Brandenburg, J. E.

This paper reports experimental and theoretical progress on the GEM unification theory. In theoretical progress, the derivation of the GEM theory using it in a fully covariant form is achieved based on the principle of self-cancellation of the ZPF EM stress-momentum tensor. This derivation reveals that the final Gravity-EM system obeys a Helmholtz-like equation resembling that governing sound propagation. Finally an improved derivation of the formula for the Newton Gravitation constant is shown, qresulting in the formula G = e2/(4πɛ0 me mp) α exp (-2 (α-.86/σ2…) = 6.673443 x10-11 N-m2 kg-2 that agrees with experimental values to 3 parts per 100,000. Experiments have found parity violating weight reductions in gyroscopes driven by rotating EM fields. These experiments appear to confirm gravity modification using electromagnetism predicted by the GEM theory through the Vacuum Bernoulli Equation.

Friedrich Nietzsche's mental illness--general paralysis of the insane vs. frontotemporal dementia.

PubMed

Orth, M; Trimble, M R

2006-12-01

For a long time it was thought that Nietzsche suffered from general paralysis of the insane (GPI). However, this diagnosis has been questioned recently, and alternative diagnoses have been proposed. We have charted Friedrich Nietzsche's final fatal illness, and viewed the differential diagnosis in the light of recent neurological understandings of dementia syndromes. It is unclear that Nietzsche ever had syphilis. He lacked progressive motor and other neurological features of a progressive syphilitic central nervous system (CNS) infection and lived at least 12 years following the onset of his CNS signs, which would be extremely rare for patients with untreated GPI. Finally, his flourish of productivity in 1888 would be quite uncharacteristic of GPI, but in keeping with reports of burgeoning creativity at some point in the progression of frontotemporal dementia (FTD). We suggest that Nietzsche did not have GPI, but died from a chronic dementia, namely FTD.

Perspectives on Progress: The School-to-Work National Customer Dialogues. Final Report.

ERIC Educational Resources Information Center

Public Forum Inst., Washington, DC.

"Perspectives on Progress: The School-to-Work (STW) National Customer Dialogues" was a series of six regional and two national discussions that were held between December 1999 and July 2000 to gather the views of more than 700 employers, educators, labor union representatives, students, parents, community-based organizations, and state…

Stage Theory and Research on Tobacco, Alcohol, and Other Drug Use.

ERIC Educational Resources Information Center

Werch, Chudley E.; Anzalone, Debra

1995-01-01

Examines the conceptual and empirical foundations of individual drug use stage development and progression related to tobacco, alcohol, and other drugs. Research examining interdrug use progression among youths supports the idea of a generally invariant sequence, involving nonuse to legal drug use, marijuana, and finally other illegal drug use.…

Development of Career Progression Systems for Employees in the Foodservice Industry. Final Report.

ERIC Educational Resources Information Center

National Restaurant Association, Chicago, IL.

Firms representing four segments of the foodservice industry (institutional foodservice (9 jobs), commercial restaurants (19 jobs), hotel foodservice (100 jobs), and airline foodservice (10 jobs), participated in a career and training study to test the feasibility of designing and implementing career progression (c.p.) systems within these…

Progress Report: Access and Persistence of Minority Students in the Arizona Universities.

ERIC Educational Resources Information Center

Cotera, Augustus S.; And Others

One of the working papers in the final report of the Arizona Board of Regents' Task Force on Excellence, Efficiency and Competitiveness, this report presents statistical information on the progress of minority student access and persistence in the three Arizona Universities, Arizona's community colleges, and the Arizona Department of Education.…

THE U.S. ENVIRONMENTAL PROTECTION AGENCY PARTICULATE MATTER HEALTH EFFECTS RESEARCH CENTERS PROGRAM: A MIDCOURSE REPORT OF STATUS, PROGRESS, AND PLANS. (R827351)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Commentary 2 to Cox and Little: radiation-induced oncogenic transformation: the interplay between dose, dose protraction, and radiation quality

NASA Technical Reports Server (NTRS)

Brenner, D. J.; Hall, E. J.

1992-01-01

There is now a substantial body of evidence for end points such as oncogenic transformation in vitro, and carcinogenesis and life shortening in vivo, suggesting that dose protraction leads to an increase in effectiveness relative to a single, acute exposure--at least for radiations of medium linear energy transfer (LET) such as neutrons. Table I contains a summary of the pertinent data from studies in which the effect is seen. [table: see text] This phenomenon has come to be known as the "inverse dose rate effect," because it is in marked contrast to the situation at low LET, where protraction in delivery of a dose of radiation, either by fractionation or low dose rate, results in a decreased biological effect; additionally, at medium and high LET, for radiobiological end points such as clonogenic survival, the biological effectiveness is independent of protraction. The quantity and quality of the published reports on the "inverse dose rate effect" leaves little doubt that the effect is real, but the available evidence indicates that the magnitude of the effect is due to a complex interplay between dose, dose rate, and radiation quality. Here, we first summarize the available data on the inverse dose rate effect and suggest that it follows a consistent pattern in regard to dose, dose rate, and radiation quality; second, we describe a model that predicts these features; and, finally, we describe the significance of the effect for radiation protection.

MicroRNA-150 directly targets MUC4 and suppresses growth and malignant behavior of pancreatic cancer cells.

PubMed

Srivastava, Sanjeev K; Bhardwaj, Arun; Singh, Seema; Arora, Sumit; Wang, Bin; Grizzle, William E; Singh, Ajay P

2011-12-01

Pancreatic cancer (PC) has the worst prognosis among all cancers due to its late diagnosis and lack of effective therapies. Therefore, identification of novel gene targets, which are differentially expressed in PC and functionally involved in malignant phenotypes, is critical to achieve early diagnosis and development of effective therapeutic strategies. We have shown previously that MUC4, an aberrantly overexpressed transmembrane mucin, promotes growth, invasion and metastasis of PC cells, thus underscoring its potential as a clinical target. Here, we report a novel microRNA (miRNA)-mediated mechanism underlying aberrant expression of MUC4 in PC. We demonstrate that the 3' untranslated region of MUC4 contains a highly conserved miRNA-150 (miR-150) binding motif and its direct interaction with miR-150 downregulates endogenous MUC4 protein levels. We also show that miR-150-mediated MUC4 downregulation is associated with a concomitant decrease in human epidermal growth factor receptor 2 and its phosphorylated form, leading to reduced activation of downstream signaling. Furthermore, our findings demonstrate that miR-150 overexpression inhibits growth, clonogenicity, migration and invasion and enhances intercellular adhesion in PC cells. Finally, our data reveal a downregulated expression of miR-150 in malignant pancreatic tissues, which is inversely associated with MUC4 protein levels. Altogether, these findings establish miR-150 as a novel regulator of MUC4 and a tumor suppressor miRNA in PC.

MicroRNA-150 directly targets MUC4 and suppresses growth and malignant behavior of pancreatic cancer cells

PubMed Central

Srivastava, Sanjeev K.; Bhardwaj, Arun; Singh, Seema; Arora, Sumit; Wang, Bin; Grizzle, William E.; Singh, Ajay P.

2011-01-01

Pancreatic cancer (PC) has the worst prognosis among all cancers due to its late diagnosis and lack of effective therapies. Therefore, identification of novel gene targets, which are differentially expressed in PC and functionally involved in malignant phenotypes, is critical to achieve early diagnosis and development of effective therapeutic strategies. We have shown previously that MUC4, an aberrantly overexpressed transmembrane mucin, promotes growth, invasion and metastasis of PC cells, thus underscoring its potential as a clinical target. Here, we report a novel microRNA (miRNA)-mediated mechanism underlying aberrant expression of MUC4 in PC. We demonstrate that the 3′ untranslated region of MUC4 contains a highly conserved miRNA-150 (miR-150) binding motif and its direct interaction with miR-150 downregulates endogenous MUC4 protein levels. We also show that miR-150-mediated MUC4 downregulation is associated with a concomitant decrease in human epidermal growth factor receptor 2 and its phosphorylated form, leading to reduced activation of downstream signaling. Furthermore, our findings demonstrate that miR-150 overexpression inhibits growth, clonogenicity, migration and invasion and enhances intercellular adhesion in PC cells. Finally, our data reveal a downregulated expression of miR-150 in malignant pancreatic tissues, which is inversely associated with MUC4 protein levels. Altogether, these findings establish miR-150 as a novel regulator of MUC4 and a tumor suppressor miRNA in PC. PMID:21983127

Selective Targeting of Brain Tumors with Gold Nanoparticle-Induced Radiosensitization

PubMed Central

Joh, Daniel Y.; Sun, Lova; Stangl, Melissa; Al Zaki, Ajlan; Murty, Surya; Santoiemma, Phillip P.; Davis, James J.; Baumann, Brian C.; Alonso-Basanta, Michelle; Bhang, Dongha; Kao, Gary D.; Tsourkas, Andrew; Dorsey, Jay F.

2013-01-01

Successful treatment of brain tumors such as glioblastoma multiforme (GBM) is limited in large part by the cumulative dose of Radiation Therapy (RT) that can be safely given and the blood-brain barrier (BBB), which limits the delivery of systemic anticancer agents into tumor tissue. Consequently, the overall prognosis remains grim. Herein, we report our pilot studies in cell culture experiments and in an animal model of GBM in which RT is complemented by PEGylated-gold nanoparticles (GNPs). GNPs significantly increased cellular DNA damage inflicted by ionizing radiation in human GBM-derived cell lines and resulted in reduced clonogenic survival (with dose-enhancement ratio of ∼1.3). Intriguingly, combined GNP and RT also resulted in markedly increased DNA damage to brain blood vessels. Follow-up in vitro experiments confirmed that the combination of GNP and RT resulted in considerably increased DNA damage in brain-derived endothelial cells. Finally, the combination of GNP and RT increased survival of mice with orthotopic GBM tumors. Prior treatment of mice with brain tumors resulted in increased extravasation and in-tumor deposition of GNP, suggesting that RT-induced BBB disruption can be leveraged to improve the tumor-tissue targeting of GNP and thus further optimize the radiosensitization of brain tumors by GNP. These exciting results together suggest that GNP may be usefully integrated into the RT treatment of brain tumors, with potential benefits resulting from increased tumor cell radiosensitization to preferential targeting of tumor-associated vasculature. PMID:23638079

Realgar nanoparticles versus ATO arsenic compounds induce in vitro and in vivo activity against multiple myeloma.

PubMed

Cholujova, Danka; Bujnakova, Zdenka; Dutkova, Erika; Hideshima, Teru; Groen, Richard W; Mitsiades, Constantine S; Richardson, Paul G; Dorfman, David M; Balaz, Peter; Anderson, Kenneth C; Jakubikova, Jana

2017-12-01

Multiple myeloma (MM), a B cell malignancy characterized by clonal proliferation of plasma cells in the bone marrow, remains incurable despite the use of novel and conventional therapies. In this study, we demonstrated MM cell cytotoxicity triggered by realgar (REA; As 4 S 4 ) nanoparticles (NREA) versus Arsenic trioxide (ATO) against MM cell lines and patient cells. Both NREA and ATO showed in vivo anti-MM activity, resulting in significantly decreased tumour burden. The anti-MM activity of NREA and ATO is associated with apoptosis, evidenced by DNA fragmentation, depletion of mitochondrial membrane potential, cleavage of caspases and anti-apoptotic proteins. NREA induced G 2 /M cell cycle arrest and modulation of cyclin B1, p53 (TP53), p21 (CDKN1A), Puma (BBC3) and Wee-1 (WEE1). Moreover, NREA induced modulation of key regulatory molecules in MM pathogenesis including JNK activation, c-Myc (MYC), BRD4, and histones. Importantly, NREA, but not ATO, significantly depleted the proportion and clonogenicity of the MM stem-like side population, even in the context of the bone marrow stromal cells. Finally, our study showed that both NREA and ATO triggered synergistic anti-MM activity when combined with lenalidomide or melphalan. Taken together, the anti-MM activity of NREA was more potent compared to ATO, providing the preclinical framework for clinical trials to improve patient outcome in MM. © 2017 John Wiley & Sons Ltd.

The Assessment of Parameters Affecting the Quality of Cord Blood by the Appliance of the Annexin V Staining Method and Correlation with CFU Assays

PubMed Central

Radke, Teja Falk; Barbosa, David; Duggleby, Richard Charles; Saccardi, Riccardo; Querol, Sergio; Kögler, Gesine

2013-01-01

The assessment of nonviable haematopoietic cells by Annexin V staining method in flow cytometry has recently been published by Duggleby et al. Resulting in a better correlation with the observed colony formation in methylcellulose assays than the standard ISHAGE protocol, it presents a promising method to predict cord blood potency. Herein, we applied this method for examining the parameters during processing which potentially could affect cord blood viability. We could verify that the current standards regarding time and temperature are sufficient, since no significant difference was observed within 48 hours or in storage at 4°C up to 26°C. However, the addition of DMSO for cryopreservation alone leads to an inevitable increase in nonviable haematopoietic stem cells from initially 14.8% ± 4.3% to at least 30.6% ± 5.5%. Furthermore, CFU-assays with varied seeding density were performed in order to evaluate the applicability as a quantitative method. The results revealed that only in a narrow range reproducible clonogenic efficiency (ClonE) could be assessed, giving at least a semiquantitative estimation. We conclude that both Annexin V staining method and CFU-assays with defined seeding density are reliable means leading to a better prediction of the final potency. Especially Annexin V, due to its fast readout, is a practical tool for examining and optimising specific steps in processing, while CFU-assays add a functional confirmation. PMID:23533443

Completion of the 1:1,500,000-Scale Geologic Map of Western Libya Montes and Northwestern Tyrrhena Terra

NASA Astrophysics Data System (ADS)

Huff, A. E.; Skinner, J. A.

2018-06-01

Final progress report on the 1:1,500,000-scale mapping of western Libya Montes and northwestern Tyrrhena Terra. The final unit names, labels, and descriptions are reported as well as the methodology for age determinations and brief geologic history.

40 CFR 123.45 - Noncompliance and program reporting by the Director.

Code of Federal Regulations, 2014 CFR

2014-07-01

... (CONTINUED) WATER PROGRAMS STATE PROGRAM REQUIREMENTS Transfer of Information and Permit Review § 123.45... schedule report for final compliance or a monitoring report. This applies when the permittee has failed to submit a final compliance schedule progress report, pretreatment report, or a Discharge Monitoring Report...

40 CFR 123.45 - Noncompliance and program reporting by the Director.

Code of Federal Regulations, 2012 CFR

2012-07-01

... (CONTINUED) WATER PROGRAMS STATE PROGRAM REQUIREMENTS Transfer of Information and Permit Review § 123.45... schedule report for final compliance or a monitoring report. This applies when the permittee has failed to submit a final compliance schedule progress report, pretreatment report, or a Discharge Monitoring Report...

40 CFR 123.45 - Noncompliance and program reporting by the Director.

Code of Federal Regulations, 2011 CFR

2011-07-01

... (CONTINUED) WATER PROGRAMS STATE PROGRAM REQUIREMENTS Transfer of Information and Permit Review § 123.45... schedule report for final compliance or a monitoring report. This applies when the permittee has failed to submit a final compliance schedule progress report, pretreatment report, or a Discharge Monitoring Report...

40 CFR 123.45 - Noncompliance and program reporting by the Director.

Code of Federal Regulations, 2013 CFR

2013-07-01

... (CONTINUED) WATER PROGRAMS STATE PROGRAM REQUIREMENTS Transfer of Information and Permit Review § 123.45... schedule report for final compliance or a monitoring report. This applies when the permittee has failed to submit a final compliance schedule progress report, pretreatment report, or a Discharge Monitoring Report...

Progressive Fracture of Fiber Composite Builtup Structures

NASA Technical Reports Server (NTRS)

Gotsis, Pascal K.; Chamis, Christos C.; Minnetyan, Levon

1996-01-01

The damage progression and fracture of builtup composite structures was evaluated by using computational simulation to examine the behavior and response of a stiffened composite (0 +/- 45/90)(sub s6) laminate panel subjected to a bending load. The damage initiation, growth, accumulation, progression, and propagation to structural collapse were simulated. An integrated computer code (CODSTRAN) was augmented for the simulation of the progressive damage and fracture of builtup composite structures under mechanical loading. Results showed that damage initiation and progression have a significant effect on the structural response. Also investigated was the influence of different types of bending load on the damage initiation, propagation, and final fracture of the builtup composite panel.

Lactate calcium salt affects the viability of colorectal cancer cells via betaine homeostasis.

PubMed

Jang, Yeong-Su; Jo, Young-Kwon; Sim, Jae Jun; Ji, Eunhee; Jeong, Keun-Yeong; Kim, Hwan Mook

2016-02-15

Betaine plays an important role in cellular homeostasis. However, the physiological roles of betaine-γ-aminobutyric acid (GABA) transporter (BGT-1) are still being disputed in cancer. In this study, we tried to find the possibility of the antitumor effect on colorectal cancer (CRC) cell via lactate calcium salt (CaLa)-induced BGT-1 downregulation. The CRC cell viability and clonogenic assay was performed using different doses of BGT-1 inhibitor. The expression level of BGT-1 was measured following the treatment of 2.5mM CaLa. Betaine was treated to confirm the resistance of the antitumor activity by CaLa. Tumor growth was also measured using a xenograft animal model. Long-term exposure of 2.5mM CaLa clearly decreased the expression of BGT-1 in the CRC cells. As a result of the downregulation of BGT-1 expression, the clonogenic ability of CRC cells was also decreased in the 2.5mM CaLa-treated group. Reversely, the number of colonies and cell viability was increased by combination treatment with betaine and 2.5mM CaLa, as compared with a single treatment of 2.5mM CaLa. Tumor growth was significantly inhibited in the xenograft model depending on BGT-1 downregulation by 2.5mM CaLa treatment. These results support the idea that long-lasting calcium supplementation via CaLa contributes to disruption of betaine homeostasis in the CRC cells and is hypothesized to reduce the risk of CRC. In addition, it indicates the possibility of CaLa being a potential incorporating agent with existing therapeutics against CRC. Copyright © 2016 Elsevier Inc. All rights reserved.

Exosomes are released by bystander cells exposed to radiation-induced biophoton signals: Reconciling the mechanisms mediating the bystander effect

PubMed Central

Fernandez-Palomo, Cristian; McNeill, Fiona E.; Seymour, Colin B.; Rainbow, Andrew J.; Mothersill, Carmel E.

2017-01-01

Objective The objective of our study was to explore a possible molecular mechanism by which ultraviolet (UV) biophotons could elicit bystander responses in reporter cells and resolve the problem of seemingly mutually exclusive mechanisms of a physical UV signal & a soluble factor-mediated bystander signal. Methods The human colon carcinoma cell line, HCT116 p53 +/+, was directly irradiated with 0.5 Gy tritium beta particles to induce ultraviolet biophoton emission. Bystander cells were not directly irradiated but were exposed to the emitted UV biophotons. Medium was subsequently harvested from UV-exposed bystander cells. The exosomes extracted from this medium were incubated with reporter cell populations. These reporter cells were then assayed for clonogenic survival and mitochondrial membrane potential with and without prior treatment of the exosomes with RNase. Results Clonogenic cell survival was significantly reduced in reporter cells incubated with exosomes extracted from cells exposed to secondarily-emitted UV. These exosomes also induced significant mitochondrial membrane depolarization in receiving reporter cells. Conversely, exosomes extracted from non-UV-exposed cells did not produce bystander effects in reporter cells. The treatment of exosomes with RNase prior to their incubation with reporter cells effectively abolished bystander effects in reporter cells and this suggests a role for RNA in mediating the bystander response elicited by UV biophotons and their produced exosomes. Conclusion This study supports a role for exosomes released from UV biophoton-exposed bystander cells in eliciting bystander responses and also indicates a reconciliation between the UV-mediated bystander effect and the bystander effect which has been suggested in the literature to be mediated by soluble factors. PMID:28278290

Modulation of ionizing radiation-induced apoptosis and cell cycle arrest by all-trans retinoic acid in promyelocytic leukemia cells (HL-60).

PubMed

Mareková, M; Vávrová, J; Vokurková, D; Psutka, J

2003-01-01

Acute promyelocytic leukemia is characterized by a block of myeloid differentiation. The incubation of cells with 1 micromol/l all-trans retinoic acid (ATRA) for 72 h induced differentiation of HL-60 cells and increased the number of CD11b positive cells. Morphological and functional changes were accompanied by a loss of proliferative capacity. Differentiation caused by preincubation of leukemic cells HL-60 with ATRA is accompanied by loss of clonogenicity (control cells: 870 colonies/10(3) cells, cells preincubated with ATRA: 150 colonies/10(3) cells). D0 for undifferentiated cells was 2.35 Gy, for ATRA differentiated cells 2.46 Gy. Statistical comparison of clonogenity curves indicated that in the whole range 0.5-10 Gy the curves are not significantly different, however, in the range 0.5-3 Gy ATRA differentiated cells were significantly more radioresistant than non-differentiated cells. When HL-60 cells preincubated with 1 micromol/l ATRA were irradiated by a sublethal dose of 6 Gy, more marked increase of apoptotic cells number was observed 24 h after irradiation and the surviving cells were mainly in the G1 phase of the cell cycle, while only irradiated cells were accumulated in G(2) phase. Our results imply that preincubation of cells with ATRA accelerates apoptosis occurrence (24 h) after irradiation by high sublethal dose of 6 Gy. Forty-eight hours after 6 Gy irradiation, late apoptotic cells were found in the group of ATRA pretreated cells, as determined by APO2.7 positivity. This test showed an increased effect (considering cell death induction) in comparison to ATRA or irradiation itself.

Concomitant administration of radiation with eribulin improves the survival of mice harboring intracerebral glioblastoma.

PubMed

Miki, Shunichiro; Imamichi, Shoji; Fujimori, Hiroaki; Tomiyama, Arata; Fujimoto, Kenji; Satomi, Kaishi; Matsushita, Yuko; Matsuzaki, Sanae; Takahashi, Masamichi; Ishikawa, Eiichi; Yamamoto, Tetsuya; Matsumura, Akira; Mukasa, Akitake; Nishikawa, Ryo; Masutomi, Kenkichi; Narita, Yoshitaka; Masutani, Mitsuko; Ichimura, Koichi

2018-05-14

Glioblastoma is the most common and devastating type of malignant brain tumor. We recently found that eribulin suppresses glioma growth in vitro and in vivo and that eribulin is efficiently transferred into mouse brain tumors at a high concentration. Eribulin is a non-taxane microtubule inhibitor approved for breast cancer and liposarcoma. Cells arrested in M-phase by chemotherapeutic agents such as microtubule inhibitors are highly sensitive to radiation-induced DNA damage. Several recent case reports demonstrated the clinical benefits of eribulin combined with radiation therapy for metastatic brain tumors. In this study, we investigated the efficacy of a combined eribulin and radiation treatment on human glioblastoma cells. The glioblastoma cell lines U87MG, U251MG, U118MG, and SJ28 cells, a patient-derived sphere culture cell line, were used to determine the radiosensitizing effect of eribulin using western blotting, flow cytometry, and clonogenic assay. Subcutaneous and intracerebral glioma xenografts were generated in mice to assess the efficacy of the combined treatment. The combination of eribulin and radiation enhanced DNA damage in vitro. The clonogenic assay of U87MG demonstrated the radiosensitizing effect of eribulin. The concomitant eribulin and radiation treatment significantly prolonged the survival of mice harboring intracerebral glioma xenografts compared with eribulin or radiation alone (p<0.0001). In addition, maintenance administration of eribulin after the concomitant treatment further controlled brain tumor growth. Aberrant microvasculature was decreased in these tumors. Concomitant treatment with eribulin and radiation followed by maintenance administration of eribulin may serve as a novel therapeutic strategy for glioblastomas. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Protective Role of Hsp27 Protein Against Gamma Radiation-Induced Apoptosis and Radiosensitization Effects of Hsp27 Gene Silencing in Different Human Tumor Cells

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

Aloy, Marie-Therese; Hospices Civils de Lyon, Service de Radiotherapie, Centre Hospitalier Lyon-Sud, Pierre-Benite; Hadchity, Elie

Purpose: The ability of heat shock protein 27 (Hsp27) to protect cells from stressful stimuli and its increased levels in tumors resistant to anticancer therapeutics suggest that it may represent a target for sensitization to radiotherapy. In this study, we investigate the protective role of Hsp27 against radiation-induced apoptosis and the effect of its attenuation in highly expressing radioresistant cancer cell lines. Methods and Materials: We examined clonogenic death and the kinetics of apoptotic events in different tumor cell lines overexpressing or underexpressing Hsp27 protein irradiated with photons. The radiosensitive Jurkat cell line, which does not express Hsp27 constitutively ormore » in response to {gamma}-rays, was stably transfected with Hsp27 complementary DNA. Attenuation of Hsp27 expression was accomplished by antisense or RNAi (interfering RNA) strategies in SQ20B head-and-neck squamous carcinoma, PC3 prostate cancer, and U87 glioblastoma radioresistant cells. Results: We measured concentration-dependent protection against the cytotoxic effects of radiation in Jurkat-Hsp27 cells, which led to a 50% decrease in apoptotic cells at 48 hours in the highest expressing cells. Underlying mechanisms leading to radiation resistance involved a significant increase in glutathione levels associated with detoxification of reactive oxygen species, a delay in mitochondrial collapse, and caspase activation. Conversely, attenuation of Hsp27 in SQ20B cells, characterized by their resistance to apoptosis, sensitizes cells to irradiation. This was emphasized by increased apoptosis, decreased glutathione basal level, and clonogenic cell death. Sensitization to irradiation was confirmed in PC3 and U87 radioresistant cells. Conclusion: Hsp27 gene therapy offers a potential adjuvant to radiation-based therapy of resistant tumors.« less

Overexpression of microRNA-95-3p suppresses brain metastasis of lung adenocarcinoma through downregulation of cyclin D1.

PubMed

Hwang, Su Jin; Lee, Hye Won; Kim, Hye Ree; Song, Hye Jin; Lee, Dong Heon; Lee, Hong; Shin, Chang Hoon; Joung, Je-Gun; Kim, Duk-Hwan; Joo, Kyeung Min; Kim, Hyeon Ho

2015-08-21

Despite great efforts to improve survival rates, the prognosis of lung cancer patients is still very poor, mainly due to high invasiveness. We developed brain metastatic PC14PE6/LvBr4 cells through intracardiac injection of lung adenocarcinoma PC14PE6 cells. Western blot and RT-qPCR analyses revealed that PC14PE6/LvBr4 cells had mesenchymal characteristics and higher invasiveness than PC14PE6 cells. We found that cyclin D1 was upregulated, miR-95-3p was inversely downregulated, and pri-miR-95 and its host gene, ABLIM2, were consistently decreased in PC14PE6/LvBr4 cells. MiR-95-3p suppressed cyclin D1 expression through direct binding to the 3' UTR of cyclin D1 mRNA and suppressed invasiveness, proliferation, and clonogenicity of PC14PE6/LvBr4 cells. Ectopic cyclin D1 reversed miR-95-3p-mediated inhibition of invasiveness and clonogenicity, demonstrating cyclin D1 downregulation is involved in function of miR-95-3p. Using bioluminescence imaging, we found that miR-95-3p suppressed orthotopic tumorigenicity and brain metastasis in vivo and increased overall survival and brain metastasis-free survival. Consistent with in vitro metastatic cells, the levels of miR-95-3p, pri-miR-95, and ABLIM2 mRNA were decreased in brain metastatic tissues compared with lung cancer tissues and higher cyclin D1 expression was involved in poor prognosis. Taken together, our results demonstrate that miR-95-3p is a potential therapeutic target for brain metastasis of lung adenocarcinoma cells.

TRPM8 is required for survival and radioresistance of glioblastoma cells

PubMed Central

Klumpp, Dominik; Frank, Stephanie C.; Klumpp, Lukas; Sezgin, Efe C.; Eckert, Marita; Edalat, Lena; Bastmeyer, Martin; Zips, Daniel; Ruth, Peter; Huber, Stephan M.

2017-01-01

TRPM8 is a Ca2+-permeable nonselective cation channel belonging to the melastatin sub-group of the transient receptor potential (TRP) family. TRPM8 is aberrantly overexpressed in a variety of tumor entities including glioblastoma multiforme where it reportedly contributes to tumor invasion. The present study aimed to disclose further functions of TRPM8 in glioma biology in particular upon cell injury by ionizing radiation. To this end, TCGA data base was queried to expose the TRPM8 mRNA abundance in human glioblastoma specimens and immunoblotting was performed to analyze the TRPM8 protein abundance in primary cultures of human glioblastoma. Moreover, human glioblastoma cell lines were irradiated with 6 MV photons and TRPM8 channels were targeted pharmacologically or by RNA interference. TRPM8 abundance, Ca2+ signaling and resulting K+ channel activity, chemotaxis, cell migration, clonogenic survival, DNA repair, apoptotic cell death, and cell cycle control were determined by qRT-PCR, fura-2 Ca2+ imaging, patch-clamp recording, transfilter migration assay, wound healing assay, colony formation assay, immunohistology, flow cytometry, and immunoblotting. As a result, human glioblastoma upregulates TRPM8 channels to variable extent. TRPM8 inhibition or knockdown slowed down cell migration and chemotaxis, attenuated DNA repair and clonogenic survival, triggered apoptotic cell death, impaired cell cycle and radiosensitized glioblastoma cells. Mechanistically, ionizing radiation activated and upregulated TRPM8-mediated Ca2+ signaling that interfered with cell cycle control probably via CaMKII, cdc25C and cdc2. Combined, our data suggest that TRPM8 channels contribute to spreading, survival and radioresistance of human glioblastoma and, therefore, might represent a promising target in future anti-glioblastoma therapy. PMID:29221175

RAD51 potentiates synergistic effects of chemotherapy with PCI-24781 and cis-diamminedichloroplatinum on gastric cancer

PubMed Central

He, Wei-Ling; Li, Yu-Huang; Hou, Wei-Jian; Ke, Zun-Fu; Chen, Xin-Lin; Lu, Li-Ya; Cai, Shi-Rong; Song, Wu; Zhang, Chang-Hua; He, Yu-Long

2014-01-01

AIM: To explore the efficacy of PCI-24781, a broad-spectrum, hydroxamic acid-derived histone deacetylase inhibitor, in the treatment of gastric cancer (GC). METHODS: With or without treatment of PCI-24781 and/or cis-diamminedichloroplatinum (CDDP), GC cell lines were subjected to functional analysis, including cell growth, apoptosis and clonogenic assays. Chromatin immunoprecipitation and luciferase reporter assays were used to determine the interacting molecules and the activity of the enzyme. An in vivo study was carried out in GC xenograft mice. Cell culture-based assays were represented as mean ± SD. ANOVA tests were used to assess differences across groups. All pairwise comparisons between tumor weights among treatment groups were made using the Tukey-Kramer method for multiple comparison adjustment to control experimental-wise type I error rates. Significance was set at P < 0.05. RESULTS: PCI-24781 significantly reduced the growth of the GC cells, enhanced cell apoptosis and suppressed clonogenicity, and these effects synergized with the effects of CDDP. PCI-24781 modulated the cell cycle and significantly reduced the expression of RAD51, which is related to homologous recombination. Depletion of RAD51 augmented the biological functions of PCI-24781, CDDP and the combination treatment, whereas overexpressing RAD51 had the opposite effects. Increased binding of the transcription suppressor E2F4 on the RAD51 promoter appeared to play a major role in these processes. Furthermore, significant suppression of tumor growth and weight in vivo was obtained following PCI-24781 treatment, which synergized with the anticancer effect of CDDP. CONCLUSION: These data suggest that RAD51 potentiates the synergistic effects of chemotherapy with PCI-24781 and CDDP on GC. PMID:25110436

MMPs-Mediated ECM Remodeling

PubMed

Mali, Aniket V; Joshi, Asavari A; Hegde, Mahabaleshwar V; Kadam, Shivajirao S

2017-04-01

Background: To enhance their own survival, tumor cells can manipulate their microenvironment through remodeling of the extra cellular matrix (ECM). The urokinase-type plasminogen activator (uPA) system catalyzes plasmin production which further mediates activation of matrix metalloproteinases (MMPs) and plays an important role in breast cancer invasion and metastasis through ECM remodeling. This provides a potential target for therapeutic intervention of breast cancer treatment. Enterolactone (EL) is derived from dietary flax lignans in the human body and is known to have anti-breast cancer activity. We here investigated molecular and cellular mechanisms of EL action on the uPA-plasmin- MMPs system. Methods: MTT and trypan blue dye exclusion assays, anchorage-dependent clonogenic assays and wound healing assays were carried out to study effects on cell proliferation and viability, clonogenicity and migration capacity, respectively. Real-time PCR was employed to study gene expression and gelatin zymography was used to assess MMP-2 and MMP-9 activities. All data were statistically analysed and presented as mean ± SEM values. Results: All the findings collectively demonstrated anticancer and antimetastatic potential of EL with antiproliferative, antimigratory and anticlonogenic cellular mechanisms. EL was found to exhibit multiple control of plasmin activation by down-regulating uPA expression and also up-regulating its natural inhibitor, PAI-1, at the mRNA level. Further, EL was found to down-regulate expression of MMP-2 and MMP-9 genes, and up-regulate TIMP-1 and TIMP-2; natural inhibitors of MMP-2 and MMP-9, respectively. This may be as a consequence of inhibition of plasmin activation, resulting in robust control over migration and invasion of breast cancer cells during metastasis. Conclusions: EL suppresses proliferation, migration and metastasis of MDA-MB-231 breast cancer cells by inhibiting induced ECM remodeling by the ‘uPA-plasmin-MMPs system’. Creative Commons Attribution License

Cryopreservation of cord blood CD34+ cells before or after thrombopoietin expansion differentially affects early platelet recovery in NOD SCID mice.

PubMed

van Hensbergen, Yvette; van der Garde, Mark; Brand, Anneke; Slot, Manon C; de Graaf-Dijkstra, Alice; Watt, Suzanne; Zwaginga, Jaap Jan

2015-07-01

Expansion of human cord blood (CB) CD34+ cells with thrombopoietin (TPO) can accelerate delayed platelet (PLT) recovery after transplantation into immunodeficient mice. Clinical implementation, however, will depend on practical and effective protocols. The best timing of TPO expansion in relation to cryopreservation in this respect is unknown. In this study, we evaluated whether the order of cryopreservation and TPO expansion affected the expansion rate and numbers of clonogenic hematopoietic progenitor cells in vitro or PLT and longer-term hematopoietic repopulation in NOD SCID mice in vivo. Our results demonstrate higher expansion rates and the generation of higher numbers of multilineage and megakaryocytic progenitors (granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units and megakaryocyte colony-forming units) in vitro when freshly isolated CB CD34+ cells are first cultured with TPO and then cryopreserved and thawed as compared to TPO expansion after CD34+ cell cryopreservation. In contrast, the cells produced with the latter strategy showed higher expression of CD62L and a superior stromal cell-derived factor-1α-mediated migration. This might play a role in an also observed superior early PLT recovery after transplantation of these cells into NOD SCID mice. The hematopoietic engraftment in the marrow 6 weeks after transplantation was not different between the two strategies. Although TPO expansion before cryopreservation would yield higher nucleated cell and clonogenic myeloid and megakaryocyte cell numbers and enable earlier availability, CB TPO expansion after cryopreservation is likely to be clinically more effective, despite the lower number of cells obtained after expansion. Moreover, the latter strategy is logistically more feasible. © 2015 AABB.

Adamantanyl-Histone Deacetylase Inhibitor H6CAHA Exhibits Favorable Pharmacokinetics and Augments Prostate Cancer Radiation Sensitivity

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

Konsoula, Zacharoula; Cao Hong; Velena, Alfredo

2011-04-01

Purpose: To evaluate pharmacological properties of H6CAHA, an adamantyl-hydroxamate histone deacetylase inhibitor, and to investigate its effect on prostate cancer cells following exposure to {gamma}-radiation in vitro and in vivo. Methods and Materials: H6CAHA was assessed for in vitro solubility, lipophilicity and growth inhibition, and in vivo plasma pharmacokinetics. The effect of H6CAHA on radiation clonogenic survival and DNA damage repair was evaluated in human prostate cancer (PC3, DU145, LNCaP) and nonmalignant control epithelial (RWPE1 and 267B1) cell lines. The effect of this agent on the growth of prostate cancer xenografts was also assessed in mice. Results: H6CAHA demonstrated goodmore » solubility and permeability profiles and preferentially inhibited the growth of prostate cancer cells over nonmalignant cells. Plasma pharmacokinetics revealed that the area under the curve of H6CAHA was 8.08 {+-} 0.91 {mu}M x h, and its half-life was 11.17 {+-} 0.87 h. Radiation clonogenic assays revealed that H6CAHA decreased the survival of prostate cancer cells at the dose that exerted limited effect on normal cells. Concomitantly, delayed DNA damage repair following combination treatment was evident in cancer cells, indicated by the prolonged appearance of {gamma}H2AX and Rad51 foci and suppression of DNA damage repair genes (ATM, BRCA1, and BRCA2). Combined modality of H6CAHA (daily intraperitoneal injections for 10 days) with {gamma}-radiation (10 x 2 Gy) completely blocked the growth of PC3 tumor xenografts (p < 0.001) over 60 days. Conclusion: These results support the potential therapeutic value of H6CAHA in combination with radiation and support the rationale for further clinical investigation.« less

Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates

PubMed Central

Bajpai, Vivek K.; Mistriotis, Panagiotis; Loh, Yuin-Han; Daley, George Q.; Andreadis, Stelios T.

2012-01-01

Aims Smooth muscle cells (SMC) play an important role in vascular homeostasis and disease. Although adult mesenchymal stem cells (MSC) have been used as a source of contractile SMC, they suffer from limited proliferation potential and culture senescence, particularly when originating from older donors. By comparison, human induced pluripotent stem cells (hiPSC) can provide an unlimited source of functional SMC for autologous cell-based therapies and for creating models of vascular disease. Our goal was to develop an efficient strategy to derive functional, contractile SMC from hiPSC. Methods and results We developed a robust, stage-wise, feeder-free strategy for hiPSC differentiation into functional SMC through an intermediate stage of multipotent MSC, which could be coaxed to differentiate into fat, bone, cartilage, and muscle. At this stage, the cells were highly proliferative and displayed higher clonogenic potential and reduced senescence when compared with parental hair follicle mesenchymal stem cells. In addition, when exposed to differentiation medium, the myogenic proteins such as α-smooth muscle actin, calponin, and myosin heavy chain were significantly upregulated and displayed robust fibrillar organization, suggesting the development of a contractile phenotype. Indeed, tissue constructs prepared from these cells exhibited high levels of contractility in response to receptor- and non-receptor-mediated agonists. Conclusion We developed an efficient stage-wise strategy that enabled hiPSC differentiation into contractile SMC through an intermediate population of clonogenic and multipotent MSC. The high yield of MSC and SMC derivation suggests that our strategy may facilitate an acquisition of the large numbers of cells required for regenerative medicine or for studying vascular disease pathophysiology. PMID:22941255

Reduction in clonogenic survival of sodium-iodide symporter (NIS)-positive cells following intracellular uptake of (99m)Tc versus (188)Re.

PubMed

Freudenberg, Robert; Wendisch, Maria; Runge, Roswitha; Wunderlich, Gerd; Kotzerke, Jörg

2012-12-01

Cellular radionuclide uptake increases the heterogeneity of absorbed dose to biological structures. Dose increase depends on uptake yield and emission characteristics of radioisotopes. We used an in vitro model to compare the impact of cellular uptake of (188)Re-perrhenate and (99m)Tc-pertechnetate on cellular survival. Rat thyroid PC Cl3 cells in culture were incubated with (188)Re or (99m)Tc in the presence or absence of perchlorate for 1 hour. Clonogenic cell survival was measured by colony formation. In addition, intracellular radionuclide uptake was quantified. Dose effect curves were established for (188)Re and (99m)Tc for various extra- and intracellular distributions of the radioactivity. In the presence of perchlorate, no uptake of radionuclides was detected and (188)Re reduced cell survival more efficiently than (99m)Tc. A(37), the activity that is necessary to yield 37% cell survival was 14 MBq/ml for (188)Re and 480 MBq/ml for (99m)Tc. In the absence of perchlorate, both radionuclides showed similar uptakes; however, A(37) was reduced by 30% for the beta-emitter and by 95% for (99m)Tc. The dose D(37) that yields 37% cell survival was between 2.3 and 2.8 Gy for both radionuclides. Uptake of (188)Re and (99m)Tc decreased cell survival. Intracellular (99m)Tc yielded a dose increase that was higher compared to (188)Re due to emitted Auger and internal conversion-electrons. Up to 5 Gy there was no difference in radiotoxicity of (188)Re and (99m)Tc. At doses higher than 5 Gy intracellular (99m)Tc became less radiotoxic than (188)Re, probably due to a non-uniform lognormal radionuclide uptake.

A new strategy for umbilical cord blood collection developed at the first Colombian public cord blood bank increases total nucleated cell content.

PubMed

Vanegas, Diana; Triviño, Lady; Galindo, Cristian; Franco, Leidy; Salguero, Gustavo; Camacho, Bernardo; Perdomo-Arciniegas, Ana-María

2017-09-01

The total nucleated cell dosage of umbilical cord blood (UCB) is an important factor in determining successful allogeneic hematopoietic stem cell transplantation after a minimum human leukocyte antigen donor-recipient match. The northern South American population is in need of a new-generation cord blood bank that cryopreserves only units with high total nucleated cell content, thereby increasing the likelihood of use. Colombia set up a public cord blood bank in 2014; and, as a result of its research for improving high total nucleated cell content, a new strategy for UCB collection was developed. Data from 2933 collected and 759 cryopreserved cord blood units between 2014 and 2015 were analyzed. The correlation of donor and collection variables with cellularity was evaluated. Moreover, blood volume, cell content, CD34+ count, clonogenic capacity, and microbial contamination were assessed comparing the new method, which combines in utero and ex utero techniques, with the conventional strategies. Multivariate analysis confirmed a correlation between neonatal birth weight and cell content. The new collection method increased total nucleated cell content in approximately 26% and did not alter pre-cryopreservation and post-thaw cell recovery, viability, or clonogenic ability. Furthermore, it showed a remarkably low microbial contamination rate (1.2%). The strategy for UCB collection developed at the first Colombian public cord blood bank increases total nucleated cell content and does not affect unit quality. The existence of this bank is a remarkable breakthrough for Latin-American patients in need of this kind of transplantation. © 2017 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

Wee-1 Kinase Inhibition Overcomes Cisplatin Resistance Associated with High-Risk TP53 Mutations in Head and Neck Cancer through Mitotic Arrest Followed by Senescence

PubMed Central

Osman, Abdullah A.; Monroe, Marcus M.; Ortega Alves, Marcus V.; Patel, Ameeta A.; Katsonis, Panagiotis; Fitzgerald, Alison L.; Neskey, David M.; Frederick, Mitchell J.; Woo, Sang Hyeok; Caulin, Carlos; Hsu, Teng-Kuei; McDonald, Thomas O.; Kimmel, Marek; Meyn, Raymond E.; Lichtarge, Olivier; Myers, Jeffrey N.

2015-01-01

Although cisplatin has played a role in “standard-of-care” multimodality therapy for patients with advanced squamous cell carcinoma of the head and neck (HNSCC), the rate of treatment failure remains particularly high for patients receiving cisplatin whose tumors have mutations in the TP53 gene. We found that cisplatin treatment of HNSCC cells with mutant TP53 leads to arrest of cells in the G2 phase of the cell cycle, leading us to hypothesize that the wee-1 kinase inhibitor MK-1775 would abrogate the cisplatin-induced G2 block and thereby sensitize isogenic HNSCC cells with mutant TP53 or lacking p53 expression to cisplatin. We tested this hypothesis using clonogenic survival assays, flow cytometry, and in vivo tumor growth delay experiments with an orthotopic nude mouse model of oral tongue cancer. We also used a novel TP53 mutation classification scheme to identify which TP53 mutations are associated with limited tumor responses to cisplatin treatment. Clonogenic survival analyses indicate that nanomolar concentration of MK-1775 sensitizes HNSCC cells with high-risk mutant p53 to cisplatin. Consistent with its ability to chemosensitize, MK-1775 abrogated the cisplatin-induced G2 block in p53-defective cells leading to mitotic arrest associated with a senescence-like phenotype. Furthermore, MK-1775 enhanced the efficacy of cisplatin in vivo in tumors harboring TP53 mutations. These results indicate that HNSCC cells expressing high-risk p53 mutations are significantly sensitized to cisplatin therapy by the selective wee-1 kinase inhibitor, supporting the clinical evaluation of MK-1775 in combination with cisplatin for the treatment of patients with TP53 mutant HNSCC. PMID:25504633

Comparative study of autophagy inhibition by 3MA and CQ on Cytarabine‑induced death of leukaemia cells.

PubMed

Palmeira dos Santos, Caroline; Pereira, Gustavo J S; Barbosa, Christiano M V; Jurkiewicz, Aron; Smaili, Soraya S; Bincoletto, Claudia

2014-06-01

As the molecular mechanisms of Cytarabine,one of the most important drugs used in the leukaemia’s treatment, are only partially understood and the role of autophagy on leukaemia development and treatment is only recently being investigated, in this study, by using Chloroquine (CQ) and 3-methyladenine (3MA) as autophagy inhibitors, we aim to evaluate the contribution of an autophagic mechanism to Cytarabine (AraC)-induced death of HL60 leukaemia cells. Trypan blue exclusion and AnnexinV/PI assays were used to evaluate HL60 cell death under AraC treatment in the presence or absence of 3MA and CQ. Western blotting and immunofluorescence experiments were performed to show the involvement of apoptosis and autophagy protein expressions. Phenotypic characterization of HL60-treated cells was performed by using immunophenotyping. Clonogenic assays were applied to analyse clonal function of HL60-treated cells. We observed that although autophagy inhibition by 3MA, but not CQ, increased the death of HL60 AraC cells after 24 h of treatment, no significant differences between AraC and AraC + 3MA-treated groups were observed by using clonogenic assay. In addition, increased number of immature (CD34(+)/CD38(−)Lin(−/low)) HL60 cells was found in AraC and AraC-3MA groups when compared with control untreated cells. Although AraC anti-leukaemia effects could be potentiated by 3MA autophagy inhibition after 24 h of exposure, leukaemia cell resistance, the main causes of treatment failure, is also promoted by autophagy initial stage impairment by 3MA, denoting the complex role of autophagy in leukaemia cells’ response to chemotherapy.

The role of morphine on rat neural stem cells viability, neuro-angiogenesis and neuro-steroidgenesis properties.

PubMed

Abdyazdani, Nima; Nourazarian, Alireza; Nozad Charoudeh, Hojjatollah; Kazemi, Masoumeh; Feizy, Navid; Akbarzade, Maryam; Mehdizadeh, Amir; Rezaie, Jafar; Rahbarghazi, Reza

2017-01-01

A lack of comprehensive data exists on the effect of morphine on neural stem cell neuro-steroidogenesis and neuro-angiogenesis properties. We, herein, investigated the effects of morphine (100μM), naloxone (100μM) and their combination on rat neural stem cells viability, clonogenicity and Ki-67 expression over a period of 72h. Any alterations in the total fatty acids profile under treatment protocols were elucidated by direct transesterification method. We also monitored the expression of p53, aromatase and 5-alpha reductase by real-time PCR assay. To examine angiogenic capacity, in vitro tubulogenesis and the level of VE-cadherin transcript were investigated during neural to endothelial differentiation under the experimental procedure. Cells supplemented with morphine displayed reduced survival (p<0.01) and clonogenicity (p<0.001). Flow cytometric analysis showed a decrease in Ki-67 during 72h. Naloxone potentially blunted morphine-induced all effects. The normal levels of fatty acids, including saturated and unsaturated were altered by naloxone and morphine supplements. Following 48h, the up-regulation of p53, aromatase and 5-alpha reductase genes occurred in morphine-primed cells. Using three-dimensional culture models of angiogenesis and real time PCR assay, we showed morphine impaired the tubulogenesis properties of neural stem cells (p<0.001) by the inhibition of trans-differentiation into vascular cells and led to decrease of in VE-cadherin expression. Collectively, morphine strongly impaired the healthy status of neural stem cells by inducing p53 and concurrent elevation of aromatase and 5-alpha reductase activities especially during early 48h. Also, neural stem cells-being exposed to morphine lost their potency to elicit angiogenesis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Down-regulation of let-7 microRNA increased K-ras expression in lung damage induced by radon.

PubMed

Chen, Zhihai; Wang, Dapeng; Gu, Chao; Liu, Xing; Pei, Weiwei; Li, Jianxiang; Cao, Yi; Jiao, Yang; Tong, Jian; Nie, Jihua

2015-09-01

Radon has long been recognized as a human carcinogen leading to lung cancer, but the underlying mechanisms remain obscure. Recent studies have shown that the let-7 microRNA and K-ras play an important role in the development of various cancers. However, the exact role between let-7 and K-ras in radon induced lung damage has not been explored so far. In the present study, wistar rats and human bronchial epithelial (HBE) cells were long-term exposed to radon, and then alterations in histological pathology of rat lung tissue, ROS, antioxidant enzymes activities and clonogenic formation in HBE cells, as well as changes in let-7 and K-ras expression were determined to observe the adverse effects induced by radon. The results showed that long-term exposure to radon produced severe lung damage in rats, significantly increased ROS production and clonogenic formation ratios and decreased SOD activities in HBE cells. In addition, an obvious down-regulation of let-7 and up-regulation of K-ras were also revealed both in mRNA and in protein level in lung tissue of rats and HBE cells exposed to radon. Furthermore, a significant down-regulation of K-ras was then confirmed in both let-7b-3p and let-7a-2-3p transfected HBE cells. Taken together, the present results propose an involvement of let-7 microRNA and K-ras in radon induced lung damage both in vivo and in vitro, which may thus be of potential value in early diagnosis and therapy of radon-induced lung tumorgenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

On the probability of cure for heavy-ion radiotherapy

NASA Astrophysics Data System (ADS)

Hanin, Leonid; Zaider, Marco

2014-07-01

The probability of a cure in radiation therapy (RT)—viewed as the probability of eventual extinction of all cancer cells—is unobservable, and the only way to compute it is through modeling the dynamics of cancer cell population during and post-treatment. The conundrum at the heart of biophysical models aimed at such prospective calculations is the absence of information on the initial size of the subpopulation of clonogenic cancer cells (also called stem-like cancer cells), that largely determines the outcome of RT, both in an individual and population settings. Other relevant parameters (e.g. potential doubling time, cell loss factor and survival probability as a function of dose) are, at least in principle, amenable to empirical determination. In this article we demonstrate that, for heavy-ion RT, microdosimetric considerations (justifiably ignored in conventional RT) combined with an expression for the clone extinction probability obtained from a mechanistic model of radiation cell survival lead to useful upper bounds on the size of the pre-treatment population of clonogenic cancer cells as well as upper and lower bounds on the cure probability. The main practical impact of these limiting values is the ability to make predictions about the probability of a cure for a given population of patients treated to newer, still unexplored treatment modalities from the empirically determined probability of a cure for the same or similar population resulting from conventional low linear energy transfer (typically photon/electron) RT. We also propose that the current trend to deliver a lower total dose in a smaller number of fractions with larger-than-conventional doses per fraction has physical limits that must be understood before embarking on a particular treatment schedule.

Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

PubMed

Antonietti, Patrick; Linder, Benedikt; Hehlgans, Stephanie; Mildenberger, Iris C; Burger, Michael C; Fulda, Simone; Steinbach, Joachim P; Gessler, Florian; Rödel, Franz; Mittelbronn, Michel; Kögel, Donat

2017-01-01

Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR. ©2016 American Association for Cancer Research.

Evaluation of preventive and therapeutic activity of novel non-steroidal anti-inflammatory drug, CG100649, in colon cancer: Increased expression of TNF-related apoptosis-inducing ligand receptors enhance the apoptotic response to combination treatment with TRAIL.

PubMed

Woo, Jong Kyu; Kang, Ju-Hee; Jang, Yeong-Su; Ro, Seonggu; Cho, Joong Myung; Kim, Hwan-Mook; Lee, Sang-Jin; Oh, Seung Hyun

2015-04-01

Non-steroidal anti-inflammatory drugs (NSAIDs) have been suggested as the potential new class of preventive or therapeutic antitumor agents. The aim of the present study was to evaluate the antitumor activity of the novel NSAID, CG100649. CG100649 is a novel NSAID dual inhibitor for COX-2 and carbonic anhydrase (CA)-I/-II. In the present study, we investigated the alternative mechanism by which CG100649 mediated suppression of the colon cancer growth and development. The anchorage‑dependent and -independent clonogenic assay showed that CG100649 inhibited the clonogenicity of human colon cancer cells. The flow cytometric analysis showed that CG100649 induced the G2/M cell cycle arrest in colon cancer cells. Animal studies showed that CG100649 inhibited the tumor growth in colon cancer xenograft in nude mice. Furthermore, quantitative PCR and FACS analysis demonstrated that CG100649 upregulated the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors (DR4 and DR5) but decreased the expression of decoy receptors (DcR1 and DcR2) in colon cancer cells. The results showed that CG100649 treatment sensitized TRAIL‑mediated growth suppression and apoptotic cell death. The combination treatment resulted in significant repression of the intestinal polyp formation in APCmin/+ mice. Our data clearly demonstrated that CG100649 contains preventive and therapeutic activity for colon cancer. The present study may be useful for identification of the potential benefit of the NSAID CG100649, for the achievement of a better treatment response in colon cancer.

Synergistic Effect of Simvastatin Plus Radiation in Gastric Cancer and Colorectal Cancer: Implications of BIRC5 and Connective Tissue Growth Factor

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

Lim, Taekyu; Lee, Inkyoung; Kim, Jungmin

Purpose: We investigated the synergistic effect of simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor plus radiation therapy, on the proliferation and survival of gastric cancer (GC) and colorectal cancer (CRC) cells. We also studied several genes involved in the simvastatin/radiation-induced effects. Methods and Materials: Gastric cancer (AGS, SNU601, MKN1, and MKN28) and CRC (CoLo320, SW48, HT29, and HCT8) cell lines were treated with 0.2 μM simvastatin alone, or in combination with 0 to 4 Gy of radiation, and subjected to clonogenic survival and proliferation assays in vitro. To assess the molecular mechanism of the combination treatment, we performed microarray analysis, immunoblot assays, small interferingmore » RNA knockdown experiments, and plasmid rescue assays. The antitumoral effects of simvastatin and radiation were evaluated in vivo using xenograft models. Results: The combination therapy of simvastatin plus radiation inhibited basal clonogenic survival and proliferation of GC and CRC cells in vitro. Simvastatin suppressed the expression of BIRC5 and CTGF genes in these cancer cells. In vivo, the combined treatment with simvastatin and radiation significantly reduced the growth of xenograft tumors compared with treatment with radiation alone. Conclusion: We suggest that simvastatin has a synergistic effect with radiation on GC and CRC through the induction of apoptosis, which may be mediated by a simultaneous inhibition of BIRC5 and CTGF expression. A clinical trial of simvastatin in combination with radiation in patients with GC or CRC is warranted.« less

A Mitochondria-Targeted Nitroxide/Hemigramicidin S Conjugate Protects Mouse Embryonic Cells Against Gamma Irradiation

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

Jiang Jianfei; Belikova, Natalia A.; Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA

2008-03-01

Purpose: To evaluate the in vitro radioprotective effect of the mitochondria-targeted hemigramicidin S-conjugated 4-amino-2,2,6,6-tetramethyl-piperidine-N-oxyl (hemi-GS-TEMPO) 5-125 in {gamma}-irradiated mouse embryonic cells and adenovirus-12 SV40 hybrid virus transformed human bronchial epithelial cells BEAS-2B and explore the mechanisms involved in its radioprotective effect. Methods and Materials: Cells were incubated with 5-125 before (10 minutes) or after (1 hour) {gamma}-irradiation. Superoxide generation was determined by using dihydroethidium assay, and lipid oxidation was quantitated by using a fluorescence high-performance liquid chromatography-based Amplex Red assay. Apoptosis was characterized by evaluating the accumulation of cytochrome c in the cytosol and externalization of phosphatidylserine on the cellmore » surface. Cell survival was measured by means of a clonogenic assay. Results: Treatment (before and after irradiation) of cells with 5-125 at low concentrations (5, 10, and 20 {mu}M) effectively suppressed {gamma}-irradiation-induced superoxide generation, cardiolipin oxidation, and delayed irradiation-induced apoptosis, evaluated by using cytochrome c release and phosphatidylserine externalization. Importantly, treatment with 5-125 increased the clonogenic survival rate of {gamma}-irradiated cells. In addition, 5-125 enhanced and prolonged {gamma}-irradiation-induced G{sub 2}/M phase arrest. Conclusions: Radioprotection/mitigation by hemi-GS-TEMPO likely is caused by its ability to act as an electron scavenger and prevent superoxide generation, attenuate cardiolipin oxidation in mitochondria, and hence prevent the release of proapoptotic factors from mitochondria. Other mechanisms, including cell-cycle arrest at the G{sub 2}/M phase, may contribute to the protection.« less

Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

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

Wei, Yan; Department of Otolaryngology, Head and Neck Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou; Li, Yuan

2011-04-15

Human skeletal muscle contains an accessible adult stem-cell compartment in which differentiated myofibers are maintained and replaced by a self-renewing stem cell pool. Previously, studies using mouse models have established a critical role for resident stem cells in skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate for extended periods of time as floating clusters of rounded cells, termed 'myospheres' or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional properties of these cells were determined usingmore » reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times, despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types.« less

Co-targeting deoxyribonucleic acid-dependent protein kinase and poly(adenosine diphosphate-ribose) polymerase-1 promotes accelerated senescence of irradiated cancer cells.

PubMed

Azad, Arun; Bukczynska, Patricia; Jackson, Susan; Haupt, Ygal; Haput, Ygal; Cullinane, Carleen; McArthur, Grant A; Solomon, Benjamin

2014-02-01

To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs, and accelerated senescence after radiation. Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination. Copyright © 2014. Published by Elsevier Inc.

Tumourigenicity and radiation resistance of mesenchymal stem cells.

PubMed

D'Andrea, Filippo P; Horsman, Michael R; Kassem, Moustapha; Overgaard, Jens; Safwat, Akmal

2012-05-01

Cancer stem cells are believed to be more radiation resistant than differentiated tumour cells of the same origin. It is not known, however, whether normal nontransformed adult stem cells share the same radioresistance as their cancerous counterpart. Nontumourigenic (TERT4) and tumourigenic (TRET20) cell lines, from an immortalised mesenchymal stem cell line, were grown in culture prior to irradiation and gene expression analysis. Radiation resistance was measured using a clonogenic assay. Differences in gene expression between the two cell lines, both under nontreated and irradiated conditions, were assessed with microarrays (Affymetrix Human Exon 1.0 ST array). The cellular functions affected by the altered gene expressions were assessed through gene pathway mapping (Ingenuity Pathway Analysis). Based on the clonogenic assay the nontumourigenic cell line was found to be more sensitive to radiation than the tumourigenic cell line. Using the exon chips, 297 genes were found altered between untreated samples of the cell lines whereas only 16 genes responded to radiation treatment. Among the genes with altered expression between the untreated samples were PLAU, PLAUR, TIMP3, MMP1 and LOX. The pathway analysis based on the alteration between the untreated samples indicated cancer and connective tissue disorders. This study has shown possible common genetic events linking tumourigenicity and radiation response. The PLAU and PLAUR genes are involved in apoptosis evasion while the genes TIMP3, MMP1 and LOX are involved in regulation of the surrounding matrix. The first group may contribute to the difference in radiation resistance observed and the latter could be a major contributor to the tumourigenic capabilities by degrading the intercellular matrix. These results also indicate that cancer stem cells are more radiation resistant than stem cells of the same origin.

Deficiency in clonogenic endometrial mesenchymal stem cells in obese women with reproductive failure--a pilot study.

PubMed

Murakami, Keisuke; Bhandari, Harish; Lucas, Emma S; Takeda, Satoru; Gargett, Caroline E; Quenby, Siobhan; Brosens, Jan J; Tan, Bee K

2013-01-01

The mechanisms of obesity associated reproductive complications remain poorly understood. Endometrial mesenchymal stem-cells are critical for cyclic renewal and uterine function. Recently, W5C5(+) cells, with high clonogenicity, capable of producing endometrial stroma in vivo, have been described. We sought to investigate the abundance and cloning efficiency of W5C5(+) and W5C5(-) endometrial cells in relation to Body Mass Index, age and reproductive outcome. W5C5(+) and W5C5(-) cells were purified from mid-luteal endometrial biopsies (n = 54) by magnetic bead separation and subjected to in vitro colony-forming assays. First trimester pregnancy losses were significantly higher in obese subjects (n = 12) compared to overweight (n = 20) and subjects with normal Body Mass Index (n = 22) (P<0.05, P<0.01, respectively). W5C5(+) cells (%) were significantly lower in obese subjects compared to subjects with normal Body Mass Index (P<0.05). W5C5(+) cloning efficiency was significantly lower in obese subjects compared to overweight and subjects with normal Body Mass Index (P<0.05, respectively). W5C5(-) cloning efficiency was significantly lower in obese subjects compared to subjects with normal Body Mass Index (P<0.05). Body Mass Index was significantly negatively correlated with W5C5(+) cloning efficiency and W5C5(-) cloning efficiency (P<0.01, respectively), and positively correlated with first trimester loss (P<0.01). We found no significant results with age (P>0.05). Our observations suggest that the regenerative capacity and plasticity of the endometrium of obese women is suboptimal, which in turn may account for the increased risk of reproductive complications associated with obesity.

Synergistic Effect of Ionizing Radiation and β-lapachone against RKO Human Colon Adenocarcinoma Cells

PubMed Central

Kim, Eun Jung; Ji, In-Mi; Ahn, Ki-Jung; Choi, Eun Kyung; Park, Heon-Jin; Lim, Byung Uk; Song, Chang W.

2005-01-01

Purpose To reveal the interaction between β-Lapachone (β-lap) and ionizing radiation in causing cell death in RKO human colon adenocarcinoma cells, and to elucidate the potential usefulness of combined β-lap treatment and radiotherapy for cancer treatment. Materials and Methods The cytotoxicities of various treatments were determined in vitro using clonogenic and apoptotic cell death. The changes in cell cycle distribution were studied using flow cytometry and an in vitro kinase assay. The tumor growth was studied using RKO tumors grown s.c. in the hind leg BALB/c- nuslc nude mice. Results β-lap caused clonogenic cell death and rapid apoptosis in RKO cells in vitro, in a dose dependent manner. The repair of sublethal radiation damage was almost completely inhibited when cells were maintained in β-lap during the interval between the two-dose irradiation. Flow cytometry study demonstrated that β-lap induced apoptosis, independent of the cell cycle phase, and completely prohibited the induction of radiation-induced G2 arrest in irradiated cells. The prohibition of radiation-induced G2 arrest is unclear, but may be related to the profound suppression of the p53, p21 and cyclin B1-Cdc2 kinase activities observed in cells treated with β-lap. The combination of β-lap and radiation markedly enhanced the radiation-induced growth suppression of tumors. Conclusion β-lap is cytotoxic against RKO cells, both in vitro and in vivo, and also sensitized cells to ionizing radiation by inhibiting sublethal radiation damage repair. β-lap is potentially useful as a potent anti-cancer chemotherapy drug and potent radiosensitizer against caner cells. PMID:19956501

Icotinib enhances lung cancer cell radiosensitivity in vitro and in vivo by inhibiting MAPK/ERK and AKT activation.

PubMed

Fu, Yonghong; Zhang, Sen; Wang, Dongjie; Wang, Jing

2018-05-16

Icotinib hydrochloride is a small epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that was developed by Chinese scientists. While clinical trials have revealed its efficacy in the treatment of lung cancer, very little is known about its role in enhancing radiosensitivity. In this study, we investigated the effectiveness of Icotinib in enhancing lung cancer cell radiosensitivity and have detailed its underlying molecular mechanism. The lung cancer cell line H1650 was pretreated with or without Icotinib for 24 hours before radiation, and clonogenic survival assay was performed. Cell apoptosis was also analyzed by flow cytometry, while western blotting was performed to examine the activation of EGFR and its downstream kinases in H1650 cells after Icotinib and radiation treatment. Furthermore, a xenograft animal model was established to evaluate the radiosensitivity of Icotinib in vivo and to confirm its mechanism. Our results demonstrate that pretreatment with Icotinib reduced clonogenic survival after radiation, inhibited EGFR activation, and increased radiation-induced apoptosis in H1650 cells. The phosphorylation of protein kinase B (AKT), extracellular regulated protein kinase 1/2 (ERK1/2), and EGFR was inhibited after Icotinib and radiation combination treatment in vitro and in vivo compared with individual treatments. Combination treatment also affected the expression of the DNA repair protein H2A histone family member X (γ-H2AX). In conclusion, our results reveal that Icotinib enhances radiosensitivity in lung cancers in vitro and in vivo and the mechanism of this may involve blocking the EGFR-AKT and MAPK-ERK pathways and limiting DNA repair. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Exosomes are released by bystander cells exposed to radiation-induced biophoton signals: Reconciling the mechanisms mediating the bystander effect.

PubMed

Le, Michelle; Fernandez-Palomo, Cristian; McNeill, Fiona E; Seymour, Colin B; Rainbow, Andrew J; Mothersill, Carmel E

2017-01-01

The objective of our study was to explore a possible molecular mechanism by which ultraviolet (UV) biophotons could elicit bystander responses in reporter cells and resolve the problem of seemingly mutually exclusive mechanisms of a physical UV signal & a soluble factor-mediated bystander signal. The human colon carcinoma cell line, HCT116 p53 +/+, was directly irradiated with 0.5 Gy tritium beta particles to induce ultraviolet biophoton emission. Bystander cells were not directly irradiated but were exposed to the emitted UV biophotons. Medium was subsequently harvested from UV-exposed bystander cells. The exosomes extracted from this medium were incubated with reporter cell populations. These reporter cells were then assayed for clonogenic survival and mitochondrial membrane potential with and without prior treatment of the exosomes with RNase. Clonogenic cell survival was significantly reduced in reporter cells incubated with exosomes extracted from cells exposed to secondarily-emitted UV. These exosomes also induced significant mitochondrial membrane depolarization in receiving reporter cells. Conversely, exosomes extracted from non-UV-exposed cells did not produce bystander effects in reporter cells. The treatment of exosomes with RNase prior to their incubation with reporter cells effectively abolished bystander effects in reporter cells and this suggests a role for RNA in mediating the bystander response elicited by UV biophotons and their produced exosomes. This study supports a role for exosomes released from UV biophoton-exposed bystander cells in eliciting bystander responses and also indicates a reconciliation between the UV-mediated bystander effect and the bystander effect which has been suggested in the literature to be mediated by soluble factors.

Differential response of DU145 and PC3 prostate cancer cells to ionizing radiation: role of reactive oxygen species, GSH and Nrf2 in radiosensitivity.

PubMed

Jayakumar, Sundarraj; Kunwar, Amit; Sandur, Santosh K; Pandey, Badri N; Chaubey, Ramesh C

2014-01-01

Radioresistance is the major impediment in radiotherapy of many cancers including prostate cancer, necessitating the need to understand the factors contributing to radioresistance in tumor cells. In the present study, the role of cellular redox and redox sensitive transcription factor, Nrf2 in the radiosensitivity of prostate cancer cell lines PC3 and DU145, has been investigated. Differential radiosensitivity of PC3 and DU145 cells was assessed using clonogenic assay, flow cytometry, and comet assay. Their redox status was measured using DCFDA and DHR probes. Expression of Nrf2 and its dependent genes was measured by EMSA and real time PCR. Knockdown studies were done using shRNA transfection. PC3 and DU145 cells differed significantly in their radiosensitivity as observed by clonogenic survival, apoptosis and neutral comet assays. Both basal and inducible levels of ROS were higher in PC3 cells than that of DU145 cells. DU145 cells showed higher level of basal GSH content and GSH/GSSG ratio than that of PC3 cells. Further, significant increase in both basal and induced levels of Nrf2 and its dependent genes was observed in DU145 cells. Knock-down experiments and pharmacological intervention studies revealed the involvement of Nrf2 in differential radio-resistance of these cells. Cellular redox status and Nrf2 levels play a causal role in radio-resistance of prostate cancer cells. The pivotal role Nrf2 has been shown in the radioresistance of tumor cells and this study will further help in exploiting this factor in radiosensitization of other tumor cell types. © 2013.

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

Azad, Arun, E-mail: arun.azad@bccancer.bc.ca; Department of Pathology, St. Vincent's Hospital, University of Melbourne, Parkville, Victoria; Bukczynska, Patricia

Purpose: To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. Methods and Materials: The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs,more » and accelerated senescence after radiation. Results: Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Conclusion: Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination.« less

Mocetinostat combined with gemcitabine for the treatment of leiomyosarcoma: Preclinical correlates

PubMed Central

Braggio, Danielle; Zewdu, Abeba; Casadei, Lucia; Batte, Kara; Bid, Hemant Kumar; Koller, David; Yu, Peter; Iwenofu, Obiajulu Hans; Strohecker, Anne; Choy, Edwin; Lev, Dina; Pollock, Raphael

2017-01-01

Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastatic disease. Chemotherapeutic intervention has demonstrated to have modest clinical efficacy with no curative potential in LMS patients. Previously, we demonstrated pan-HDAC inhibition to have a superior effect in various complex karyotypic sarcomas. In this study, our goal is to evaluate the therapeutic efficacy of mocetinostat alone and in combination with gemcitabine in LMS. Human leiomyosarcoma (LMS) cell lines were used for in vitro and in vivo studies. Compounds tested included the class I HDAC inhibitor, mocetinostat, and nucleoside analog, gemcitabine. MTS and clonogenic assays were used to evaluate the effect of mocetinostat on LMS cell growth. Cleaved caspase 3/7 analysis was used to determine the effects of mocetinostat on apoptosis. Compusyn software was used to determine in vitro synergy studies for the combination of mocetinostat plus gemcitabine. A LMS xenograft model in SCID mice was used to test the impact of mocetinostat alone, gemcitabine alone and the combination of mocetinostat plus gemcitabine. Mocetinostat abrogated LMS cell growth and clonogenic potential, and enhanced apoptosis in LMS cell lines. The combination of mocetinostat plus gemcitabine exhibited a synergistic effect in LMS cells in vitro. Similarly, mocetinostat combined with gemcitabine resulted in superior anti-LMS effects in vivo. Mocetinostat reduced the expression of gemcitabine-resistance markers RRM1, RRM2, and increased the expression of gemcitabine-sensitivity marker, hENT1, in LMS cells. LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of mocetinostat combined with gemcitabine for the treatment of LMS. PMID:29186204

Straight A's: Public Education Policy and Progress. Volume 11, Number 8

ERIC Educational Resources Information Center

Amos, Jason, Ed.

2011-01-01

"Straight A's: Public Education Policy and Progress" is a biweekly newsletter that focuses on education news and events both in Washington, DC and around the country. The following articles are included in this issue: (1) Last Cut Is the Deepest: Final FY 2011 Spending Agreement Cuts Spending by Nearly $40 Billion, Includes More Cuts for Education…

A Visualization System for Predicting Learning Activities Using State Transition Graphs

ERIC Educational Resources Information Center

Okubo, Fumiya; Shimada, Atsushi; Taniguchi, Yuta

2017-01-01

In this paper, we present a system for visualizing learning logs of a course in progress together with predictions of learning activities of the following week and the final grades of students by state transition graphs. Data are collected from 236 students attending the course in progress and from 209 students attending the past course for…

STAGE-DEPENDENCE, SEASONAL CHANGES AND EFFECTS OF CADMIUM ON CELL CYCLE AND APOPTOTIC ACTIVITIES DURING THE SPERMATOGENIC PROGRESSION IN THE DOGFISH SHARK (SQUALUS ACANTHIAS). (R825434)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Jet Crackle

DTIC Science & Technology

2015-06-23

DISTRIBUTION/AVAILABILITY STATEMENT DISTRIBUTION A 13. SUPPLEMENTARY NOTES 14. ABSTRACT Fighter jets and other aircraft with high specific thrust engines...interim, memorandum, master’s thesis , progress, quarterly, research, special, group study, etc. 3. DATES COVERED. Indicate the time during which the...State the type of report, such as final, technical, interim, memorandum, master’s thesis , progress, quarterly, research, special, group study, etc

[Research progress of thermal control system for extravehicular activity space suit].

PubMed

Wu, Z Q; Shen, L P; Yuan, X G

1999-08-01

New research progress of thermal control system for oversea Extravehicular Activity (EVA) space suit is presented. Characteristics of several thermal control systems are analyzed in detail. Some research tendencies and problems are discussed, which are worthwhile to be specially noted. Finally, author's opinion about thermal control system in the future is put forward.

Reading Achievement in Disadvantaged Children as a Consequence of Non Verbal Perceptual Training. Final Technical Progress Report.

ERIC Educational Resources Information Center

Elkind, David; Deblinger, Jo Ann

The theoretical orientation based on perceptual development, proposed by Piaget in 1961, is the starting point of this investigation. According to Piaget, the perception of the young child is "centered" on dominant aspects of the field. With maturity, perception becomes "decentered" and progressively freed from the field. The…

Straight A's: Public Education Policy and Progress. Volume 9, Number 23

ERIC Educational Resources Information Center

Alliance for Excellent Education, 2009

2009-01-01

"Straight A's: Public Education Policy and Progress" is a biweekly newsletter that focuses on education news and events both in Washington, DC and around the country. The following articles are included in this issue: (1) Two Omnibus Bills and a Partridge in a Pear Tree?: Final Vote on Education Appropriations Bill Could Come Before…

Tom Green County Library Literacy Project. Final Performance Report.

ERIC Educational Resources Information Center

Vavricka, D. Karen

The final report of the Tom Green Country Library System (Texas) literacy project details progress toward achievement of 11 objectives. Objectives of the literacy outreach program were to: (1) increase Hispanic enrollment; (2) increase Black enrollment; (3) provide free child care for 4 students to attend 50 tutoring sessions; (4) provide…

TREMATODES IN SNAILS NEAR RACCOON LATRINES SUGGEST A FINAL HOST ROLE FOR THIS MAMMAL IN CALIFORNIA SALT MARSHES. (R828676C001)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

George A. Olah, Carbocation and Hydrocarbon Chemistry

Science.gov Websites

. Final Technical Report. [HF:BF{sub 2}/H{sub 2}] , DOE Technical Report, 1980 Superacid Catalyzed Coal Conversion Chemistry. 1st and 2nd Quarterly Technical Progress Reports, September 1, 1983-March 30, 1984 , DOE Technical Report, 1984 Superacid Catalyzed Coal Conversion Chemistry. Final Technical Report

THE DEVELOPMENT AND PRESENTATION OF FOUR COLLEGE COURSES BY COMPUTER TELEPROCESSING. FINAL REPORT.

ERIC Educational Resources Information Center

MITZEL, HAROLD E.

THIS IS A FINAL REPORT ON THE DEVELOPMENT AND PRESENTATION OF FOUR COLLEGE COURSES BY COMPUTER TELEPROCESSING FROM APRIL 1964 TO JUNE 1967. IT OUTLINES THE PROGRESS MADE TOWARDS THE PREPARATION, DEVELOPMENT, AND EVALUATION OF MATERIALS FOR COMPUTER PRESENTATION OF COURSES IN AUDIOLOGY, MANAGEMENT ACCOUNTING, ENGINEERING ECONOMICS, AND MODERN…

40 CFR 62.14565 - How do I comply with the increment of progress for achieving final compliance?

Code of Federal Regulations, 2013 CFR

2013-07-01

... DESIGNATED FACILITIES AND POLLUTANTS Federal Plan Requirements for Commercial and Industrial Solid Waste Incineration Units That Commenced Construction On or Before November 30, 1999 Compliance Schedule and... complete retrofit construction of control devices, as specified in the final control plan, so that, when...

PFBC HGCU Test Facility. Technical progress report No. 24, Third quarter, CY 1995

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

NONE

This is the twenty-fourth and final Technical Progress Report submitted to the Department of Energy (DOE) in connection with the cooperative agreement between the DOE and Ohio Power Company for the Tidd PFBC Hot Gas Clean Up Test Facility. This report covers the work completed during the Third Quarter of CY 1995. All activity this quarter was directed toward the completion of the program final report. A draft copy of the final report was forwarded to DOE during this quarter, and DOE submitted their comments on the report to AEPSC. DOE requested that Westinghouse write an appendix to the reportmore » covering the performance of the fail-safe regenerator devices during Tad operation, and Westinghouse subsequently prepared the appendix. Additional DOE comments were incorporated into the report, and it will be issued in camera-ready form by the end of October, 1995, which is the program end date. Appendix 1 presents the results of filter candle posttest examination by Westinghouse performed on selected filter candles following final shutdown of the system.« less

Development of a Conceptual Model of Disease Progression for Use in Economic Modeling of Chronic Obstructive Pulmonary Disease.

PubMed

Tabberer, Maggie; Gonzalez-McQuire, Sebastian; Muellerova, Hana; Briggs, Andrew H; Rutten-van Mölken, Maureen P M H; Chambers, Mike; Lomas, David A

2017-05-01

To develop and validate a new conceptual model (CM) of chronic obstructive pulmonary disease (COPD) for use in disease progression and economic modeling. The CM identifies and describes qualitative associations between disease attributes, progression and outcomes. A literature review was performed to identify any published CMs or literature reporting the impact and association of COPD disease attributes with outcomes. After critical analysis of the literature, a Steering Group of experts from the disciplines of health economics, epidemiology and clinical medicine was convened to develop a draft CM, which was refined using a Delphi process. The refined CM was validated by testing for associations between attributes using data from the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE). Disease progression attributes included in the final CM were history and occurrence of exacerbations, lung function, exercise capacity, signs and symptoms (cough, sputum, dyspnea), cardiovascular disease comorbidities, 'other' comorbidities (including depression), body composition (body mass index), fibrinogen as a biomarker, smoking and demographic characteristics (age, gender). Mortality and health-related quality of life were determined to be the most relevant final outcome measures for this model, intended to be the foundation of an economic model of COPD. The CM is being used as the foundation for developing a new COPD model of disease progression and to provide a framework for the analysis of patient-level data. The CM is available as a reference for the implementation of further disease progression and economic models.

Isolation, Culture, and Motility Measurements of Epidermal Melanocytes from GFP-Expressing Reporter Mice.

PubMed

Dagnino, Lina; Crawford, Melissa

2018-03-27

In this article, we provide a method to isolate primary epidermal melanocytes from reporter mice, which also allow targeted gene inactivation. The mice from which these cells are isolated are bred into a Rosa26 mT/mG reporter background, which results in GFP expression in the targeted melanocytic cell population. These cells are isolated and cultured to >95% purity. The cells can be used for gene expression studies, clonogenic experiments, and biological assays, such as capacity for migration. Melanocytes are slow moving cells, and we also provide a method to measure motility using individual cell tracking and data analysis.

Micronucleus formation induced by dielectric barrier discharge plasma exposure in brain cancer cells

NASA Astrophysics Data System (ADS)

Kaushik, Nagendra K.; Uhm, Hansup; Ha Choi, Eun

2012-02-01

Induction of micronucleus formation (cytogenetic damage) in brain cancer cells upon exposure of dielectric barrier discharge plasma has been investigated. We have investigated the influence of exposure and incubation times on T98G brain cancer cells by using growth kinetic, clonogenic, and micronucleus formation assay. We found that micronucleus formation rate directly depends on the plasma exposure time. It is also shown that colony formation capacity of cells has been inhibited by the treatment of plasma at all doses. Cell death and micronucleus formation are shown to be significantly elevated by 120 and 240 s exposure of dielectric barrier discharge plasma.

Delayed luminescence in a multiparameter approach to evaluation and reduction of radiobiological risks

NASA Astrophysics Data System (ADS)

Grasso, Rosaria; Cammarata, Francesco Paolo; Minafra, Luigi; Marchese, Valentina; Russo, Giorgio; Manti, Lorenzo; Musumeci, Francesco; Scordino, Agata

2017-07-01

In the framework of the research project ETHICS "Pre-clinical experimental and theoretical studies to improve treatment and protection by charged particles" funded by the National Nuclear Physics Institute, Italy, we studied the phenomenon called delayed luminescence emitted by non-tumorigenic breast epithelial MCF10A cell line after proton irradiation at different doses (0.5, 2, 6, 9 Gy). The aim is to found possible correlations between delayed luminescence and in vitro damaging induced by ion irradiation. The first results of this research show that the delayed luminescence kinetics is proton dose dependent. An interesting correlation between delayed luminescence and clonogenic potential was observed.

Measuring and Perceiving Changes in Oral Complexity, Accuracy and Fluency: Examining Instructed Learners' Short-Term Gains

ERIC Educational Resources Information Center

Tonkyn, Alan Paul

2012-01-01

This paper reports a case study of the nature and extent of progress in speaking skills made by a group of upper intermediate instructed learners, and also assessors' perceptions of that progress. Initial and final interview data were analysed using several measures of Grammatical and Lexical Complexity, Language Accuracy and Fluency. These…

Research, Development and Validation of the Daily Demand Computer Schedule 360/50. Final Report.

ERIC Educational Resources Information Center

Ovard, Glen F.; Rowley, Vernon C.

A study was designed to further the research, development and validation of the Daily Demand Computer Schedule (DDCS), a system by which students can be rescheduled daily for facilitating their individual continuous progress through the curriculum. It will allow teachers to regroup students as needed based upon that progress, and will make time a…

Research in the Theory of Condensed Matter and Elementary Particles: Final Report, September 1, 1984 - November 30, 1987

DOE R&D Accomplishments Database

Friedan, D.; Kadanoff, L.; Nambu, Y.; Shenker, S.

1988-04-01

Progress is reported in the field of condensed matter physics in the area of two-dimensional critical phenomena, specifically results allowing complete classification of all possible two-dimensional critical phenomena in a certain domain. In the field of high energy physics, progress is reported in string and conformal field theory, and supersymmetry.

National Assessment of Educational Progress Grade 12 Preparedness Research College Course Content Analysis Study: Final Report

ERIC Educational Resources Information Center

Educational Policy Improvement Center, 2014

2014-01-01

The National Assessment Governing Board is an independent, bipartisan organization that sets policy for the National Assessment of Educational Progress (NAEP). The Governing Board established the NAEP Program of 12th Grade Preparedness Research to assess what NAEP can report on the academic preparedness of 12th grade students entering college and…

Using Curriculum Based Measures To Identify and Monitor Progress in an Adult Basic Education Program. Final Report.

ERIC Educational Resources Information Center

Bean, Rita M.; And Others

The purpose of a project was to develop and test curriculum-based procedures and measures to monitor and assess the reading and writing progress of adults in a basic education program. The most efficient, reliable, and feasible measure of reading performance from beginning reading level through eighth-grade level was the repeated oral reading…

Abiraterone acetate for patients with metastatic castration-resistant prostate cancer progressing after chemotherapy: final analysis of a multicentre, open-label, early-access protocol trial.

PubMed

Sternberg, Cora N; Castellano, Daniel; Daugaard, Gedske; Géczi, Lajos; Hotte, Sebastien J; Mainwaring, Paul N; Saad, Fred; Souza, Ciro; Tay, Miah H; Garrido, José M Tello; Galli, Luca; Londhe, Anil; De Porre, Peter; Goon, Betty; Lee, Emma; McGowan, Tracy; Naini, Vahid; Todd, Mary B; Molina, Arturo; George, Daniel J

2014-10-01

In the final analysis of the phase 3 COU-AA-301 study, abiraterone acetate plus prednisone significantly prolonged overall survival compared with prednisone alone in patients with metastatic castration-resistant prostate cancer progressing after chemotherapy. Here, we present the final analysis of an early-access protocol trial that was initiated after completion of COU-AA-301 to enable worldwide preapproval access to abiraterone acetate in patients with metastatic castration-resistant prostate cancer progressing after chemotherapy. We did a multicentre, open-label, early-access protocol trial in 23 countries. We enrolled patients who had metastatic castration-resistant prostate cancer progressing after taxane chemotherapy. Participants received oral doses of abiraterone acetate (1000 mg daily) and prednisone (5 mg twice a day) in 28-day cycles until disease progression, development of sustained side-effects, or abiraterone acetate becoming available in the respective country. The primary outcome was the number of adverse events arising during study treatment and within 30 days of discontinuation. Efficacy measures (time to prostate-specific antigen [PSA] progression and time to clinical progression) were gathered to guide treatment decisions. We included in our analysis all patients who received at least one dose of abiraterone acetate. This study is registered with ClinicalTrials.gov, number NCT01217697. Between Nov 17, 2010, and Sept 30, 2013, 2314 patients were enrolled into the early-access protocol trial. Median follow-up was 5·7 months (IQR 3·5-10·6). 952 (41%) patients had a grade 3 or 4 treatment-related adverse event, and grade 3 or 4 serious adverse events were recorded in 585 (25%) people. The most common grade 3 and 4 adverse events were hepatotoxicity (188 [8%]), hypertension (99 [4%]), cardiac disorders (52 [2%]), osteoporosis (31 [1%]), hypokalaemia (28 [1%]), and fluid retention or oedema (23 [1%]). 172 (7%) patients discontinued the study because of adverse events (64 [3%] were drug-related), as assessed by the investigator, and 171 (7%) people died. The funder assessed causes of death, which were due to disease progression (85 [4%]), an unrelated adverse experience (72 [3%]), and unknown reasons (14 [1%]). Of the 86 deaths not attributable to disease progression, 18 (<1%) were caused by a drug-related adverse event, as assessed by the investigator. Median time to PSA progression was 8·5 months (95% CI 8·3-9·7) and median time to clinical progression was 12·7 months (11·8-13·8). No new safety signals or unexpected adverse events were found in this early-access protocol trial to assess abiraterone acetate for patients with metastatic castration-resistant prostate cancer who progressed after chemotherapy. Future work is needed to ascertain the most effective regimen of abiraterone acetate to optimise patients' outcomes. Janssen Research & Development. Copyright © 2014 Elsevier Ltd. All rights reserved.

Tissue polypeptide-specific antigen (TPS) determinations before and during intermittent maximal androgen blockade in patients with metastatic prostatic carcinoma.

PubMed

Kil, P J M; Goldschmidt, H M J; Wieggers, B J A; Kariakine, O B; Studer, U E; Whelan, P; Hetherington, J; de Reijke, Th M; Hoekstra, J W; Collette, L

2003-01-01

To evaluate the prognostic significance of serially measured tissue polypeptide-specific antigen (TPS) levels in patients with metastatic prostatic carcinoma treated with intermittent maximal androgen blockade (MAB). To determine its value with respect to predicting response to treatment and time to clinical progression. Finally to compare TPS with prostate-specific antigen (PSA) measurements in terms of prognostic impact in patients with metastatic prostatic carcinoma. TPS and PSA measurements were performed before start of and monthly during intermittent MAB in 68 patients participating in EORTC protocol 30954. Both TPS and PSA were measured in serum. Fifty-six patients from eight centers were included in the final analysis because at least three TPS values were available. TPS and PSA values were correlated with clinical course of the disease. Median follow-up was 21.3 months. Three patient groups were defined on clinical grounds: (a) clinically progressive disease (n=18); (b) clinically stable disease (n=33); and (c) patients who did not reach a predefined nadir PSA value following 9 months of treatment (n=5). Pretreatment TPS was significantly higher in the clinically progressive patients than in the other patient groups (p=0.0041). When grouping patients according to their pretreatment TPS values (cut-off value of 100 U/l) the pretreatment TPS value (>100 U/l) proved to be a statistically significant prognostic factor with respect to time to progression: elevated TPS was associated with a 3.8 increased risk for progressive disease (p=0.0055). Pretreatment PSA (>100 ng/ml) was of no prognostic value for time to progression. In five patients increase of TPS coincided with or preceded clinical progression during treatment, whereas PSA remained normal. Additional value of pretreatment TPS measurements in metastatic prostate cancer patients is found in defining the patients with rapid clinical progression. Following MAB an increase in TPS signifies clinical progression even if PSA is found to remain normal.

Monitoring the Hazards of Silicic Volcanoes with Remote Sensing

NASA Technical Reports Server (NTRS)

Fink, Jonathan; Wessels, Rick; Eisinger, Chris; Ramsey, Michael; Hellman, Melanie; Kuhn, Sally

2004-01-01

This report details the final progress on the Solid Earth and Natural Hazards project: Monitoring of Hazards of Silicic Volcanoes with Remote Sensing (SENH99-0000-0159). The original award went to Arizona State University (ASU) with Dr. Jonathan Fink as the P.I. and Dr. Michael Ramsey as the Co-I. In May 2000, Dr. Ramsey left ASU to take a tenure-track faculty position at the University of Pittsburgh. The principle investigators and NASA Headquarters agreed to split the grant award at the HQ level and therefore avoid the double overhead charges that would arise from a university subcontract. The objectives of the science were divided, and coordinated yearly progress reports have been submitted from each University. This report details the final progress on work carried out at Arizona State. A report by Dr. Ramsey at the University of Pittsburgh has already been submitted. The work from both institutions is closely related and this report will reflect that connection.

Final Progress Report for Collaborative Research: Aging of Black Carbon during Atmospheric Transport: Understanding Results from the DOE’s 2010 CARES and 2012 ClearfLo Campaigns

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

Mazzoleni, Claudio; Subramanian, R.

2016-08-31

Over the course of this project, we have analyzed data and samples from the Carbonaceous Aerosol and Radiative Effects Study (CARES) and the Clear air for London (ClearfLo) campaign, as well as conducted or participated in laboratory experiments designed to better understand black carbon mixing state and climate-relevant properties. The laboratory campaigns took place at the Pacific Northwest National Laboratory and Carnegie Mellon University to study various climate-relevant aerosol properties of different sources of soot mixing with secondary organic aerosol precursors. Results from some of these activities were summarized in the previous progress report. This final report presents the manuscriptsmore » that have been published (many in the period since the last progress report), lists presentations at different conferences based on grant-related activities, and presents some results that are likely to be submitted for publication in the near future.« less

A program for undergraduate research into the mechanisms of sensory coding and memory decay

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

Calin-Jageman, R J

This is the final technical report for this DOE project, entitltled "A program for undergraduate research into the mechanisms of sensory coding and memory decay". The report summarizes progress on the three research aims: 1) to identify phyisological and genetic correlates of long-term habituation, 2) to understand mechanisms of olfactory coding, and 3) to foster a world-class undergraduate neuroscience program. Progress on the first aim has enabled comparison of learning-regulated transcripts across closely related learning paradigms and species, and results suggest that only a small core of transcripts serve truly general roles in long-term memory. Progress on the second aimmore » has enabled testing of several mutant phenotypes for olfactory behaviors, and results show that responses are not fully consistent with the combinitoral coding hypothesis. Finally, 14 undergraduate students participated in this research, the neuroscience program attracted extramural funding, and we completed a successful summer program to enhance transitions for community-college students into 4-year colleges to persue STEM fields.« less

Progressive Vector Quantization on a massively parallel SIMD machine with application to multispectral image data

NASA Technical Reports Server (NTRS)

Manohar, Mareboyana; Tilton, James C.

1994-01-01

A progressive vector quantization (VQ) compression approach is discussed which decomposes image data into a number of levels using full search VQ. The final level is losslessly compressed, enabling lossless reconstruction. The computational difficulties are addressed by implementation on a massively parallel SIMD machine. We demonstrate progressive VQ on multispectral imagery obtained from the Advanced Very High Resolution Radiometer instrument and other Earth observation image data, and investigate the trade-offs in selecting the number of decomposition levels and codebook training method.

Validation of Technical Recommendations. Final Report. ISSOE Managing Student Progress.

ERIC Educational Resources Information Center

Ridley, Dennis; And Others

This report is organized to provide a complete, logical presentation of the major steps taken in the third phase of research on dynamics of dissemination of the Instructional Support System for Occupational Education (ISSOE). It reports on the confirmation and validation of the Phase II Final Report on Dissemination Issues and the Phase III…

77 FR 26316 - Progress Energy Florida; Final Environmental Impact Statement for Combined Licenses for Levy...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-03

..., Rockville, Maryland, 20852 or from NRC's Agencywide Documents Access and Management System (ADAMS). ADAMS is accessible from the NRC's Web site at www.nrc.gov/reading-rm/adams.html . The ADAMS accession numbers for the... . In addition, the following four public libraries have agreed to make the final EIS available to the...

Beyond Therapy Dogs: Coordinating Large-Scale Finals Week Activities

ERIC Educational Resources Information Center

Flynn, Holly

2017-01-01

Finals week activities have become increasingly popular in academic libraries in the last few years, but what is a library to do when it is not allowed to have therapy dogs? This column examines a progression of increasingly popular activities at Michigan State University Libraries. Included is an assessment of what makes them popular, our…

Navigational Strategies of Migrating Monarch Butterflies

DTIC Science & Technology

2014-11-10

AFRL-OSR-VA-TR-2014-0339 NAVIGATIONAL STRATEGIES OF MIGRATING MONARCH BUTTERFLIES Steven Reppert UNIVERSITY OF MASSACHUSETTS Final Report 11/10/2014...Final Progress Statement to (Dr. Patrick Bradshaw) Contract/Grant Title: Navigational Strategies of Migrating Monarch Butterflies Contract...Grant #: FA9550-10-1-0480 Reporting Period: 01-Sept-10 to 31-Aug-14 Overview of accomplishments: Migrating monarch butterflies (Danaus

Application of Communications Satellites to Educational Development. Final Technical Report, September 1, 1969-August 31, 1975.

ERIC Educational Resources Information Center

Morgan, Robert P.

Research is summarized in a brief final report built around a four-section bibliography. The first section lists periodic progress reports and articles which provide an overview of the program, including articles which pertain primarily to educational rather than technical aspects of satellite utilization. Theses carried out in the fields of…

Hydroxychavicol, a betel leaf component, inhibits prostate cancer through ROS-driven DNA damage and apoptosis.

PubMed

Gundala, Sushma Reddy; Yang, Chunhua; Mukkavilli, Rao; Paranjpe, Rutugandha; Brahmbhatt, Meera; Pannu, Vaishali; Cheng, Alice; Reid, Michelle D; Aneja, Ritu

2014-10-01

Dietary phytochemicals are excellent ROS-modulating agents and have been shown to effectively enhance ROS levels beyond toxic threshold in cancer cells to ensure their selective killing while leaving normal cells unscathed. Here we demonstrate that hydroxychavicol (HC), extracted and purified from Piper betel leaves, significantly inhibits growth and proliferation via ROS generation in human prostate cancer, PC-3 cells. HC perturbed cell-cycle kinetics and progression, reduced clonogenicity and mediated cytotoxicity by ROS-induced DNA damage leading to activation of several pro-apoptotic molecules. In addition, HC treatment elicited a novel autophagic response as evidenced by the appearance of acidic vesicular organelles and increased expression of autophagic markers, LC3-IIb and beclin-1. Interestingly, quenching of ROS with tiron, an antioxidant, offered significant protection against HC-induced inhibition of cell growth and down regulation of caspase-3, suggesting the crucial role of ROS in mediating cell death. The collapse of mitochondrial transmembrane potential by HC further revealed the link between ROS generation and induction of caspase-mediated apoptosis in PC-3 cells. Our data showed remarkable inhibition of prostate tumor xenografts by ~72% upon daily oral administration of 150mg/kg bw HC by quantitative tumor volume measurements and non-invasive real-time bioluminescent imaging. HC was well-tolerated at this dosing level without any observable toxicity. This is the first report to demonstrate the anti-prostate cancer efficacy of HC in vitro and in vivo, which is perhaps attributable to its selective prooxidant activity to eliminate cancer cells thus providing compelling grounds for future preclinical studies to validate its potential usefulness for prostate cancer management. Copyright © 2014 Elsevier Inc. All rights reserved.

2'-Hydroxyflavanone: A novel strategy for targeting breast cancer.

PubMed

Singhal, Jyotsana; Nagaprashantha, Lokesh; Chikara, Shireen; Awasthi, Sanjay; Horne, David; Singhal, Sharad S

2017-09-26

Breast cancer is the most common cancer in women that is driven by cross-talk with hormonal and cellular signaling pathways. The natural phytochemicals, due to broad-spectrum anti-inflammatory and anti-cancerous properties, present with novel opportunities for targeting breast cancer. Intake of citrus fruits is known to reduce the risk for incidence of breast cancer. Hence, we tested the efficacy of citrus flavonoid 2'-hydroxyflavanone (2HF) in breast cancer. 2HF inhibited survival, clonogenic ability, cell cycle progression and induced apoptosis in breast cancer cells. 2HF also decreased VEGF levels and inhibited migratory capacity of breast cancer cells. Administration of 2HF led to regression of triple-negative MDA-MB-231 tumors in the mice xenograft model. 2HF decreased the levels of RLIP76 both in vitro studies and in vivo MDA-MB-231 xenograft model of breast cancer. Western blot and histopathological analyses of resected tumors showed a decline in the levels of survival and proliferation markers Ki67, pAkt, survivin, and cell cycle proteins CDK4 and cyclin B1. 2HF treatment led to inhibition of angiogenesis as determined by decreased VEGF levels in vitro and angiogenesis marker CD31 in vivo . 2HF reversed the pro-/anti-apoptotic ratio of BAX/BCL-2 by decreasing anti-apoptotic protein BCL-2 and increasing pro-apoptotic proteins BAX and BIM in vivo . 2HF also decreased the mesenchymal markers vimentin and fibronectin along with causing a parallel increase in pro-differentiation protein E-cadherin. Collectively, the ability of 2HF to decrease RLIP76, VEGF and regulate critical proliferative, apoptotic and differentiation proteins together provides strong rationale to further develop 2HF based interventions for targeting breast cancer.

Synergistic Antiproliferative Effects of a New Cucurbitacin B Derivative and Chemotherapy Drugs on Lung Cancer Cell Line A549.

PubMed

Marostica, Lucas Lourenço; Silva, Izabella Thaís; Kratz, Jadel Müller; Persich, Lara; Geller, Fabiana Cristina; Lang, Karen Luise; Caro, Miguel Soriano Balparda; Durán, Fernando Javier; Schenkel, Eloir Paulo; Simões, Cláudia Maria Oliveira

2015-10-19

Nonsmall cell lung cancer (NSCLC) represents an important cause of mortality worldwide due to its aggressiveness and growing resistance to currently available therapy. Cucurbitacins have emerged as novel potential anticancer agents showing strong antiproliferative effects and can be promising candidates for combined treatments with clinically used anticancer agents. This study investigates the synergistic antiproliferative effects of a new semisynthetic derivative of cucurbitacin B (DACE) with three chemotherapy drugs: cisplatin (CIS), irinotecan (IRI), and paclitaxel (PAC) on A549 cells. The most effective combinations were selected for studies of the mechanism of action. Using an in silico tool, DACE seems to act by a different mechanism of action when compared with that of different classes of drugs already used in clinical settings. DACE also showed potent synergic effects with drugs, and the most potent combinations induced G2/M cell cycle arrest by modulating survivin and p53 expression, disruption of F-actin cytoskeleton, and cell death by apoptosis. These treatments completely inhibited the clonogenic potential and did not reduce the proliferation of nontumoral lung cells (MRC-5). DACE also showed relevant antimigratory and anti-invasive effects, and combined treatments modulated cell migration signaling pathways evolved with metastasis progression. The effects of DACE associated with drugs was potentiated by the oxidant agent l-buthionine-sulfoximine (BSO), and attenuated by N-acetilcysteine (NAC), an antioxidant agent. The antiproliferative effects induced by combined treatments were attenuated by a pan-caspase inhibitor, indicating that the effects of these treatments are dependent on caspase activity. Our data highlight the therapeutic potential of DACE used in combination with known chemotherapy drugs and offer important insights for the development of more effective and selective therapies against lung cancer.

DNA Polymerase β as a Novel Target for Chemotherapeutic Intervention of Colorectal Cancer

PubMed Central

Jaiswal, Aruna S.; Banerjee, Sanjeev; Aneja, Ritu; Sarkar, Fazlul H.; Ostrov, David A.; Narayan, Satya

2011-01-01

Chemoprevention presents a major strategy for the medical management of colorectal cancer. Most drugs used for colorectal cancer therapy induce DNA-alkylation damage, which is primarily repaired by the base excision repair (BER) pathway. Thus, blockade of BER pathway is an attractive option to inhibit the spread of colorectal cancer. Using an in silico approach, we performed a structure-based screen by docking small-molecules onto DNA polymerase β (Pol-β) and identified a potent anti-Pol-β compound, NSC-124854. Our goal was to examine whether NSC-124854 could enhance the therapeutic efficacy of DNA-alkylating agent, Temozolomide (TMZ), by blocking BER. First, we determined the specificity of NSC-124854 for Pol-β by examining in vitro activities of APE1, Fen1, DNA ligase I, and Pol-β-directed single nucleotide (SN)- and long-patch (LP)-BER. Second, we investigated the effect of NSC-124854 on the efficacy of TMZ to inhibit the growth of mismatch repair (MMR)-deficient and MMR-proficient colon cancer cell lines using in vitro clonogenic assays. Third, we explored the effect of NSC-124854 on TMZ-induced in vivo tumor growth inhibition of MMR-deficient and MMR-proficient colonic xenografts implanted in female homozygous SCID mice. Our data showed that NSC-124854 has high specificity to Pol-β and blocked Pol-β-directed SN- and LP-BER activities in in vitro reconstituted system. Furthermore, NSC-124854 effectively induced the sensitivity of TMZ to MMR-deficient and MMR-proficient colon cancer cells both in vitro cell culture and in vivo xenograft models. Our findings suggest a potential novel strategy for the development of highly specific structure-based inhibitor for the prevention of colonic tumor progression. PMID:21311763

The pro-apoptotic and anti-invasive effects of hypericin-mediated photodynamic therapy are enhanced by hyperforin or aristoforin in HT-29 colon adenocarcinoma cells.

PubMed

Šemeláková, Martina; Mikeš, Jaromír; Jendželovský, Rastislav; Fedoročko, Peter

2012-12-05

Photodynamic therapy is a rapidly-developing anti-cancer approach for the treatment of various types of malignant as well as non-malignant diseases. In this study, hypericin-mediated photodynamic therapy (HY-PDT) in sub-optimal dose was combined with hyperforin (HP) or its stable derivative aristoforin (AR) in an effort to improve efficacy on the cellular level. The logic of this combination is based on the fact that both bioactive compounds naturally occur in plants of Hypericum sp. At relatively low concentrations up to 5 μM, hyperforin and aristoforin were able to stimulate onset of apoptosis in HT-29 colon adenocarcinoma cells exposed to HY-PDT, inhibit cell cycle progression, suppress expression of matrixmetalloproteinases-2/-9 together with cell adhesivity, thereby affecting the clonogenic potential of the cells. As the action of aristoforin was more pronounced, in line with our assumption, these changes were also linked in this case with hypericin accumulation and increased ROS generation leading to dissipation of mitochondrial membrane potential in a significant portion of the cells, as well as activation of caspase-3. Comparison of HT-29 cells to another colon adenocarcinoma-derived cell line HCT-116 demonstrated significant differences in sensitivity of different cell lines to PDT, however, accumulated effect of HY-PDT with HP/AR proved similar in both tested cell lines. The presented data may help to elucidate the mechanisms of action for different bioactive constituents of St. John's wort, which are increasingly recognized as being able to regulate a variety of pathobiological processes, thus possessing potential therapeutic properties. Copyright © 2012 Elsevier B.V. All rights reserved.

Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

PubMed Central

Sahara, Makoto; Hansson, Emil M; Wernet, Oliver; Lui, Kathy O; Später, Daniela; Chien, Kenneth R

2014-01-01

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC+CD31+CD34+CD14−KDRhigh endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR+ mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs. PMID:24810299

FANCD2 monoubiquitination and activation by hexavalent chromium [Cr(VI)] exposure

PubMed Central

Vilcheck, Susan K.; Ceryak, Susan; O’Brien, Travis J.; Patierno, Steven R.

2007-01-01

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by congenital abnormalities, progressive bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA crosslinking agents. FA is a genetically heterogeneous disease with at least 11 complementation groups. The eight cloned FA proteins interact in a common pathway with established DNA-damage-response proteins, including BRCA1 and ATM. Six FA proteins (A, C, E, F, G, and L) regulate the monoubiquitination of FANCD2 after DNA damage by crosslinking agents, which targets FANCD2 to BRCA1 nuclear foci containing BRCA2 (FANCD1) and RAD51. Some forms of hexavalent chromium [Cr(VI)] are implicated as respiratory carcinogens and induce several types of DNA lesions, including DNA interstrand crosslinks. We have shown that FA-A fibroblasts are hypersensitive to both Cr(VI)-induced apoptosis and clonogenic lethality. Here we show that Cr(VI) treatment induced monoubiquitination of FANCD2 in normal human fibroblasts, providing the first molecular evidence of Cr(VI)-induced activation of the FA pathway. FA-A fibroblasts demonstrated no FANCD2 monoubiquitination, in keeping with the requirement of FA-A for this modification. We also found that Cr(VI) treatment induced significantly more S-phase-dependent DNA double strand breaks (DSBs), as measured by γ-H2AX expression, in FA-A fibroblasts compared to normal cells. However, and notably, DSBs were repaired equally in both normal and FA-A fibroblasts during recovery from Cr(VI) treatment. While previous research on FA has defined the genetic causes of this disease, it is critical in terms of individual risk assessment to address how cells from FA patients respond to genotoxic insult. PMID:16893675

The MET/AXL/FGFR Inhibitor S49076 Impairs Aurora B Activity and Improves the Antitumor Efficacy of Radiotherapy.

PubMed

Clémenson, Céline; Chargari, Cyrus; Liu, Winchygn; Mondini, Michele; Ferté, Charles; Burbridge, Mike F; Cattan, Valérie; Jacquet-Bescond, Anne; Deutsch, Eric

2017-10-01

Several therapeutic agents targeting HGF/MET signaling are under clinical development as single agents or in combination, notably with anti-EGFR therapies in non-small cell lung cancer (NSCLC). However, despite increasing data supporting a link between MET, irradiation, and cancer progression, no data regarding the combination of MET-targeting agents and radiotherapy are available from the clinic. S49076 is an oral ATP-competitive inhibitor of MET, AXL, and FGFR1-3 receptors that is currently in phase I/II clinical trials in combination with gefitinib in NSCLC patients whose tumors show resistance to EGFR inhibitors. Here, we studied the impact of S49076 on MET signaling, cell proliferation, and clonogenic survival in MET-dependent (GTL16 and U87-MG) and MET-independent (H441, H460, and A549) cells. Our data show that S49076 exerts its cytotoxic activity at low doses on MET-dependent cells through MET inhibition, whereas it inhibits growth of MET-independent cells at higher but clinically relevant doses by targeting Aurora B. Furthermore, we found that S49076 improves the antitumor efficacy of radiotherapy in both MET-dependent and MET-independent cell lines in vitro and in subcutaneous and orthotopic tumor models in vivo In conclusion, our study demonstrates that S49076 has dual antitumor activity and can be used in combination with radiotherapy for the treatment of both MET-dependent and MET-independent tumors. These results support the evaluation of combined treatment of S49076 with radiation in clinical trials without patient selection based on the tumor MET dependency status. Mol Cancer Ther; 16(10); 2107-19. ©2017 AACR . ©2017 American Association for Cancer Research.

Hydroxychavicol, a betel leaf component, inhibits prostate cancer through ROS-driven DNA damage and apoptosis

PubMed Central

Gundala, Sushma Reddy; Yang, Chunhua; Mukkavilli, Rao; Paranjpe, Rutugandha; Brahmbhatt, Meera; Pannu, Vaishali; Cheng, Alice; Reid, Michelle D.; Aneja, Ritu

2015-01-01

Dietary phytochemicals are excellent ROS-modulating agents and have been shown to effectively enhance ROS levels beyond toxic threshold in cancer cells to ensure their selective killing while leaving normal cells unscathed. Here we demonstrate that hydroxychavicol (HC), extracted and purified from Piper betel leaves, significantly inhibits growth and proliferation via ROS generation in human prostate cancer, PC-3 cells. HC perturbed cell-cycle kinetics and progression, reduced clonogenicity and mediated cytotoxicity by ROS-induced DNA damage leading to activation of several pro-apoptotic molecules. In addition, HC treatment elicited a novel autophagic response as evidenced by the appearance of acidic vesicular organelles and increased expression of autophagic markers, LC3-IIb and beclin-1. Interestingly, quenching of ROS with tiron, an antioxidant, offered significant protection against HC-induced inhibition of cell growth and down regulation of caspase-3, suggesting the crucial role of ROS in mediating cell death. The collapse of mitochondrial transmembrane potential by HC further revealed the link between ROS generation and induction of caspase-mediated apoptosis in PC-3 cells. Our data showed remarkable inhibition of prostate tumor xenografts by ~72% upon daily oral administration of 150 mg/kg bw HC by quantitative tumor volume measurements and non-invasive real-time bioluminescent imaging. HC was well-tolerated at this dosing level without any observable toxicity. This is the first report to demonstrate the anti-prostate efficacy of HC in vitro and in vivo, which is perhaps attributable to its selective prooxidant activity to eliminate cancer cells thus providing compelling grounds for future preclinical studies to validate its potential usefulness for prostate cancer management. PMID:25064160

Transition in Survival From Low-Dose Hyper-Radiosensitivity to Increased Radioresistance Is Independent of Activation of ATM SER1981 Activity

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

Krueger, Sarah A.; Collis, Spencer J.; Joiner, Michael C.

2007-11-15

Purpose: The molecular basis of low-dose hyper-radiosensitivity (HRS) is only partially understood. The aim of this study was to define the roles of ataxia telangiectasia mutated (ATM) activity and the downstream ATM-dependent G{sub 2}-phase cell cycle checkpoint in overcoming HRS and triggering radiation resistance. Methods and Materials: Survival was measured using a high-resolution clonogenic assay. ATM Ser1981 activation was measured by Western blotting. The role of ATM was determined in survival experiments after molecular (siRNA) and chemical (0.4 mM caffeine) inhibition and chemical (20 {mu}g/mL chloroquine, 15 {mu}M genistein) activation 4-6 h before irradiation. Checkpoint responsiveness was assessed in eightmore » cell lines of differing HRS status using flow cytometry to quantify the progression of irradiated (0-2 Gy) G{sub 2}-phase cells entering mitosis, using histone H3 phosphorylation analysis. Results: The dose-response pattern of ATM activation was concordant with the transition from HRS to radioresistance. However, ATM activation did not play a primary role in initiating increased radioresistance. Rather, a relationship was discovered between the function of the downstream ATM-dependent early G{sub 2}-phase checkpoint and the prevalence and overcoming of HRS. Four cell lines that exhibited HRS failed to show low-dose (<0.3-Gy) checkpoint function. In contrast, four HRS-negative cell lines exhibited immediate cell cycle arrest for the entire 0-2-Gy dose range. Conclusion: Overcoming HRS is reliant on the function of the early G{sub 2}-phase checkpoint. These data suggest that clinical exploitation of HRS could be achieved by combining radiotherapy with chemotherapeutic agents that modulate this cell cycle checkpoint.« less

miR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27Kip1.

PubMed

Galardi, Silvia; Mercatelli, Neri; Giorda, Ezio; Massalini, Simone; Frajese, Giovanni Vanni; Ciafrè, Silvia Anna; Farace, Maria Giulia

2007-08-10

MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of several types of cancers. Here we show that miR-221 and miR-222, encoded in tandem on chromosome X, are overexpressed in the PC3 cellular model of aggressive prostate carcinoma, as compared with LNCaP and 22Rv1 cell line models of slowly growing carcinomas. In all cell lines tested, we show an inverse relationship between the expression of miR-221 and miR-222 and the cell cycle inhibitor p27(Kip1). We recognize two target sites for the microRNAs in the 3' untranslated region of p27 mRNA, and we show that miR-221/222 ectopic overexpression directly results in p27 down-regulation in LNCaP cells. In those cells, we demonstrate that the ectopic overexpression of miR-221/222 strongly affects their growth potential by inducing a G(1) to S shift in the cell cycle and is sufficient to induce a powerful enhancement of their colony-forming potential in soft agar. Consistently, miR-221 and miR-222 knock-down through antisense LNA oligonucleotides increases p27(Kip1) in PC3 cells and strongly reduces their clonogenicity in vitro. Our results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27(Kip1), and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27(Kip1) down-regulation.

Effects of TGF-β signaling blockade on human A549 lung adenocarcinoma cell lines.

PubMed

Xu, Cheng-Cheng; Wu, Lei-Ming; Sun, Wei; Zhang, Ni; Chen, Wen-Shu; Fu, Xiang-Ning

2011-01-01

Transforming growth factor β (TGF-β) is overexpressed in a wide variety of cancer types including lung adenocarcinoma (LAC), and the TGF-β signaling pathway plays an important role in tumor development. To determine whether blockade of the TGF-β signaling pathway can inhibit the malignant biological behavior of LAC, RNA interference (RNAi) technology was used to silence the expression of TGF-β receptor, type II (TGFβRII) in the LAC cell line, A549, and its effects on cell proliferation, invasion and metastasis were examined. Three specific small interfering RNAs (siRNAs) designed for targeting human TGFβRII were transfected into A549 cells. The expression of TGFβRII was detected by Western blot analysis. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The invasion and metastasis of A549 cells were investigated using the wound healing and Matrigel invasion assays. The expression of PI3K, phosphorylated Smad2, Smad4, Akt, Erk1/2, P38 and MMPs was detected by Western blot analysis. The TGFβRII siRNA significantly reduced the expression of TGFβRII in A549 cells. The knockdown of TGFβRII in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis. In addition to the Smad-dependent pathway, independent pathways including the Erk MAPK, PI3K/Akt and p38 MAPK pathways, as well as the expression of MMPs and VEGF, were inhibited. In conclusion, TGF-β signaling is required for LAC progression. Therefore, the blockade of this signaling pathway by the down-regulation of TGFβRII using SiRNA may provide a potential gene therapy for LAC.

SIRT1 deacetylase is overexpressed in human melanoma and its small molecule inhibition imparts anti-proliferative response via p53 activation.

PubMed

Wilking, Melissa J; Singh, Chandra; Nihal, Minakshi; Zhong, Weixiong; Ahmad, Nihal

2014-12-01

Melanoma causes more deaths than any other skin cancer, and its incidence in the US continues to rise. Current medical therapies are insufficient to control this deadly neoplasm, necessitating the development of new target-based approaches. The objective of this study was to determine the role and functional significance of the class III histone deacetylase SIRT1 in melanoma. We have found that SIRT1 is overexpressed in clinical human melanoma tissues and human melanoma cell lines (Sk-Mel-2, WM35, G361, A375, and Hs294T) compared to normal skin and normal melanocytes, respectively. In addition, treatment of melanoma cell lines A375, Hs294T, and G361 with Tenovin-1, a small molecule SIRT1 inhibitor, resulted in a significant decrease in cell growth and cell viability. Further, Tenovin-1 treatment also resulted in a marked decrease in the clonogenic survival of melanoma cells. Further experiments showed that the anti-proliferative response of Tenovin-1 was accompanied by an increase in the protein as well as activity of the tumor suppressor p53. This increase in p53 activity was substantiated by an increase in the protein level of its downstream target p21. Overall, these data suggest that small molecule inhibition of SIRT1 causes anti-proliferative effects in melanoma cells. SIRT1 appears to be acting through the activity of the tumor suppressor p53, which is not mutated in the majority of melanomas. However, future detailed studies are needed to further explore the role and mechanism of SIRT1 in melanoma development and progression and its usefulness in melanoma treatment.

Specific telomere dysfunction induced by GRN163L increases radiation sensitivity in breast cancer cells

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

Gomez-Millan, Jaime; Goldblatt, Erin M.; Gryaznov, Sergei M.

Purpose: Telomerase is expressed in 80-90% of tumor cells, but is absent in most somatic cells. The absence of telomerase activity results in progressive telomere shortening, leading to cellular senescence or death through deoxyribonucleic acid (DNA) damage signals. In addition, a role for telomerase in DNA damage repair has also been suggested. A specific telomerase inhibitor, GRN163L that is complementary to the template region of the telomerase ribonucleic acid component (hTR). We hypothesized that exposure to GRN163L, either through immediate inhibition of telomerase activity or through eventual telomere shortening and dysfunction, may enhance radiation sensitivity. Our goal was to testmore » whether the treatment with GRN163L enhances sensitivity to irradiation (IR) in MDA-MB-231 breast cancer cells. Methods and Materials: The MDA-MB-231 breast cancer cells were treated with or without GRN163L for 2-42 days. Inhibition of telomerase activity and shortening of telomeres were confirmed. Cells were then irradiated and clonogenic assays were performed to show cell survival differences. In vivo studies using MDA-MB-231 xenografts were performed to corroborate the in vitro results. Results: We show that cells with shortened telomeres due to GRN163L enhance the effect on IR reducing survival by an additional 30% (p < 0.01). These results are confirmed in vivo, with a significant decrease in tumor growth in mice exposed to GRN163L. Conclusions: We found that GRN163L is a promising adjuvant treatment in combination with radiation therapy that may improve the therapeutic index by enhancing the radiation sensitivity. These studies prompt further investigation as to whether this combination can be applied to other cancers and the clinic.« less

Dasatinib synergises with irinotecan to suppress hepatocellular carcinoma via inhibiting the protein synthesis of PLK1.

PubMed

Xu, Li; Zhu, Yuanrun; Shao, Jinjin; Chen, Min; Yan, Hao; Li, Guanqun; Zhu, Yi; Xu, Zhifei; Yang, Bo; Luo, Peihua; He, Qiaojun

2017-04-11

Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumour and has poor prognosis. Currently, systematic chemotherapy is the only approach to prolong survival. Thus the development of new treatment regimens is urgently needed to improve the therapeutic efficacy. Our study intended to assess the combination of dasatinib and irinotecan against HCC and made an effort to develop a potential medical choice for advanced HCC patients. We used SRB colorimetric assay and clonogenic assay to assess antitumour effect in vitro and HCC xenograft model to assess antitumour effect in vivo. We applied flow cytometry and western blotting to explore the mechanism of the combined therapy. Knockdown and overexpression of PLK1 are also applied for validation. We confirmed that dasatinib has synergistic effect with irinotecan (or SN38) on HCC both in vitro and in vivo. The effect is due to arisen apoptosis rate of HCC cells that is accompanied by mitochondria dysfunction. The enhanced antitumour efficacy of SN38 could be explained by additional inhibition of PLK1, which is triggered by dasatinib. Unlike existed PLK1 inhibitors, dasatinib does not inhibit PLK1 activity in a direct way. Instead, we found that dasatinib reduces PLK1 level by interfering with its protein synthesis progress. We validated that this kind of downregulation of PLK1 level has a key role in the synergistic effect of the two agents. Dasatinib is able to reinforce the anti-HCC efficacy of irinotecan/SN38 by downregulation of PLK1 synthesis. The combination of the two agents might be a potential medical choice for HCC therapy.

A liposomal formulation of the synthetic curcumin analog EF24 (Lipo-EF24) inhibits pancreatic cancer progression: towards future combination therapies.

PubMed

Bisht, Savita; Schlesinger, Martin; Rupp, Alexander; Schubert, Rolf; Nolting, Jens; Wenzel, Jörg; Holdenrieder, Stefan; Brossart, Peter; Bendas, Gerd; Feldmann, Georg

2016-07-11

Pancreatic cancer is one of the most lethal of human malignancies known to date and shows relative insensitivity towards most of the clinically available therapy regimens. 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), a novel synthetic curcumin analog, has shown promising in vitro therapeutic efficacy in various human cancer cells, but insufficient water solubility and systemic bioavailability limit its clinical application. Here, we describe nano-encapsulation of EF24 into pegylated liposomes (Lipo-EF24) and evaluation of these particles in preclinical in vitro and in vivo model systems of pancreatic cancer. Transmission electron microscopy and size distribution studies by dynamic light scattering confirmed intact spherical morphology of the formed liposomes with an average diameter of less than 150 nm. In vitro, treatment with Lipo-EF24 induced growth inhibition and apoptosis in MIAPaCa and Pa03C pancreatic cancer cells as assessed by using cell viability and proliferation assays, replating and soft agar clonogenicity assays as well as western blot analyses. Lipo-EF24 potently suppressed NF-kappaB nuclear translocation by inhibiting phosphorylation and subsequent degradation of its inhibitor I-kappa-B-alpha. In vivo, synergistic tumor growth inhibition was observed in MIAPaCa xenografts when Lipo-EF24 was given in combination with the standard-of-care cytotoxic agent gemcitabine. In line with in vitro observations, western blot analysis revealed decreased phosphorylation of I-kappa-B-alpha in excised Lipo-EF24-treated xenograft tumor tissues. Due to its promising therapeutic efficacy and favorable toxicity profile Lipo-EF24 might be a promising starting point for development of future combinatorial therapeutic regimens against pancreatic cancer.

Transformational Solar Array Final Report

NASA Technical Reports Server (NTRS)

Gaddy, Edward; Ballarotto, Mihaela; Drabenstadt, Christian; Nichols, John; Douglas, Mark; Spence, Brian; Stall, Richard A.; Sulyma, Chris; Sharps, Paul

2017-01-01

We have made outstanding progress in the Base Phase towards achieving the final NASA Research Announcement (NRA) goals. Progress is better than anticipated due to the lighter than predicted mass of the IMM solar cells. We look forward to further improvements in the IMM cell performance during Option I and Option II; so, we have confidence that the first four items listed in the table will improve to better than the NRA goals. The computation of the end of life blanket efficiency is uncertain because we have extrapolated the radiation damage from room temperature measurements. The last three items listed in the Table were not intended to be accomplished during the Base Phase; they will be achieved during Option I and Option II.

Quantum Manybody Physics with Rydberg Polaritons

DTIC Science & Technology

2016-06-22

report, such as final, technical, interim, memorandum, master’s thesis, progress, quarterly, research , special, group study, etc. 3. DATES COVERED...for public release. Over the course of this grant, we have seen tremendous progress, both theoretically and experimentally , in our control of photonic...shown in multiple stages of construction at left and below, spans three optical tables in two rooms: One for the experimental control system

Implementing Program Integrity from an HBCU Perspective: Progress, Problems, and Prospects. An Analysis of Part H, Program Integrity - TRIAD of the Higher Education Act and the Final Rules and Related Interim Final Regulations Published in the "Federal Register" on April 29, 1994.

ERIC Educational Resources Information Center

Blakely, William A.

This paper examines the impact and implications for the nation's 104 historically black colleges and universities (HBCUs) of the final regulations published in the April 29, 1994 "Federal Register" and traces both the legislative and regulatory history of Part H of the Higher Education Act provisions. The analysis addresses specific…

National Ocean Sciences Bowl in 2014: A National Competition for High School Ocean Science Education

DTIC Science & Technology

2015-03-31

the 2014 National Finals Competition. The Finals were held May 1-4, 2014 in Seattle, WA with a theme of ocean acidification . A longitudinal study and...Washington (UW) in Seattle, WA on May 1-4, 2014. The theme for the 2014 Finals Competition was ocean acidification , exploring the progressive increase in...and environmental and societal effects of ocean acidification . They became more aware of ocean acidification’s potential to disrupt ecosystems in a

Process development for automated solar cell and module production. Task 4: Automated array assembly

NASA Technical Reports Server (NTRS)

Hagerty, J. J.

1981-01-01

Progress in the development of automated solar cell and module production is reported. The unimate robot is programmed for the final 35 cell pattern to be used in the fabrication of the deliverable modules. The mechanical construction of the automated lamination station and final assembly station phases are completed and the first operational testing is underway. The final controlling program is written and optimized. The glass reinforced concrete (GRC) panels to be used for testing and deliverables are in production. Test routines are grouped together and defined to produce the final control program.

Status and path to a final EUVL reticle-handling solution

NASA Astrophysics Data System (ADS)

He, Long; Orvek, Kevin; Seidel, Phil; Wurm, Stefan; Underwood, Jon; Betancourt, Ernie

2007-03-01

In extreme ultraviolet lithography (EUVL), the lack of a suitable material to build conventional pellicles calls for industry standardization of new techniques for protection and handling throughout the reticle's lifetime. This includes reticle shipping, robotic handling, in-fab transport, storage, and uses in atmospheric environments for metrology and vacuum environments for EUV exposure. In this paper, we review the status of the industry-wide progress in developing EUVL reticle-handling solutions. We show the industry's leading reticle carrier approaches for particle-free protection, such as improvements in conventional single carrier designs and new EUVL-specific carrier concepts, including variations on a removable pellicle. Our test indicates dual pod approach of the removable pellicle led to nearly particle-free use during a simulated life cycle, at ~50nm inspection sensitivity. We will provide an assessment of the remaining technical challenges facing EUVL reticle-handling technology. Finally, we will review the progress of the SEMI EUVL Reticle-handling Task Force in its efforts to standardize a final EUV reticle protection and handling solution.

More eyes on the prize: variability in White Americans' perceptions of progress toward racial equality.

PubMed

Brodish, Amanda B; Brazy, Paige C; Devine, Patricia G

2008-04-01

Much recent research suggests that Whites and non-Whites think differently about issues of race in contemporary America. For example, Eibach and Ehrlinger (2006) recently demonstrated that Whites perceive that more progress toward racial equality has been made as compared to non-Whites. The authors of this article sought to extend Eibach and Ehrlinger's analysis. To this end, they found that differences in Whites' and non-Whites' perceptions of racial progress can be explained by the reference points they use for understanding progress toward racial equality (Study 1). Furthermore, they demonstrated that there is variability in White people's perceptions of racial progress that can be explained by self-reported racial prejudice (Studies 1 and 2). Finally, they demonstrated that White people's perceptions of racial progress predict reactions to affirmative action (Study 2). Implications for better understanding intergroup relations and reactions to social policies are discussed.

Progressive taxation and the subjective well-being of nations.

PubMed

Oishi, Shigehiro; Schimmack, Ulrich; Diener, Ed

2012-01-01

Using data from the Gallup World Poll, we examined whether progressive taxation is associated with increased levels of subjective well-being. Consistent with Rawls's theory of justice, our results showed that progressive taxation was positively associated with the subjective well-being of nations. However, the overall tax rate and government spending were not associated with the subjective well-being of nations. Furthermore, controlling for the wealth of nations and income inequality, we found that respondents living in a nation with more-progressive taxation evaluated their lives as closer to the best possible life and reported having more positive and less negative daily experiences than did respondents living in a nation with less-progressive taxation. Finally, we found that the association between more-progressive taxation and higher levels of subjective well-being was mediated by citizens' satisfaction with public goods, such as education and public transportation.

Final Progress Report, 1961-1976,

DTIC Science & Technology

1977-06-16

shock. In addition, norepinephrine, prostaglandin E1, and carbachol were used to study changes in myocardial cyclic nucleotides, lipids, lipid turnover and sympathetic-parasympathetic nervous system interaction. (Author)

International electives in the final year of German medical school education--a student's perspective.

PubMed

Ebrahimi-Fakhari, Darius; Agrawal, Mridul; Wahlster, Lara

2014-01-01

The final year of medical school has a unique role for introducing students to their future responsibilities and challenges. At many medical schools, electives at an accredited institution abroad are a common part of the student's final year experience. International electives provide an opportunity for a personal and academic experience that will often create new perspectives on clinical medicine and research, medical education and healthcare policy. In this article the authors reflect on their experience as elective students abroad and discuss the contribution of international electives to the constant development and progress of local final year rotations. They identify key areas for improving final year electives and outline essential features for a valuable and successful final year elective.

Proceedings of a Series of Workshops on Writing Research and Development Proposals: A Final Report. Kentucky Research in Vocational Education Series, No. 2.

ERIC Educational Resources Information Center

McKinney, Floyd L., Ed.; Sims, E. Norman, Ed.

This book contains presentations made at a series of 3-day workshops held in Kentucky to prepare vocational educators to write proposals, progress reports, and final reports for research and development efforts. Workshop presentation included "Need for Research and Development in Vocational-Technical Education," by Robert Warmbrod,…

Women in Science and Technology: The Institutional Ecology Approach. Volume I: Final Research Report.

ERIC Educational Resources Information Center

Byrne, Eileen M.

This document is the final research report of the University of Queensland Women in Science and Technology in Australia (WISTA) project. The report is a policy review study conducted from 1985 to 1990, of the factors that act as critical filters or positive factors that hinder or help women's access to and progression in certain scientific and…

Correlated parameter fit of arrhenius model for thermal denaturation of proteins and cells.

PubMed

Qin, Zhenpeng; Balasubramanian, Saravana Kumar; Wolkers, Willem F; Pearce, John A; Bischof, John C

2014-12-01

Thermal denaturation of proteins is critical to cell injury, food science and other biomaterial processing. For example protein denaturation correlates strongly with cell death by heating, and is increasingly of interest in focal thermal therapies of cancer and other diseases at temperatures which often exceed 50 °C. The Arrhenius model is a simple yet widely used model for both protein denaturation and cell injury. To establish the utility of the Arrhenius model for protein denaturation at 50 °C and above its sensitivities to the kinetic parameters (activation energy E a and frequency factor A) were carefully examined. We propose a simplified correlated parameter fit to the Arrhenius model by treating E a, as an independent fitting parameter and allowing A to follow dependently. The utility of the correlated parameter fit is demonstrated on thermal denaturation of proteins and cells from the literature as a validation, and new experimental measurements in our lab using FTIR spectroscopy to demonstrate broad applicability of this method. Finally, we demonstrate that the end-temperature within which the denaturation is measured is important and changes the kinetics. Specifically, higher E a and A parameters were found at low end-temperature (50 °C) and reduce as end-temperatures increase to 70 °C. This trend is consistent with Arrhenius parameters for cell injury in the literature that are significantly higher for clonogenics (45-50 °C) vs. membrane dye assays (60-70 °C). Future opportunities to monitor cell injury by spectroscopic measurement of protein denaturation are discussed.

Inhibition of EGFR Induces a c-MET Driven Stem Cell Population in Glioblastoma

PubMed Central

Jun, Hyun Jung; Bronson, Roderick T.; Charest, Al

2015-01-01

Glioblastoma multiforme (GBM) is the most lethal form of primary brain tumors, characterized by highly invasive and aggressive tumors that are resistant to all current therapeutic options. GBMs are highly heterogeneous in nature and contain a small but highly tumorigenic and self-renewing population of stem or initiating cells (Glioblastoma stem cells or GSCs). GSCs have been shown to contribute to tumor propagation and resistance to current therapeutic modalities. Recent studies of human GBMs have elucidated the genetic alterations common in these tumors, but much remains unknown about specific signaling pathways that regulate GSCs. Here we identify a distinct fraction of cells in a genetically engineered mouse model of EGFR-driven GBM that respond to anti-EGFR therapy by inducing high levels of c-MET expression. The MET positive cells displayed clonogenic potential and long-term self-renewal ability in vitro and are capable of differentiating into multiple lineages. The MET positive GBM cells are resistant to radiation and highly tumorigenic in vivo. Activation of MET signaling led to an increase in expression of the stemness transcriptional regulators Oct4, Nanog and Klf4. Pharmacological inhibition of MET activity in GSCs prevented the activation of Oct4, Nanog and Klf4 and potently abrogated stemness. Finally, the MET expressing cells were preferentially localized in perivascular regions of mouse tumors consistent with their function as GSCs. Together, our findings indicate that EGFR inhibition in GBM induces MET activation in GSCs, which is a functional requisite for GSCs activity and thus represents a promising therapeutic target. PMID:24115218

Correlated Parameter Fit of Arrhenius Model for Thermal Denaturation of Proteins and Cells

PubMed Central

Qin, Zhenpeng; Balasubramanian, Saravana Kumar; Wolkers, Willem F.; Pearce, John A.; Bischof, John C.

2014-01-01

Thermal denaturation of proteins is critical to cell injury, food science and other biomaterial processing. For example protein denaturation correlates strongly with cell death by heating, and is increasingly of interest in focal thermal therapies of cancer and other diseases at temperatures which often exceed 50 °C. The Arrhenius model is a simple yet widely used model for both protein denaturation and cell injury. To establish the utility of the Arrhenius model for protein denaturation at 50 °C and above its sensitivities to the kinetic parameters (activation energy Ea and frequency factor A) were carefully examined. We propose a simplified correlated parameter fit to the Arrhenius model by treating Ea, as an independent fitting parameter and allowing A to follow dependently. The utility of the correlated parameter fit is demonstrated on thermal denaturation of proteins and cells from the literature as a validation, and new experimental measurements in our lab using FTIR spectroscopy to demonstrate broad applicability of this method. Finally, we demonstrate that the end-temperature within which the denaturation is measured is important and changes the kinetics. Specifically, higher Ea and A parameters were found at low end-temperature (50°C) and reduce as end-temperatures increase to 70 °C. This trend is consistent with Arrhenius parameters for cell injury in the literature that are significantly higher for clonogenics (45 – 50 °C) vs. membrane dye assays (60 –70 °C). Future opportunities to monitor cell injury by spectroscopic measurement of protein denaturation are discussed. PMID:25205396

SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy.

PubMed

Bui, Anh Thu; Laurent, Fanny; Havard, Maryline; Dautry, François; Tchénio, Thierry

2015-01-01

Metastasis involves the dissemination of single or small clumps of cancer cells through blood or lymphatic vessels and their extravasation into distant organs. Despite the strong regulation of metastases development by a cell dormancy phenomenon, the dormant state of cancer cells remains poorly characterized due to the difficulty of in vivo studies. We have recently shown in vitro that clonogenicity of prostate cancer cells is regulated by a dormancy phenomenon that is strongly induced when cells are cultured both at low cell density and in a slightly hypertonic medium. Here, we characterized by RT-qPCR a genetic expression signature of this dormant state which combines the presence of both stemness and differentiation markers. We showed that both TFGβ/BMP signaling and redox imbalance are required for the full induction of this dormancy signature and cell quiescence. Moreover, reconstruction experiments showed that TFGβ/BMP signaling and redox imbalance are sufficient to generate a pattern of genetic expression displaying all characteristic features of the dormancy signature. Finally, we observed that low cell density was sufficient to activate TGFβ/BMP signaling and to generate a slight redox imbalance thus priming cells for dormancy that can be attained with a co-stimulus like hypertonicity, most likely through an increased redox imbalance. The identification of a dual regulation of dormancy provides a framework for the interpretation of previous reports showing a restricted ability of BMP signaling to regulate cancer cell dormancy in vivo and draws attention on the role of oxidative stress in the metastatic process.

Signal detection in global mean temperatures after "Paris": an uncertainty and sensitivity analysis

NASA Astrophysics Data System (ADS)

Visser, Hans; Dangendorf, Sönke; van Vuuren, Detlef P.; Bregman, Bram; Petersen, Arthur C.

2018-02-01

In December 2015, 195 countries agreed in Paris to hold the increase in global mean surface temperature (GMST) well below 2.0 °C above pre-industrial levels and to pursue efforts to limit the temperature increase to 1.5 °C. Since large financial flows will be needed to keep GMSTs below these targets, it is important to know how GMST has progressed since pre-industrial times. However, the Paris Agreement is not conclusive as regards methods to calculate it. Should trend progression be deduced from GCM simulations or from instrumental records by (statistical) trend methods? Which simulations or GMST datasets should be chosen, and which trend models? What is pre-industrial and, finally, are the Paris targets formulated for total warming, originating from both natural and anthropogenic forcing, or do they refer to anthropogenic warming only? To find answers to these questions we performed an uncertainty and sensitivity analysis where datasets and model choices have been varied. For all cases we evaluated trend progression along with uncertainty information. To do so, we analysed four trend approaches and applied these to the five leading observational GMST products. We find GMST progression to be largely independent of various trend model approaches. However, GMST progression is significantly influenced by the choice of GMST datasets. Uncertainties due to natural variability are largest in size. As a parallel path, we calculated GMST progression from an ensemble of 42 GCM simulations. Mean progression derived from GCM-based GMSTs appears to lie in the range of trend-dataset combinations. A difference between both approaches appears to be the width of uncertainty bands: GCM simulations show a much wider spread. Finally, we discuss various choices for pre-industrial baselines and the role of warming definitions. Based on these findings we propose an estimate for signal progression in GMSTs since pre-industrial.

Hypofractionated stereotactic radiation therapy in three to five fractions for vestibular schwannoma.

PubMed

Morimoto, Masahiro; Yoshioka, Yasuo; Kotsuma, Tadayuki; Adachi, Kana; Shiomi, Hiroya; Suzuki, Osamu; Seo, Yuji; Koizumi, Masahiko; Kagawa, Naoki; Kinoshita, Manabu; Hashimoto, Naoya; Ogawa, Kazuhiko

2013-08-01

To retrospectively examine the outcomes of hypofractionated stereotactic radiation therapy in three to five fractions for vestibular schwannomas. Twenty-five patients with 26 vestibular schwannomas were treated with hypofractionated stereotactic radiation therapy using a CyberKnife. The vestibular schwannomas of 5 patients were associated with type II neurofibromatosis. The median follow-up time was 80 months (range: 6-167); the median planning target volume was 2.6 cm(3) (0.3-15.4); and the median prescribed dose (≥D90) was 21 Gy in three fractions (18-25 Gy in three to five fractions). Progression was defined as ≥2 mm 3-dimensional post-treatment tumor enlargement excluding transient expansion. Progression or any death was counted as an event in progression-free survival rates, whereas only progression was counted in progression-free rates. The 7-year progression-free survival and progression-free rates were 78 and 95%, respectively. Late adverse events (≥3 months) with grades based on Common Terminology Criteria for Adverse Events, v4.03 were observed in 6 patients: Grade 3 hydrocephalus in one patient, Grade 2 facial nerve disorders in two and Grade 1-2 tinnitus in three. In total, 12 out of 25 patients maintained pure tone averages ≤50 dB before hypofractionated stereotactic radiation therapy, and 6 of these 12 patients (50%) maintained pure tone averages at this level at the final audiometric follow-up after hypofractionated stereotactic radiation therapy. However, gradient deterioration of pure tone average was observed in 11 of these 12 patients. The mean pure tone averages before hypofractionated stereotactic radiation therapy and at the final follow-up for the aforementioned 12 patients were 29.8 and 57.1 dB, respectively. Treating vestibular schwannomas with hypofractionated stereotactic radiation therapy in three to five fractions may prevent tumor progression with tolerable toxicity. However, gradient deterioration of pure tone average was observed.

The McDonnell Douglas geophysical observatory program progress report 13 Conjugate point riometer program

NASA Technical Reports Server (NTRS)

Baker, M. B.

1975-01-01

This report, the thirteenth and final progress report on the McDonnell Douglas Geophysical Observatory Program, discusses history of the program from 1962 through 1973, and results of the research carried out in 1974. Topic areas covered include: Station operation; Ionospheric work; Solar studies, Magnetospheric studies; Satellite measurements; International participation; and, 1974 research on solar activity, ATS-6 studies, magnetospheric physics, and station operation.

7 CFR 1499.16 - Appeals.

Code of Federal Regulations, 2010 CFR

2010-01-01

... AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS FOOD FOR PROGRESS PROGRAM § 1499.16 Appeals. A participant... opportunity to have a hearing before a final decision is made regarding its appeal. ...

Moses Lake Fishery Restoration Project; Factors Affecting the Recreational Fishery in Moses Lake Washington, Annual Report 2002-2003.

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

Burgess, Dave

2003-11-01

This annual report is a precursor to the final technical report we will be writing the next contract period. Consequently, this report, covering the period between September 27, 2002, and September 26, 2003, represents a progress report towards the final technical report we anticipate completing by September 26, 2004. Sample analysis and field work have progressed well and we anticipate no further delays. There are 4 objectives: (1) To quantify secondary production Moses Lake; (2) To quantify the influence of predation on target fishes in Moses Lake; (3) To quantify mortality of selected fished in Moses Lake; and (4) Tomore » assess effects of habitat changes from shoreline development and carp on the fish community in Moses Lake.« less

Bioactive Encapsulation for Military Food Applications: Request for Enhanced Nano and Micro Particle Fabrication and Characterization Facilities

DTIC Science & Technology

2016-01-25

2013 21-Jul-2014 Approved for Public Release; Distribution Unlimited Final Report: Bioactive Encapsulation for Military Food Applications: Request for...reviewed journals: Number of Papers published in non peer-reviewed journals: Final Report: Bioactive Encapsulation for Military Food Applications...Total Number: ...... Inventions (DD882) Scientific Progress Equipment was purchased. Technology Transfer 1 Bioactive Encapsulation for Military Food

Programs for Children with Specific Learning Disabilities. P.L. 91-230, Title VI-G Formal Final Evaluation. (Statistical Analysis of Data).

ERIC Educational Resources Information Center

Murphy, Philip J.

The paper reports the final evaluation of a program for approximately 143 learning disabled (LD) students (grades 6-to-12) from six school districts. A number of test instruments were used to evaluate student progress during the program, including the Wide Range Achievement Test (WRAT), the Durrell Analysis of Reading Difficulty, and the…

Medical students in their final six months of training: progress in self-perceived clinical competence, and relationship between experience and confidence in practical skills.

PubMed

Lai, N M; Sivalingam, N; Ramesh, J C

2007-11-01

We evaluated the progress in the self-perceived competence of medical students in a range of common clinical, practical and personal skills, in their final six months of training. The study was conducted on 65 final-year medical students undertaking their senior clerkship training at International Medical University, Malaysia. Questionnaire surveys were conducted at the beginning and the end of the six-month period, with 44 items covering clinical, practical, personal skills and readiness to work. Correlations were performed for experience and self-perceived competence, with the respective skills. 64 students returned the first survey and 63 returned the second survey. When the two survey results were compared, significant increases were found in self-perceived competence for the majority of the skills examined. The items with no significant improvement were divided into those which the students were already proficient in before senior clerkship, and those in which experience and confidence remained poor at the end of training. There were significant, but moderate, correlations between the experience and confidence of all common practical skills (correlation coefficients: 0.348-0.522, p-value is less than 0.001 for all items). At the end of training, students were, in general, more prepared to work as house officers (mean rating in the first survey: 3.05, second survey: 3.97, p-value is less than 0.001). Significant progresses in clinical experience and confidence can be observed in the final stages of medical training. The findings of inadequate improvements in some skills call for dedicated training sessions and strengthening of on-site supervision.

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

Mascle, J.; Blarez, E.

The authors present a marine study of the eastern Ivory Coast-Ghana continental margins which they consider one of the most spectacular extinct transform margins. This margin has been created during Early-Lower Cretaceous time and has not been submitted to any major geodynamic reactivation since its fabric. Based on this example, they propose to consider during the evolution of the transform margin four main and successive stages. Shearing contact is first active between two probably thick continental crusts and then between progressively thinning continental crusts. This leads to the creation of specific geological structures such as pull-apart graben, elongated fault lineaments,more » major fault scarps, shear folds, and marginal ridges. After the final continental breakup, a hot center (the mid-oceanic ridge axis) is progressively drifting along the newly created margin. The contact between two lithospheres of different nature should necessarily induce, by thermal exchanges, vertical crustal readjustments. Finally, the transform margin remains directly adjacent to a hot but cooling oceanic lithosphere; its subsidence behavior should then progressively be comparable to the thermal subsidence of classic rifted margins.« less

A Comparison of Functional and Structural Measures for Identifying Progression of Glaucoma

PubMed Central

Xin, Daiyan; Greenstein, Vivienne C.; Ritch, Robert; Liebmann, Jeffrey M.; De Moraes, Carlos Gustavo

2011-01-01

Purpose. To compare glaucoma progression by functional and structural tests. Methods. The authors prospectively studied 33 glaucoma patients (55 eyes); 20 eyes (15 patients) had disc hemorrhage, and 35 eyes (18 patients) had exfoliation glaucoma. The following tests were performed at two baseline and three follow-up examinations: frequency doubling perimetry (FDT), 24-2 Humphrey visual fields (HVF), multifocal visual evoked potentials (mfVEP), and optical coherence tomography (OCT). To identify progression, the baseline measurements were averaged and compared to those obtained at the final examination. Stereophotographs of the optic disc were obtained at baseline and compared with those at the final examination. Results. Patients were followed up for 21.1 ± 1.8 months. For HVF there were significant changes in mean deviation (MD) in eight (14.5%) eyes but in pattern standard deviation (P/SD) in only two (3.6%) eyes. For FDT, there were significant changes in MD in 13 (23.6%) eyes. Five eyes showed changes in MD for HVF and FDT. For mfVEP, there was an increase in abnormal points in nine (16.4%) eyes. Six of these eyes did not show significant HVF or FDT changes. For OCT, RNFL average thickness values were significantly decreased in nine (16.4%) eyes. Nine (16.4%) eyes showed progression on stereophotography; four of these eyes did not show significant changes on OCT and functional tests. Conclusions. Each test showed evidence of progression in some eyes. However, agreement among tests and stereophotography regarding which eyes showed progression was poor, illustrating the importance of following up patients with a combination of functional and structural tests. PMID:20847115

Progression of Inflammatory Bowel Disease to Cancer: Is the Patient Better Off without Lymphatic Vessels or Nodes (or Angiopoietin 2)?

DTIC Science & Technology

2013-12-01

carcinoma (CRC) in IBD patients and experimental models. Nonetheless, the pathogenic link, interrelationship, and practical clinical application of these...and are continuing final data analysis and latest imaging studies. This project has potentially high impact because of the substantial incidence of...pathogenic link, interrelationship, and practical clinical application of these various theories of progression have remained elusive. We proposed that

Prevention of Organ Injury in Exertional Heat Stroke: Preclinical Evaluation of a New Class of NSAIDs

DTIC Science & Technology

2017-10-01

and integration of data from 3 centers. PROGRESS: All of year one goals were completed except for completion of the manuscripts, which are...samples from mice, integrate data collection from the 3 laboratories and prepare final reports and manuscripts of experimental outcomes. PROGRESS...the later recovery period. These data suggest some abnormalities in glucose metabolism, particularly in male mice, during the recovery from EHS

Slow Progress In Finalizing Measles And Rubella Elimination In The European Region.

PubMed

Biellik, Robin; Davidkin, Iria; Esposito, Susanna; Lobanov, Andrey; Kojouharova, Mira; Pfaff, Günter; Santos, José Ignacio; Simpson, John; Mamou, Myriam Ben; Butler, Robb; Deshevoi, Sergei; Huseynov, Shahin; Jankovic, Dragan; Shefer, Abigail

2016-02-01

All countries in the World Health Organization European Region committed to eliminating endemic transmission of measles and rubella by 2015, and disease incidence has decreased dramatically. However, there was little progress between 2012 and 2013, and the goal will likely not be achieved on time. Genuine political commitment, increased technical capacity, and greater public awareness are urgently needed, especially in Western Europe. Project HOPE—The People-to-People Health Foundation, Inc.

Uncertainty and Variability in Physiologically-Based ...

EPA Pesticide Factsheets

EPA announced the availability of the final report, Uncertainty and Variability in Physiologically-Based Pharmacokinetic (PBPK) Models: Key Issues and Case Studies. This report summarizes some of the recent progress in characterizing uncertainty and variability in physiologically-based pharmacokinetic models and their predictions for use in risk assessment. This report summarizes some of the recent progress in characterizing uncertainty and variability in physiologically-based pharmacokinetic models and their predictions for use in risk assessment.

Recent work on gaseous detonations

NASA Astrophysics Data System (ADS)

Nettleton, M. A.

The paper reviews recent progress in the field of gaseous detonations, with sections on shock diffraction and reflection, the transition to detonation, hybrid, spherically-imploding, and galloping and stuttering fronts, their structure, their transmission and quenching by additives, the critical energy for initiation and detonation of more unusual fuels. The final section points out areas where our understanding is still far from being complete and contains some suggestions of ways in which progress might be made.

Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

PubMed

Ding, Xia; He, Zhuohao; Zhou, Kechun; Cheng, Ju; Yao, Hailan; Lu, Dongliang; Cai, Rong; Jin, Yening; Dong, Bin; Xu, Yinghui; Wang, Yizheng

2010-07-21

Patients with glioblastoma multiforme, the most aggressive form of glioma, have a median survival of approximately 12 months. Calcium (Ca(2+)) signaling plays an important role in cell proliferation, and some members of the Ca(2+)-permeable transient receptor potential canonical (TRPC) family of channel proteins have demonstrated a role in the proliferation of many types of cancer cells. In this study, we investigated the role of TRPC6 in cell cycle progression and in the development of human glioma. TRPC6 protein and mRNA expression were assessed in glioma (n = 33) and normal (n = 17) brain tissues from patients and in human glioma cell lines U251, U87, and T98G. Activation of TRPC6 channels was tested by platelet-derived growth factor-induced Ca(2+) imaging. The effect of inhibiting TRPC6 activity or expression using the dominant-negative mutant TRPC6 (DNC6) or RNA interference, respectively, was tested on cell growth, cell cycle progression, radiosensitization of glioma cells, and development of xenografted human gliomas in a mouse model. The green fluorescent protein (GFP) and wild-type TRPC6 (WTC6) were used as controls. Survival of mice bearing xenografted tumors in the GFP, DNC6, and WTC6 groups (n = 13, 15, and 13, respectively) was compared using Kaplan-Meier analysis. All statistical tests were two-sided. Functional TRPC6 was overexpressed in human glioma cells. Inhibition of TRPC6 activity or expression attenuated the increase in intracellular Ca(2+) by platelet-derived growth factor, suppressed cell growth and clonogenic ability, induced cell cycle arrest at the G2/M phase, and enhanced the antiproliferative effect of ionizing radiation. Cyclin-dependent kinase 1 activation and cell division cycle 25 homolog C expression regulated the cell cycle arrest. Inhibition of TRPC6 activity also reduced tumor volume in a subcutaneous mouse model of xenografted human tumors (P = .014 vs GFP; P < .001 vs WTC6) and increased mean survival in mice in an intracranial model (P < .001 vs GFP or WTC6). In this preclinical model, TRPC6 channels were essential for glioma development via regulation of G2/M phase transition. This study suggests that TRPC6 might be a new target for therapeutic intervention of human glioma.

15 CFR 923.94 - Application for program development or implementation grants.

Code of Federal Regulations, 2010 CFR

2010-01-01

... AND COASTAL RESOURCE MANAGEMENT COASTAL ZONE MANAGEMENT PROGRAM REGULATIONS Applications for Program...) The cumulative progress toward meeting the requirements for preliminary or final approval of a coastal...

15 CFR 923.94 - Application for program development or implementation grants.

Code of Federal Regulations, 2011 CFR

2011-01-01

... AND COASTAL RESOURCE MANAGEMENT COASTAL ZONE MANAGEMENT PROGRAM REGULATIONS Applications for Program...) The cumulative progress toward meeting the requirements for preliminary or final approval of a coastal...

Sensitizing effects of gallium citrate on hyperthermic cell killing in vitro.

PubMed

Miyazaki, N; Nakano, H; Kawakami, N; Kugotani, M; Nishihara, K; Aoki, Y; Shinohara, K

2000-01-01

The lethal effects of gallium citrate in combination with heat were studied using four cell lines, L5178Y, FM3A, P388 and HeLa. Cells were incubated with different concentrations (0.2 2 mM) of gallium citrate at 37 degrees C for 24 h and heated at a range of temperatures from 40-44 degrees C for various time periods up to 6 h in the absence of gallium citrate. Survival and cell viability were determined by clonogenic assay and the dye-exclusion test, respectively. All of the cell lines tested were insensitive to heat below 41 degrees C, but were very sensitive to heat above 43 degrees C. Gallium citrate was cytotoxic to these cell lines at different levels: P388 and HeLa were far more sensitive than L5178Y and FM3A. The killing effects of heat at 41 degrees C were greatly enhanced by gallium citrate in L5178Y and P388 cells. The Arrhenius analysis for the lethal effect of heat, determined by clonogenic assay, in L5178Y cells showed that the transition temperature was remarkably decreased for the gallium-treated cells from approximately 43 degrees C to 41 degrees C. The mechanism for this decrease in the transition temperature may be attributable to the additional effects of gallium citrate on energy metabolism. Preincubation with 0.05 mM gallium citrate at 37 degrees C for 7 days also enhanced heat sensitization at 41 degrees C in L5178Y. This preincubation condition may correspond to the condition for the continuous infusion of gallium that is clinically used for cancer treatment. In contrast, treatment with gallium did not greatly enhance the sensitivity of FM3A or HeLa cells to heat at 41 degrees C, but the effects of gallium were significant.

Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

PubMed Central

Avanzini, Maria Antonietta; Bernardo, Maria Ester; Cometa, Angela Maria; Perotti, Cesare; Zaffaroni, Nadia; Novara, Francesca; Visai, Livia; Moretta, Antonia; Del Fante, Claudia; Villa, Raffaella; Ball, Lynne M.; Fibbe, Willem E.; Maccario, Rita; Locatelli, Franco

2009-01-01

Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. Conclusions Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications. PMID:19773264

Functional Role of CLIC1 Ion Channel in Glioblastoma-Derived Stem/Progenitor Cells

PubMed Central

2013-01-01

Background Chloride channels are physiologically involved in cell division and motility. Chloride intracellular channel 1 (CLIC1) is overexpressed in a variety of human solid tumors compared with normal tissues, suggesting a potential involvement of CLIC1 in the regulation of tumorigenesis. This led us to investigate the role of CLIC1 in gliomagenesis. Methods We used the neurosphere system to isolate stem/progenitor cells from human glioblastomas (GBMs). CLIC1 targeting in GBM neurospheres was achieved by both lentiviral-mediated short-hairpin RNA transduction and CLIC1 antibody treatment, and its effect on stem-like properties was analyzed in vitro by proliferation and clonogenic assays and in vivo by orthotopic injection in immunocompromised mice. Channel activity was studied by perforated patch clamp technique. Differences in expression were analyzed by analysis of variance with Tamhane’s multiple comparison test. Kaplan–Meier analyses and log-rank test were used to assess survival. All statistical tests were two-sided. Results CLIC1 was statistically significantly overexpressed in GBMs compared with normal brain tissues (P < .001) with a better survival of patients with CLIC1 low-expressing tumors (CLIC1low vs CLIC1high survival: χ2 = 74.35; degrees of freedom = 1; log-rank P < .001). CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment. CLIC1 silencing reduced proliferative (P < .01), clonogenic (P < .01), and tumorigenic capacity (P < .05) of stem/progenitor cells. The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient–derived neurospheres. Conclusions Reduced gliomagenesis after CLIC1 targeting in tumoral stem/progenitor cells and the finding that CLIC1 expression is inversely associated with patient survival suggest CLIC1 as a potential target and prognostic biomarker. PMID:24115360

Sulforaphane down-regulates SKP2 to stabilize p27(KIP1) for inducing antiproliferation in human colon adenocarcinoma cells.

PubMed

Chung, Yuan-Kai; Chi-Hung Or, Richard; Lu, Chien-Hsing; Ouyang, Wei-Ting; Yang, Shu-Yi; Chang, Chia-Che

2015-01-01

Sulforaphane is a cruciferous vegetable-derived isothiocyanate with promising chemopreventive and therapeutic activities. Induction of proliferation arrest and apoptosis principally contribute to sulforaphane's anticancer activity, but the precise molecular mechanisms remain elusive. The oncoprotein SKP2 is a key component of the SKP1-CULLIN1-F-box (SCF) E3 ligase complex and is responsible for directing SCF-mediated degradation of cyclin-dependent kinase inhibitor p27(KIP1) to promote cell proliferation. We herein provide the first evidence supporting the critical involvement of the SKP2-p27(KIP1) axis in sulforaphane-induced antiproliferation in various human colon adenocarcinoma cell lines. Specifically, sulforaphane markedly suppressed the levels of bromodeoxyuridine (BrdU) incorporation and clonogenicity in all tested cell lines, illustrating the antiproliferative effect of sulforaphane. Of note, sulforaphane-induced antiproliferation was accompanied with down-regulation of SKP2, leading to the stabilization and thus up-regulation of p27(KIP1). Additionally, sulforaphane was found to down-regulate SKP2 mainly through transcriptional repression, as sulforaphane lowered SKP2 mRNA expression and the SKP2 promoter activity. Furthermore, sulforaphane treatment led to the activation of both AKT and ERK, thus ruling out the possibility that sulforaphane down-regulates SKP2 by inhibiting AKT or ERK. Notably, sulforaphane-elicited suppression of BrdU incorporation and clonogenicity were significantly rescued in the context of SKP2 overexpression or p27(KIP1) depletion, therefore highlighting the important role of SKP2 down-regulation and the ensuing stabilization of p27(KIP1) in sulforaphane-induced antiproliferation. Collectively, these data expand our molecular understanding about how sulforaphane elicits proliferation arrest, but also implicate the application of sulforaphane in therapeutic modalities targeting SKP2. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Cord blood-derived CD34+ hematopoietic cells with low mitochondrial mass are enriched in hematopoietic repopulating stem cell function.

PubMed

Romero-Moya, Damia; Bueno, Clara; Montes, Rosa; Navarro-Montero, Oscar; Iborra, Francisco J; López, Luis Carlos; Martin, Miguel; Menendez, Pablo

2013-07-01

The homeostasis of the hematopoietic stem/progenitor cell pool relies on a fine-tuned balance between self-renewal, differentiation and proliferation. Recent studies have proposed that mitochondria regulate these processes. Although recent work has contributed to understanding the role of mitochondria during stem cell differentiation, it remains unclear whether the mitochondrial content/function affects human hematopoietic stem versus progenitor function. We found that mitochondrial mass correlates strongly with mitochondrial membrane potential in CD34(+) hematopoietic stem/progenitor cells. We, therefore, sorted cord blood CD34(+) cells on the basis of their mitochondrial mass and analyzed the in vitro homeostasis and clonogenic potential as well as the in vivo repopulating potential of CD34(+) cells with high (CD34(+) Mito(High)) versus low (CD34(+) Mito(Low)) mitochondrial mass. The CD34(+) Mito(Low) fraction contained 6-fold more CD34(+)CD38(-) primitive cells and was enriched in hematopoietic stem cell function, as demonstrated by its significantly greater hematopoietic reconstitution potential in immuno-deficient mice. In contrast, the CD34(+) Mito(High) fraction was more enriched in hematopoietic progenitor function with higher in vitro clonogenic capacity. In vitro differentiation of CD34(+) Mito(Low) cells was significantly delayed as compared to that of CD34(+) Mito(High) cells. The eventual complete differentiation of CD34(+) Mito(Low) cells, which coincided with a robust expansion of the CD34(-) differentiated progeny, was accompanied by mitochondrial adaptation, as shown by significant increases in ATP production and expression of the mitochondrial genes ND1 and COX2. In conclusion, cord blood CD34(+) cells with low levels of mitochondrial mass are enriched in hematopoietic repopulating stem cell function whereas high levels of mitochondrial mass identify hematopoietic progenitors. A mitochondrial response underlies hematopoietic stem/progenitor cell differentiation and proliferation of lineage-committed CD34(-) cells.

Radioresistance in murine solid tumors induced by interleukin-1

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

Braunschweiger, P.G.; Basrur, V.; Santos, O.

1996-02-01

Interleukin-1 (IL-1) has radioprotective activity in hematopoietic lineages and in other normal cell renewal systems, but little is known about the effects of IL-1{alpha} on the radiosensitivity of tumor cell populations. The present studies were conducted to investigate the effects of IL-1{alpha} on the radiosensitivity of clonogenic cells in RIF-1 and SCC-7 tumors. Radioresistance was detected within 2-4 h after administration of IL-1{alpha} (0.5 {mu}g/mouse, ip) and characterized by increases in D{sub 0}, D{sub q}, {alpha}/{Beta} and SF2. This radioresistance was similar to that seen in tumors rendered totally hypoxic before X irradiation. Tirapazamine, a hypoxic cell cytotoxin, and IL-1{alpha}more » had synergistic schedule-dependent antitumor activity in vivo, suggesting that IL-1-induced radioresistance in vivo is due to hypoxia. Radioresistance induced by IL-1{alpha} was transient, and the data suggested reoxygenation within 12 h. In vitro, IL-1{alpha} had no direct effect on the radiosensitivity of SCC-7 cells in tissue culture under aerobic conditions. However, an increase in D{sub 0}, {alpha}/{Beta} and SF2 was seen in clonogenic tumor cells from primary cultures treated with IL-1{alpha} under aerobic conditions. Superoxide dismutase and catalase prevented the induction of radioresistance by IL-1{alpha} in vitro, suggesting that oxidative responses from tumor macrophages after administration of IL-1{alpha} may be responsible for induced radioresistance by IL-1 in vitro. Although oxidant stress induced by IL-1 may play an important role in the activity of IL-1{alpha} both in vivo and in vitro in our models, the mechanisms by which such responses modulate tumor radiosensitivity in vivo and in vitro are likely quite different. 32 refs., 6 figs., 1 tab.« less

A standard dose of radiation for microscopic disease is not appropriate

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

Marks, L.B.

1990-12-15

Elective irradiation of sites of potential occult tumor spread is often part of a patient's radiation therapy program. The required radiation dose (D) depends on the probability that occult disease exists (P(occ)), the number of sites at risk (A), the number of tumor clonogens present (Ni), their radiation sensitivity, and the desired control rate. An exponential model of cell survival is used to quantify the importance of these factors. Control Probability = (1 - Pocc x (1 - e-Ni x (SF2)D/2))A; SF2 = surviving fraction after 2 Gy. Implications for clinical radiation therapy include: 1. Since the number of clonogensmore » in an occult site may vary from 10 degrees to 10(8), Ni is the major determinant of the required dose. The intrinsic radiation sensitivity of the clonogens (SF2) is also extremely important in determining the dose. Other factors are less influential since they vary less. 2. The variability of Ni (8 logs) is larger than the variation in cell number seen with gross disease (1 cm3 versus 1000 cm3, 3 logs). When Ni approximately 10(8), the required dose approaches that needed for small volume gross disease (10(9) cells, 1 cm3). 3. The dose prescribed to elective sites should reflect the risk of occult disease based on the primary tumor site, stage, and grade. 4. Regions where clinicoradiologic evaluation is difficult (e.g., pelvis and obese neck) require higher doses because macroscopic tumor deposits may exist. 5. Relatively low doses (10 to 30 Gy) are often thought to be inadequate for microscopic tumor. However, similar doses have been reported to sterilize microscopic tumor in ovarian, rectal, bladder, breast, and head and neck carcinomas. Relatively low doses should not be discounted since they may be useful in select cases when normal tissue tolerances and/or previous irradiation treatment limit the radiation dose.« less

Enterolactone Suppresses Proliferation, Migration and Metastasis of MDA-MB-231 Breast Cancer Cells Through Inhibition of uPA Induced Plasmin Activation and MMPs-Mediated ECM Remodeling

PubMed Central

Mali, Aniket V; Joshi, Asavari A; Hegde, Mahabaleshwar V; Kadam, Shivajirao S

2017-01-01

Background: To enhance their own survival, tumor cells can manipulate their microenvironment through remodeling of the extra cellular matrix (ECM). The urokinase-type plasminogen activator (uPA) system catalyzes plasmin production which further mediates activation of matrix metalloproteinases (MMPs) and plays an important role in breast cancer invasion and metastasis through ECM remodeling. This provides a potential target for therapeutic intervention of breast cancer treatment. Enterolactone (EL) is derived from dietary flax lignans in the human body and is known to have anti-breast cancer activity. We here investigated molecular and cellular mechanisms of EL action on the uPA-plasmin-MMPs system. Methods: MTT and trypan blue dye exclusion assays, anchorage-dependent clonogenic assays and wound healing assays were carried out to study effects on cell proliferation and viability, clonogenicity and migration capacity, respectively. Real-time PCR was employed to study gene expression and gelatin zymography was used to assess MMP-2 and MMP-9 activities. All data were statistically analysed and presented as mean ± SEM values. Results: All the findings collectively demonstrated anticancer and antimetastatic potential of EL with antiproliferative, antimigratory and anticlonogenic cellular mechanisms. EL was found to exhibit multiple control of plasmin activation by down-regulating uPA expression and also up-regulating its natural inhibitor, PAI-1, at the mRNA level. Further, EL was found to down-regulate expression of MMP-2 and MMP-9 genes, and up-regulate TIMP-1 and TIMP-2; natural inhibitors of MMP-2 and MMP-9, respectively. This may be as a consequence of inhibition of plasmin activation, resulting in robust control over migration and invasion of breast cancer cells during metastasis. Conclusions: EL suppresses proliferation, migration and metastasis of MDA-MB-231 breast cancer cells by inhibiting induced ECM remodeling by the ‘uPA-plasmin-MMPs system’. PMID:28545187

Role of Na+/Ca2+ Exchangers in Therapy Resistance of Medulloblastoma Cells.

PubMed

Pelzl, Lisann; Hosseinzadeh, Zohreh; Al-Maghout, Tamer; Singh, Yogesh; Sahu, Itishri; Bissinger, Rosi; Schmidt, Sebastian; Alkahtani, Saad; Stournaras, Christos; Toulany, Mahmoud; Lang, Florian

2017-01-01

Alterations of cytosolic Ca2+-activity ([Ca2+]i) are decisive in the regulation of tumor cell proliferation, migration and survival. Transport processes participating in the regulation of [Ca2+]i include Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether medulloblastoma cells express Na+/Ca2+-exchangers, whether expression differs between therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells, and whether Na+/Ca2+-exchangers participate in the regulation of cell survival. In therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells transcript levels were estimated by RT-PCR, protein abundance by Western blotting, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/ Ca2+-exchanger activity from the increase of [Ca2+]i (Δ[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free by Na+ free and Ca2+ containing (2 mM) extracellular perfusate as well as cell death from PI -staining and annexin-V binding in flow cytometry. The transcript levels of NCX3, NCKX2, and NCKX5, protein abundance of NCX3, slope and peak of Δ[Ca2+]i as well as Ica were significantly lower in therapy sensitive D283 than in therapy resistant UW228-3 medulloblastoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i, and augmented the ionizing radiation-induced apoptosis but did not significantly modify clonogenicity of medulloblastoma cells. Apoptosis was further enhanced by NCX3 silencing. Na+/Ca2+-exchanger activity significantly counteracts apoptosis but does not significantly affect clonogenicity after radiation of medulloblastoma cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

Differential Efficacy of Combined Therapy With Radiation and AEE788 in High and Low EGFR-Expressing Androgen-Independent Prostate Tumor Models

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

Huamani, Jessica; Willey, Christopher; Thotala, Dinesh

2008-05-01

Purpose: To determine the efficacy of combining radiation (XRT) with a dual epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor inhibitor, AEE788, in prostate cancer models with different levels of EGFR expression. Methods and Materials: Immunoblotting was performed for EGFR, phosphorylated-EGFR, and phosphorylated-AKT in prostate cancer cells. Clonogenic assays were performed on DU145, PC-3, and human umbilical vein endothelial cells treated with XRT {+-} AEE788. Tumor xenografts were established for DU145 and PC-3 on hind limbs of athymic nude mice assigned to four treatment groups: (1) control, (2) AEE788, (3) XRT, and (4) AEE788 + XRT. Tumor blood flowmore » and growth measurements were performed using immunohistochemistry and imaging. Results: AEE788 effectively decreased phosphorylated-EGFR and phosphorylated-AKT levels in DU145 and PC-3 cells. Clonogenic assays showed no radiosensitization for DU145 and PC-3 colonies treated with AEE788 + XRT. However, AEE788 caused decreased proliferation in DU145 cells. AEE788 showed a radiosensitization effect in human umbilical vein endothelial cells and increased apoptosis susceptibility. Concurrent AEE788 + XRT compared with either alone led to significant tumor growth delay in DU145 tumors. Conversely, PC-3 tumors derived no added benefit from combined-modality therapy. In DU145 tumors, a significant decrease in tumor blood flow with combination therapy was shown by using power Doppler sonography and tumor blood vessel destruction on immunohistochemistry. Maldi-spectrometry (MS) imaging showed that AEE788 is bioavailable and heterogeneously distributed in DU145 tumors undergoing therapy. Conclusions: AEE788 + XRT showed efficacy in vitro/in vivo with DU145-based cell models, whereas PC-3-based models were adequately treated with XRT alone without added benefit from combination therapy. These findings correlated with differences in EGFR expression and showed effects on both tumor cell proliferation and vascular destruction.« less

Short- and long-term cytotoxic effects of doxorubicin conjugates with dendrimers and vector protein on MCF-7/MDR1 chemoresistant breast cancer cells

NASA Astrophysics Data System (ADS)

Zamulaeva, I. A.; Matchuk, O. N.; Churyukina, K. A.; Kudryavtzev, V. A.; Yabbarov, N. G.; Nikolskaya, E. D.; Zhunina, O. A.; Kondrasheva, I. G.; Severin, E. S.

2017-09-01

The dendritic polymers (dendrimers) are perspective nanocontainers for targeted transport of anticancer drugs to tumor cells. We used polyamidoamine dendrimers of the second generation (G2) covalently conjugated with doxorubicin (Dox) and vector protein - recombinant third domain (3D) of alpha-fetoprotein. The objects of the study were MCF-7/MDR1 breast cancer cells, which demonstrated resistance to traditional anticancer agents due to high expression of P-glycoprotein. Effects of free Dox, G2 dendrimers loaded with Dox (G2-Dox), or conjugates of dendrimers with the vector protein and Dox (3D-G2-Dox) were assessed by the criteria of surviving cell number and clonogenic activity 24 hours and 11 days after treatment with the agents at Dox concentration of 2.5 μM, correspondingly. Flow cytometry was used to evaluate accumulation of Dox immediately after the treatment with the agents and removal of Dox during 24 hours of incubation in agent-free medium following by the treatment. Intracellular localization of Dox was studied using laser scanning microscopy. 3D-G2-Dox demonstrated the highest accumulation and the weakest removal from the cells in comparison with all other agents. The use of free Dox, G2-Dox, or 3D-G2-Dox resulted in a significant decrease in number of surviving cells by approximately 25-30% compared to the control (p ≤ 0.01). However, the most pronounced decrease in the clonogenic ability of cells was observed in the 3D-G2-Dox group (to 19% compared to the control, p < 0.01). Taking into account the previously obtained data on the extremely low 3D-G2-Dox accumulation in normal cells, it can be concluded that further development of 3D-G2-Dox as a possible anticancer drug is a promising way to overcome multiple drug resistance with minimal impact on normal cells.

Anti-Cancer Effects of Imperata cylindrica Leaf Extract on Human Oral Squamous Carcinoma Cell Line SCC-9 in Vitro.

PubMed

Keshava, Rohini; Muniyappa, Nagesh; Gope, Rajalakshmi; Ramaswamaiah, Ananthanarayana Saligrama

2016-01-01

Imperata cylindrica, a tall tufted grass which has multiple pharmacological applications is one of the key ingredients in various traditional medicinal formula used in India. Previous reports have shown that I. cylindrica plant extract inhibited cell proliferation and induced apoptosis in various cancer cell lines. To our knowledge, no studies have been published on the effect of I. cylindrica leaf extract on human oral cancers. The present study was undertaken in order to evaluate the anticancer properties of the leaf extract of I. cylindrica using an oral squamous cell carcinoma cell line SCC-9 as an in vitro model system. A methanol extract from dried leaves of I. cylindrica (ICL) was prepared by standard procedures. Effects of the ICL extract on the morphology of SCC-9 cells was visualized by microscopy. Cytotoxicity was determined by MTT assay. Effects of the ICL extract on colony forming ability of SCC-9 cells was evaluated using clonogenic assay. Cell cycle analysis was performed by flow cytometry and induction of apoptosis was determined by DNA fragmentation assay. The ICL extract treatment caused cytotoxicity and induced cell death in vitro in SCC-9 cells in a dose-dependent manner. This treatment also significantly reduced the clonogenic potential and inhibited cell proliferation by arresting the cell cycle in the G2/M phase. Furthermore, DNA fragmentation assays showed that the observed cell death was caused by apoptosis. This is the first report showing the anticancer activity of the methanol extracts from the leaves of I. cylindrica in human oral cancer cell line. Our data indicates that ICL extract could be considered as one of the lead compounds for the formulation of anticancer therapeutic agents to treat/manage human oral cancers. The natural abundance of I. cylindrica and its wide geographic distribution could render it one of the primary resource materials for preparation of anticancer therapeutic agents.

Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific.

PubMed

Pathak, Bhakti R; Breed, Ananya A; Nakhawa, Vaishali H; Jagtap, Dhanashree D; Mahale, Smita D

2010-09-01

The prostate secretory protein of 94 amino acids (PSP94) has been shown to interact with cysteine-rich secretory protein 3 (CRISP-3) in human seminal plasma. Interestingly, PSP94 expression is reduced or lost in the majority of the prostate tumours, whereas CRISP-3 expression is upregulated in prostate cancer compared with normal prostate tissue. To obtain a better understanding of the individual roles these proteins have in prostate tumourigenesis and the functional relevance of their interaction, we ectopically expressed either PSP94 or CRISP-3 alone or PSP94 along with CRISP-3 in three prostate cell lines (PC3, WPE1-NB26 and LNCaP) and performed growth inhibition assays. Reverse transcription-polymerase chain reaction and Western blot analysis were used to screen prostate cell lines for PSP94 and CRISP-3 expression. Mammalian expression constructs for human PSP94 and CRISP-3 were also generated and the expression, localization and secretion of recombinant protein were assayed by transfection followed by Western blot analysis and immunofluorescence assay. The effect that ectopic expression of PSP94 or CRISP-3 had on cell growth was studied by clonogenic survival assay following transfection. To evaluate the effects of co-expression of the two proteins, stable clones of PC3 that expressed PSP94 were generated. They were subsequently transfected with a CRISP-3 expression construct and subjected to clonogenic survival assay. Our results showed that PSP94 and CRISP-3 could each induce growth inhibition in a cell line specific manner. Although the growth of CRISP-3-positive cell lines was inhibited by PSP94, growth inhibition mediated by CRISP-3 was not affected by the presence or absence of PSP94. This suggests that CRISP-3 may participate in PSP94-independent activities during prostate tumourigenesis.

Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells.

PubMed

Hermans, Mirjam H A; van de Geijn, Gert-Jan; Antonissen, Claudia; Gits, Judith; van Leeuwen, Daphne; Ward, Alister C; Touw, Ivo P

2003-04-01

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Tyr704, Tyr729, Tyr744, Tyr764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation, and cell survival. However, it is unclear whether these tyrosines are equally important under more physiologic conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated G-CSF-R-deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (m0), single tyrosine "add-back" mutants, or wild-type (WT) receptors. G-CSF-induced responses were determined in primary colony assays, serial replatings, and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Tyr764 strongly enhanced proliferative responses, which was reverted by inhibition of ERK activity. Tyr729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing m0 gradually dropped compared with WT. The presence of Tyr729, but also Tyr704 and Tyr744, both involved in activation of signal transducer and activator of transcription 3 (STAT3), further reduced replating efficiencies. Conversely, Tyr764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a more than 10(4)-fold increase of colony-forming cells over m0 after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.

Differential activation of cell death and autophagy results in an increased cytotoxic potential for trifluorothymidine compared to 5-fluorouracil in colon cancer cells.

PubMed

Bijnsdorp, Irene V; Peters, Godefridus J; Temmink, Olaf H; Fukushima, Masakazu; Kruyt, Frank A

2010-05-15

Trifluorothymidine (TFT) is part of the oral drug formulation TAS-102. Both 5-fluorouracil (5-FU) and TFT can inhibit thymidylate synthase and be incorporated into DNA. TFT shows only moderate cross-resistance to 5-FU. Therefore, we examined whether mechanistic differences in cell death could underlie their different modes of action in colorectal cancer cell lines (WiDR, Lovo92 and Colo320). Drug cytotoxicity was determined by SRB- and clonogenic assays, cell death by flow cytometry (PI and annexin V), caspase cleavage by Western blotting and activity assays and in vivo activity in the hollow fiber assay. The IC(50) values of TFT were 1-6 fold lower than for 5-FU, and clonogenic survival was less than 0.9% at 3 muM TFT, while 2-20% of the cells still survived after 20 muM 5-FU. In general, TFT was a more potent inducer of apoptosis than 5-FU, although the contribution of caspases varied between the used cell lines and necrosis-like cell death was detected. Accordingly, both drugs induced caspase (Z-VAD) independent cell death and lysosomal cathepsin B was involved. Activation of autophagy recovery mechanisms was only triggered by 5-FU, but not by TFT as determined by LC3B expression and cleavage. Inhibition of autophagy by 3-MA in 5-FU exposed cells reduced cell survival. Also, in vivo TFT (as TAS-102) caused more cell death than a 5-FU formulation. We conclude that TFT and 5-FU induce cell death via both caspase-dependent and independent mechanisms. The TFT was more potent than 5-FU, because it induces higher levels of cell death and does not elicit an autophagic survival response in the cancer cell lines. This provides a strong molecular basis for further application of TFT in cancer therapy.

The Jak2 Inhibitor, G6, Alleviates Jak2-V617F-Mediated Myeloproliferative Neoplasia by Providing Significant Therapeutic Efficacy to the Bone Marrow1

PubMed Central

Kirabo, Annet; Park, Sung O; Majumder, Anurima; Gali, Meghanath; Reinhard, Mary K; Wamsley, Heather L; Zhao, Zhizhuang Joe; Cogle, Christopher R; Bisht, Kirpal S; Keserü, György M; Sayeski, Peter P

2011-01-01

We recently developed a Janus kinase 2 (Jak2) small-molecule inhibitor called G6 and found that it inhibits Jak2-V617F-mediated pathologic cell growth in vitro, ex vivo, and in vivo. However, its ability to inhibit Jak2-V617F-mediated myeloproliferative neoplasia, with particular emphasis in the bone marrow, has not previously been examined. Here, we investigated the efficacy of G6 in a transgenic mouse model of Jak2-V617F-mediated myeloproliferative neoplasia. We found that G6 provided therapeutic benefit to the peripheral blood as determined by elimination of leukocytosis, thrombocytosis, and erythrocytosis. G6 normalized the pathologically high plasma concentrations of interleukin 6 (IL-6). In the liver, G6 eliminated Jak2-V617F-driven extramedullary hematopoiesis. With respect to the spleen, G6 significantly reduced both the splenomegaly and megakaryocytic hyperplasia. In the critically important bone marrow, G6 normalized the pathologically high levels of phospho-Jak2 and phospho-signal transducer and activator of transcription 5 (STAT5). It significantly reduced the megakaryocytic hyperplasia in the marrow and completely normalized the M/E ratio. Most importantly, G6 selectively reduced the mutant Jak2 burden by 67%on average, with virtual elimination of mutant Jak2 cells in one third of all treated mice. Lastly, clonogenic assays using marrow stem cells from the myeloproliferative neoplasm mice revealed a time-dependent elimination of the clonogenic growth potential of these cells by G6. Collectively, these data indicate that G6 exhibits exceptional efficacy in the peripheral blood, liver, spleen, and, most importantly, in the bone marrow, thereby raising the possibility that this compound may alter the natural history of Jak2-V617F-mediated myeloproliferative neoplasia. PMID:22131881

Pasteurization of bone for tumour eradication prior to reimplantation – An in vitro & pre-clinical efficacy study

PubMed Central

Kode, Jyoti; Taur, Prasad; Gulia, Ashish; Jambhekar, Nirmala; Agarwal, Manish; Puri, Ajay

2014-01-01

Background & objectives: In current era of limb-salvage therapy, pasteurization of bone sarcomas is receiving growing attention as a potential extracorporeal treatment and cost-effective alternative to allografts and radiation before surgical reimplantation. Detailed in vitro and in vivo pre-clinical study to evaluate efficacy of pasteurization to eradicate malignant cells has not been reported yet. The present study was carried out to assess the efficacy of pasteurization to kill tumour cells both in vitro and in vivo. Methods: Surgically resected specimens of osteosarcomas (n=4) were cut into equal halves and one section was pasteurized by heating at 60°C to 65°C for 40 min. Paired samples before and after pasteurization were studied in vitro for DNA ploidy, evaluation of histological change and elimination of mitotic activity. These tissues were transplanted in immune-deficient NOD-SCID mice to evaluate effect on tumour-generating ability, presence of human nuclei, osteopontin and cytokine/chemokines released in tumour-transplanted mice. Results: Non-pasteurized tumour samples had viable tumour cells which exhibited significant growth in culture, increased proliferative ability and clonogenic potential while respective pasteurized tumour tissues did not grow in culture and did not exhibit clonogenicity. Flow cytometry revealed that propidium iodide positive dead cells increased significantly (P< 0.01) post pasteurization. Seven of 12 non-pasteurized tumour transplanted mice demonstrated tumour-forming ability as against 0 of 12 in pasteurized tumour transplanted mice. Solid tumour xenografts exhibited strong expression of anti-human nuclei and osteopontin by immunohistochemistry as well as secretary human interluekin-6 (IL-6) while pasteurized mice failed to express these markers. Interpretation & conclusions: This study has provided a basis to establish pasteurization as being efficacious in ensuring tumour eradication from resected bone tumour specimens. Pasteurized tumour bearing bone can thus safely be used to reconstruct large defects after tumour resection. PMID:24927346

ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

PubMed

Karayazi Atici, Ödül; Urbanska, Anna; Gopinathan, Sesha Gopal; Boutillon, Florence; Goffin, Vincent; Shemanko, Carrie S

2018-02-01

Prolactin (PRL) acts as a survival factor for breast cancer cells, but the PRL signaling pathway and the mechanism are unknown. Previously, we identified the master chaperone, heat shock protein 90 (HSP90) α, as a prolactin-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) target gene involved in survival, and here we investigated the role of HSP90 in the mechanism of PRL-induced viability in response to DNA damage. The ataxia-telangiectasia mutated kinase (ATM) protein plays a critical role in the cellular response to double-strand DNA damage. We observed that PRL increased viability of breast cancer cells treated with doxorubicin or etoposide. The increase in cellular resistance is specific to the PRL receptor, because the PRL receptor antagonist, Δ1-9-G129R-hPRL, prevented the increase in viability. Two different HSP90 inhibitors, 17-allylamino-17-demethoxygeldanamycin and BIIB021, reduced the PRL-mediated increase in cell viability of doxorubicin-treated cells and led to a decrease in JAK2, ATM, and phosphorylated ATM protein levels. Inhibitors of JAK2 (G6) and ATM (KU55933) abolished the PRL-mediated increase in cell viability of DNA-damaged cells, supporting the involvement of each, as well as the crosstalk of ATM with the PRL pathway in the context of DNA damage. Drug synergism was detected between the ATM inhibitor (KU55933) and doxorubicin and between the HSP90 inhibitor (BIIB021) and doxorubicin. Short interfering RNA directed against ATM prevented the PRL-mediated increase in cell survival in two-dimensional cell culture, three-dimensional collagen gel cultures, and clonogenic cell survival, after doxorubicin treatment. Our results indicate that ATM contributes to the PRL-JAK2-STAT5-HSP90 pathway in mediating cellular resistance to DNA-damaging agents. Copyright © 2018 Endocrine Society.