SM22{alpha}-induced activation of p16{sup INK4a}/retinoblastoma pathway promotes cellular senescence caused by a subclinical dose of {gamma}-radiation and doxorubicin in HepG2 cells

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

Kim, Tae Rim; Lee, Hee Min; Lee, So Yong

Research highlights: {yields} SM22{alpha} overexpression in HepG2 cells leads cells to a growth arrest state, and the treatment of a subclinical dose of {gamma}-radiation or doxorubicin promotes cellular senescence. {yields} SM22{alpha} overexpression elevates p16{sup INK4a} followed by pRB activation, but there are no effects on p53/p21{sup WAF1/Cip1} pathway. {yields} SM22{alpha}-induced MT-1G activates p16{sup INK4a}/pRB pathway, which promotes cellular senescence by damaging agents. -- Abstract: Smooth muscle protein 22-alpha (SM22{alpha}) is known as a transformation- and shape change-sensitive actin cross-linking protein found in smooth muscle tissue and fibroblasts; however, its functional role remains uncertain. We reported previously that SM22{alpha} overexpression confersmore » resistance against anti-cancer drugs or radiation via induction of metallothionein (MT) isozymes in HepG2 cells. In this study, we demonstrate that SM22{alpha} overexpression leads cells to a growth arrest state and promotes cellular senescence caused by treatment with a subclinical dose of {gamma}-radiation (0.05 and 0.1 Gy) or doxorubicin (0.01 and 0.05 {mu}g/ml), compared to control cells. Senescence growth arrest is known to be controlled by p53 phosphorylation/p21{sup WAF1/Cip1} induction or p16{sup INK4a}/retinoblastoma protein (pRB) activation. SM22{alpha} overexpression in HepG2 cells elevated p16{sup INK4a} followed by pRB activation, but did not activate the p53/p21{sup WAF1/Cip1} pathway. Moreover, MT-1G, which is induced by SM22{alpha} overexpression, was involved in the activation of the p16{sup INK4a}/pRB pathway, which led to a growth arrest state and promoted cellular senescence caused by damaging agents. Our findings provide the first demonstration that SM22{alpha} modulates cellular senescence caused by damaging agents via regulation of the p16{sup INK4a}/pRB pathway in HepG2 cells and that these effects of SM22{alpha} are partially mediated by MT-1G.« less

Dysregulation of the Bmi-1/p16Ink4a pathway provokes an aging-associated decline of submandibular gland function

PubMed Central

Yamakoshi, Kimi; Katano, Satoshi; Iida, Mayu; Kimura, Hiromi; Okuma, Atsushi; Ikemoto-Uezumi, Madoka; Ohtani, Naoko; Hara, Eiji; Maruyama, Mitsuo

2015-01-01

Bmi-1 prevents stem cell aging, at least partly, by blocking expression of the cyclin-dependent kinase inhibitor p16Ink4a. Therefore, dysregulation of the Bmi-1/p16Ink4a pathway is considered key to the loss of tissue homeostasis and development of associated degenerative diseases during aging. However, because Bmi-1 knockout (KO) mice die within 20 weeks after birth, it is difficult to determine exactly where and when dysregulation of the Bmi-1/p16Ink4a pathway occurs during aging in vivo. Using real-time in vivo imaging of p16Ink4a expression in Bmi-1-KO mice, we uncovered a novel function of the Bmi-1/p16Ink4a pathway in controlling homeostasis of the submandibular glands (SMGs), which secrete saliva into the oral cavity. This pathway is dysregulated during aging in vivo, leading to induction of p16Ink4a expression and subsequent declined SMG function. These findings will advance our understanding of the molecular mechanisms underlying the aging-related decline of SMG function and associated salivary gland hypofunction, which is particularly problematic among the elderly. PMID:25832744

Loss of the tumor suppressor p15Ink4b enhances myeloid progenitor formation from common myeloid progenitors.

PubMed

Rosu-Myles, Michael; Taylor, Barbara J; Wolff, Linda

2007-03-01

The tumor suppressor p15Ink4b (Ink4b) is a cell-cycle inhibitor that is inactivated in a high percentage of acute myeloid leukemia and myeloid dysplasia syndrome cases. Despite this, the role of Ink4b in hematopoiesis remains unclear. Here we examined the role of Ink4b in blood cell formation using Ink4b-deficient (Ink4b(-/-)) mice. We compared the bone marrow (BM) of Ink4b(-/-) and wild-type mice using flow cytometric, colony-forming unit and competitive repopulating assays (CRA). The proliferation, differentiation, self-renewal, and apoptosis of progenitor cells were further compared by in vitro and in vivo methods. BM from Ink4b(-/-) mice contained increased numbers of granulocyte-monocyte progenitors and Gr-1(+) cells and showed a competitive advantage over wild-type cells in myeloid cell formation by CRA. Ink4b(-/-) progenitors did not demonstrate increased proliferation, self-renewing potential, or reduced apoptosis. Instead, Ink4b(-/-) common myeloid progenitors (CMPs) showed increased myeloid progenitor formation concomitant with reduced erythroid potential. This work establishes a role for Ink4b in regulating the differentiation of CMPs and indicates that loss of Ink4b enhances the formation of myeloid progenitors.

Association between P16INK4a Promoter Methylation and Non-Small Cell Lung Cancer: A Meta-Analysis

PubMed Central

Zhu, Siwei; Hua, Feng; Zhao, Hui; Xu, Hongrui; You, Jiacong; Sun, Linlin; Wang, Weiqiang; Chen, Jun; Zhou, Qinghua

2013-01-01

Background Aberrant methylation of CpG islands acquired in tumor cells in promoter regions plays an important role in carcinogenesis. Accumulated evidence demonstrates P16INK4a gene promoter hypermethylation is involved in non-small cell lung carcinoma (NSCLC), indicating it may be a potential biomarker for this disease. The aim of this study is to evaluate the frequency of P16INK4a gene promoter methylation between cancer tissue and autologous controls by summarizing published studies. Methods By searching Medline, EMBSE and CNKI databases, the open published studies about P16INK4a gene promoter methylation and NSCLC were identified using a systematic search strategy. The pooled odds of P16INK4A promoter methylation in lung cancer tissue versus autologous controls were calculated by meta-analysis method. Results Thirty-four studies, including 2 652 NSCLC patients with 5 175 samples were included in this meta-analysis. Generally, the frequency of P16INK4A promoter methylation ranged from 17% to 80% (median 44%) in the lung cancer tissue and 0 to 80% (median 15%) in the autologous controls, which indicated the methylation frequency in cancer tissue was much higher than that in autologous samples. We also find a strong and significant correlation between tumor tissue and autologous controls of P16INK4A promoter methylation frequency across studies (Correlation coefficient 0.71, 95% CI:0.51–0.83, P<0.0001). And the pooled odds ratio of P16INK4A promoter methylation in cancer tissue was 3.45 (95% CI: 2.63–4.54) compared to controls under random-effect model. Conclusion Frequency of P16INK4a promoter methylation in cancer tissue was much higher than that in autologous controls, indicating promoter methylation plays an important role in carcinogenesis of the NSCLC. Strong and significant correlation between tumor tissue and autologous samples of P16INK4A promoter methylation demonstrated a promising biomarker for NSCLC. PMID:23577085

The tumour suppressor CDKN2A/p16INK4a regulates adipogenesis and bone marrow-dependent development of perivascular adipose tissue

PubMed Central

Wouters, Kristiaan; Deleye, Yann; Hannou, Sarah A; Vanhoutte, Jonathan; Maréchal, Xavier; Coisne, Augustin; Tagzirt, Madjid; Derudas, Bruno; Bouchaert, Emmanuel; Duhem, Christian; Vallez, Emmanuelle; Schalkwijk, Casper G; Pattou, François; Montaigne, David; Staels, Bart; Paumelle, Réjane

2017-01-01

The genomic CDKN2A/B locus, encoding p16INK4a among others, is linked to an increased risk for cardiovascular disease and type 2 diabetes. Obesity is a risk factor for both cardiovascular disease and type 2 diabetes. p16INK4a is a cell cycle regulator and tumour suppressor. Whether it plays a role in adipose tissue formation is unknown. p16INK4a knock-down in 3T3/L1 preadipocytes or p16INK4a deficiency in mouse embryonic fibroblasts enhanced adipogenesis, suggesting a role for p16INK4a in adipose tissue formation. p16INK4a-deficient mice developed more epicardial adipose tissue in response to the adipogenic peroxisome proliferator activated receptor gamma agonist rosiglitazone. Additionally, adipose tissue around the aorta from p16INK4a-deficient mice displayed enhanced rosiglitazone-induced gene expression of adipogenic markers and stem cell antigen, a marker of bone marrow-derived precursor cells. Mice transplanted with p16INK4a-deficient bone marrow had more epicardial adipose tissue compared to controls when fed a high-fat diet. In humans, p16INK4a gene expression was enriched in epicardial adipose tissue compared to other adipose tissue depots. Moreover, epicardial adipose tissue from obese humans displayed increased expression of stem cell antigen compared to lean controls, supporting a bone marrow origin of epicardial adipose tissue. These results show that p16INK4a modulates epicardial adipose tissue development, providing a potential mechanistic link between the genetic association of the CDKN2A/B locus and cardiovascular disease risk. PMID:28868898

Overexpression of the Cell Cycle Inhibitor p16INK4a Promotes a Prothrombotic Phenotype Following Vascular Injury in Mice

PubMed Central

Cardenas, Jessica C.; Owens, A. Phillip; Krishnamurthy, Janakiraman; Sharpless, Norman E.; Whinna, Herbert C.; Church, Frank C.

2011-01-01

Objective Age-associated cellular senescence is thought to promote vascular dysfunction. p16INK4a is a cell cycle inhibitor that promotes senescence and is upregulated during normal aging. In this study, we examine the contribution of p16INK4a overexpression on venous thrombosis. Methods and Results Mice overexpressing p16INK4a were studied with four different vascular injury models: (1) ferric chloride (FeCl3) and (2) Rose Bengal to induce saphenous vein thrombus formation; (3) FeCl3 and vascular ligation to examine thrombus resolution; and (4) LPS administration to initiate inflammation-induced vascular dysfunction. p16INK4a transgenic mice had accelerated occlusion times (13.1 ± 0.4 min) compared to normal controls (19.7 ± 1.1 min) in the FeCl3 model and 12.7 ± 2.0 and 18.6 ± 1.9, respectively in the Rose Bengal model. Moreover, overexpression of p16INK4a delayed thrombus resolution compared to normal controls. In response to LPS treatment, the p16INK4a transgenic mice showed enhanced thrombin generation in plasma-based calibrated automated thrombography (CAT) assays. Finally, bone marrow transplantation studies suggested increased p16INK4a expression in hematopoietic cells contributes to thrombosis, demonstrating a role for p16INK4a expression in venous thrombosis. Conclusions Venous thrombosis is augmented by overexpression of the cellular senescence gene p16INK4a. PMID:21233453

p16(INK4A) inhibits the pro-metastatic potentials of osteosarcoma cells through targeting the ERK pathway and TGF-β1.

PubMed

Silva, Gabriela; Aboussekhra, Abdelilah

2016-05-01

Extracellular signal-regulated kinase (ERK) is a downstream component of the evolutionarily conserved mitogen-activated protein kinase-signaling pathway, which controls the expression of a plethora of genes implicated in various physiological processes. This pathway is often hyper-activated by mutations or abnormal extracellular signaling in different types of human cancer, including the most common primary malignant bone tumor osteosarcomas. p16(INK4A) is an important tumor suppressor gene frequently lost in osteosarcomas, and is associated with the progression of these malignancies. We have shown, here, that the ERK1/2 protein kinase is also activated by p16(INK4A) down-regulation in osteosarcoma cells and normal human as well as mouse cells. This inhibitory effect is associated with the suppression of the upstream kinase MEK1/2, and is mediated via the repression of miR-21-5p and the consequent up-regulation of the MEK/ERK antagonist SPRY2 in osteosarcoma cells. Furthermore, we have shown that p16(INK4) inhibits the migration/invasion abilities of these cells through miR-21-5p-dependent inhibition of ERK1/2. In addition, we present clear evidence that p16(INK4) represses the paracrine pro-migratory effect of osteosarcoma cells on stromal fibroblasts through the inhibition of the TGF-β1 expression/secretion. This effect is also ERK1/2-dependent, indicating that in addition to their cell-autonomous actions, p16(INK4) and ERK1/2 have also non-cell-autonomous cancer-related functions. Together, these results indicate that the tumor suppressor p16(INK4) protein represses the carcinogenic process of osteosarcoma cells not only as a cell cycle regulator, but also as a negative regulator of pro-carcinogenic/-metastatic pathways. This indicates that targeting the ERK pathway is of utmost therapeutic value. © 2015 Wiley Periodicals, Inc.

Human papillomavirus infection and immunohistochemical p16(INK4a) expression as predictors of outcome in penile squamous cell carcinomas.

PubMed

Bezerra, Stephania M; Chaux, Alcides; Ball, Mark W; Faraj, Sheila F; Munari, Enrico; Gonzalez-Roibon, Nilda; Sharma, Rajni; Bivalacqua, Trinity J; Burnett, Arthur L; Netto, George J

2015-04-01

Approximately 50% of penile squamous cell carcinomas (SCC) are associated with high-risk human papillomavirus (HR-HPV) infection. We evaluated the correlation of p16(INK4a) expression and HR-HPV with clinicopathological features and outcome in a cohort of patients with penile SCC. Two tissue microarrays were constructed from 53 invasive penile SCC at our hospital. p16(INK4a) expression was assessed by immunohistochemistry (CINtec Kit). High-risk human papillomavirus status was assessed by in situ hybridization (INFORM HPV III family 16 probe B cocktail). High-risk human papillomavirus was detected in 8 cases (15%), and p16(INK4a) overexpression was found in 23 cases (44%). Both markers showed a significant association with histologic subtype (P = .017 and P = .01, respectively) and lymphovascular invasion (P = .015 and P = .015, respectively). Regarding outcome analyses, neither HPV infection nor p16(INK4a) overexpression significantly predicted overall survival or cancer-specific survival using Cox proportional hazards regression model. High-risk human papillomavirus positivity and p16(INK4a) overexpression were significantly associated with histologic subtype and presence of lymphovascular invasion. Human papillomavirus status was not predictive of outcome in our cohort. Copyright © 2015 Elsevier Inc. All rights reserved.

Evidence that P12, a specific variant of P16{sup INK4A}, plays a suppressive role in human pancreatic carcinogenesis

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

Poi, Ming J.; Knobloch, Thomas J.; Division of Environmental Health Sciences, College of Public Health, The Ohio State University, Columbus, OH 43210

2013-06-28

Highlights: •P12, a variant of P16{sup INK4A}, inhibits the proliferation of pancreatic cancer cells. •P12 is distinct from P16 in function and structure. •Genetic alterations of p12 are prevalent in human pancreatic carcinoma. •P12 represents a potential pancreas-specific tumor suppressor. -- Abstract: The INK4a-ARF locus plays a central role in the development of pancreatic tumors as evidenced by the fact that up to 98% of pancreatic tumor specimens harbored genetic alterations at the INK4a-ARF locus. Interestingly, in addition to the well-known P16{sup INK4A} (P16) and P14ARF tumor suppressors, the INK4a-ARF locus in pancreas encodes another protein, P12, whose structure, function,more » and contributions to pancreatic carcinogenesis remain to be elucidated. In the current study, we demonstrated that over-expression of p12 in human pancreatic cancer cells led to cell arrest at the G1 phase and such cell cycle arrest was related to down-regulation of a number of oncogenes, such as c-Jun, Fos, and SEI1. Furthermore, unlike P16, P12 did not retain any cyclin-dependent kinase 4 (CDK4)-inhibitory activity. Instead, P12 exhibited a transactivating activity not found in P16. We also examined the genetic status of p12 in a cohort of 40 pancreatic tumor specimens and found that p12 alteration was prevalent in pancreatic tumors with an incidence of 70% (28/40). These results support that P12 is a tumor suppressive protein distinct from P16, and its genetic inactivation is associated with pancreatic carcinogenesis.« less

p16(INK4a) promoter methylation and protein expression in breast fibroadenoma and carcinoma.

PubMed

Di Vinci, Angela; Perdelli, Luisa; Banelli, Barbara; Salvi, Sandra; Casciano, Ida; Gelvi, Ilaria; Allemanni, Giorgio; Margallo, Edoardo; Gatteschi, Beatrice; Romani, Massimo

2005-04-10

The potential role of p16(INK4a) methylation in breast cancer is controversial whereas there are no data on fibroadenoma. To assess if inactivation of p16(INK4a) by promoter hypermethylation occurs in this hyperproliferative benign breast lesion or, on the contrary, it is strictly related to the carcinogenic process, we have tested the different histological components of 15 cases of fibroadenoma and the intraductal and infiltrating components of 15 cases of carcinoma and their adjacent non-tumoral epithelium. All samples were obtained by laser-assisted microdissection. The relationship between promoter methylation status, immunohistochemical protein expression and ki67 proliferative activity was evaluated for each lesion. Our data demonstrate that hypermethylation of p16(INK4a) promoter is a common event occurring at similar frequency in all the different histological areas of the benign and malignant breast lesions taken into exam. Conversely, protein p16 expression, although heterogeneously distributed within the section, is considerably higher in breast carcinoma as compared to fibroadenoma in both tumoral and non-tumoral epithelia and stroma. The protein localization was almost exclusively nuclear in fibroadenoma and non-tumoral epithelia whereas, in carcinoma, the staining was both nuclear and cytoplasmic or cytoplasmic alone. Furthermore, in a subset of fibroadenoma with higher proliferative activity, p16 protein expression was substantially decreased as compared to those showing lower proliferation. We did not observe this association in carcinomas. Our data demonstrate that the hypermethylation of the p16(INK4a) promoter is not specifically associated with malignancy and that, on the contrary, the overexpression of p16 and its cytoplasmic sequestration is a feature of breast carcinoma. (c) 2004 Wiley-Liss, Inc.

p16INK4a deficiency promotes IL-4-induced polarization and inhibits proinflammatory signaling in macrophages

PubMed Central

Cudejko, Céline; Wouters, Kristiaan; Fuentes, Lucía; Hannou, Sarah Anissa; Paquet, Charlotte; Bantubungi, Kadiombo; Bouchaert, Emmanuel; Vanhoutte, Jonathan; Fleury, Sébastien; Remy, Patrick; Tailleux, Anne; Chinetti, Giulia; Dombrowicz, David; Staels, Bart; Paumelle, Réjane

2011-01-01

The CDKN2A locus, which contains the tumor suppressor gene p16INK4a, is associated with an increased risk of age-related inflammatory diseases, such as cardiovascular disease and type 2 diabetes, in which macrophages play a crucial role. Monocytes can polarize towards classically (CAMφ) or alternatively (AAMφ) activated macrophages. However, the molecular mechanisms underlying the acquisition of these phenotypes are not well defined. Here, we show that p16INK4a-deficiency (p16−/−) modulates the macrophage phenotype. Transcriptome analysis revealed that p16−/− bone marrow-derived macrophages (BMDM) exhibit a phenotype resembling interleukin (IL)-4-induced macrophage polarization. In line with this observation, p16−/− BMDM displayed a decreased response to classically polarizing IFNγ and LPS and an increased sensitivity to alternative polarization by IL-4. Furthermore, mice transplanted with p16−/− bone marrow displayed higher hepatic AAMφ marker expression levels upon Schistosoma mansoni infection, an in vivo model of AAMφ phenotype-skewing. Surprisingly, p16−/− BMDM did not display increased IL-4-induced STAT6 signaling, but decreased IFNγ-induced STAT1 and LPS-induced IKKα,β phosphorylation. This decrease correlated with decreased JAK2 phosphorylation and with higher levels of inhibitory acetylation of STAT1 and IKKα,β. These findings identify p16INK4a as a modulator of macrophage activation and polarization via the JAK2-STAT1 pathway with possible roles in inflammatory diseases. PMID:21636855

Differential p16/INK4A cyclin-dependent kinase inhibitor expression correlates with chemotherapy efficacy in a cohort of 88 malignant pleural mesothelioma patients.

PubMed

Jennings, C J; Murer, B; O'Grady, A; Hearn, L M; Harvey, B J; Kay, E W; Thomas, W

2015-06-30

Malignant pleural mesothelioma (MPM) is a rare and essentially incurable malignancy most often linked with occupational exposure to asbestos fibres. In common with other malignancies, the development and progression of MPM is associated with extensive dysregulation of cell cycle checkpoint proteins that modulate cell proliferation, apoptosis, DNA repair and senescence. The expression of cyclin-dependent kinase inhibitor p16/INK4A was evaluated by immunohistochemistry using tumour biopsy specimens from 88 MPM cases and a semi-quantitative score for p16/INK4A expression was obtained. Post-diagnosis survival and the survival benefit of chemotherapeutic intervention was correlated with p16/INK4A expression. A low, intermediate and high score for p16/INK4A expression was observed for 45 (51.1%), 28 (31.8%) and 15 (17.1%) of the MPM cases, respectively. Those cases with intermediate or high p16/INK4A tumour expression had a significantly better post-diagnosis survival than those cases whose tumours lost p16 expression (log-rank P<0.001). Those patients with sustained p16/INK4A expression who received chemotherapy also had a better survival than those treated patients whose tumours had lost p16/INK4A expression (log-rank P<0.001). Sustained p16/INK4A expression predicts better post-diagnosis survival in MPM and also better survival following chemotherapeutic intervention.

Clinical and prognosis value of the CIMP status combined with MLH1 or p16 INK4a methylation in colorectal cancer.

PubMed

Saadallah-Kallel, Amana; Abdelmaksoud-Dammak, Rania; Triki, Mouna; Charfi, Slim; Khabir, Abdelmajid; Sallemi-Boudawara, Tahia; Mokdad-Gargouri, Raja

2017-08-01

Aberrant DNA methylation of CpG islands occurred frequently in CRC and associated with transcriptional silencing of key genes. In this study, the CIMP combined with MLH1 or p16 INK4a methylation status was determined in CRC patients and correlated with clinicopathological parameters and overall survival. Our data showed that CIMP+ CRCs were identified in 32.9% of cases and that CACNAG1 is the most frequently methylated promoter. When we combined the CIMP with the MLH1 or the p16 INK4a methylation status, we found that CIMP-/MLH1-U (37.8%) and CIMP-/p16 INK4a -U (35.4%) tumors were the most frequent among the four subtypes. Statistical analysis showed that tumor location, lymphovascular invasion, TNM stage, and MSI differed among the group of patients. Kaplan-Meier analyses revealed differences in overall survival according to the CIMP combined with MLH1 or p16 INK4a methylation status. In a multivariate analysis, CIMP/MLH1 and CIMP/p16 INK4a methylation statuses were predictive of prognosis, and the OS was longer for patients with tumors CIMP-/MLH1-M, as well as CIMP-/p16 INK4a -M. Furthermore, DNMT1 is significantly overexpressed in tumors than in normal tissues as well as in CIMP+ than CIMP- tumors. Our results suggest that tumor classification based on the CIMP status combined with MLH1 or p16 INK4a methylation is useful to predict prognosis in CRC patients.

Cooperation of p27Kip1 and p18INK4c in Progestin-Mediated Cell Cycle Arrest in T-47D Breast Cancer Cells

PubMed Central

Swarbrick, Alexander; Lee, Christine S. L.; Sutherland, Robert L.; Musgrove, Elizabeth A.

2000-01-01

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G1 cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27Kip1 among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27Kip1 and few were bound to p21Cip1. In vitro, recombinant His6-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His6-p27 in vitro or p27Kip1 in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18INK4c and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18INK4c led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27Kip1 and p18INK4c cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor β and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation. PMID:10713180

Differential p16/INK4A cyclin-dependent kinase inhibitor expression correlates with chemotherapy efficacy in a cohort of 88 malignant pleural mesothelioma patients

PubMed Central

Jennings, C J; Murer, B; O'Grady, A; Hearn, L M; Harvey, B J; Kay, E W; Thomas, W

2015-01-01

Background: Malignant pleural mesothelioma (MPM) is a rare and essentially incurable malignancy most often linked with occupational exposure to asbestos fibres. In common with other malignancies, the development and progression of MPM is associated with extensive dysregulation of cell cycle checkpoint proteins that modulate cell proliferation, apoptosis, DNA repair and senescence. Methods: The expression of cyclin-dependent kinase inhibitor p16/INK4A was evaluated by immunohistochemistry using tumour biopsy specimens from 88 MPM cases and a semi-quantitative score for p16/INK4A expression was obtained. Post-diagnosis survival and the survival benefit of chemotherapeutic intervention was correlated with p16/INK4A expression. Results: A low, intermediate and high score for p16/INK4A expression was observed for 45 (51.1%), 28 (31.8%) and 15 (17.1%) of the MPM cases, respectively. Those cases with intermediate or high p16/INK4A tumour expression had a significantly better post-diagnosis survival than those cases whose tumours lost p16 expression (log-rank P<0.001). Those patients with sustained p16/INK4A expression who received chemotherapy also had a better survival than those treated patients whose tumours had lost p16/INK4A expression (log-rank P<0.001). Conclusions: Sustained p16/INK4A expression predicts better post-diagnosis survival in MPM and also better survival following chemotherapeutic intervention. PMID:26057448

Investigation of p16(INK4a) as a prognostic biomarker in oral epithelial dysplasia.

PubMed

Nankivell, Paul; Williams, Hazel; Webster, Keith; Pearson, David; High, Alec; MacLennan, Kenneth; Senguven, Burcu; McConkey, Christopher; Rabbitts, Pamela; Mehanna, Hisham

2014-04-01

Human papilloma virus is a risk factor for oropharyngeal cancer. Evidence for a similar aetiological role in the development of oral dysplasia or its transformation to oral cancer is not as clear. Meta-analyses estimate the prevalence of high-risk human papilloma virus (HPV) serotypes to be three times higher in pre-malignant lesions and cancer than in normal oral mucosa. However, this does not imply a causal relationship. Conflicting results are reported from the few studies examining the prognostic significance of HPV positivity in the development of oral cancer. We aimed to examine the ability of p16(INK4a) protein expression, a surrogate marker of HPV infection, to predict malignant progression in a large cohort of oral dysplasia patients. One hundred forty eight oral dysplasia cases underwent immunohistochemical analysis using a monoclonal antibody against p16(INK4a) . Clinical factors were also collated on each case. Slides were double scored independently by two trained observers. Univariate analyses using both logistic and Cox regression models were performed. Thirty nine of 148 cases progressed to cancer. Ten of 148 cases (7%) were p16(INK4a) positive. High grade of dysplasia (P = 0.0002) and lesion morphology (P = 0.03) were found to be prognostic of malignant progression. p16(INK4a) score was not prognostic in this cohort (P = 0.29). This did not change with a time to event analysis (P = 0.24). Few studies have assessed the aetiological role of HPV in cancer development from dysplastic lesions. Our study, using one of the largest cohorts of oral dysplasia, demonstrated a low rate of p16(INK4a) positivity and was unable to confirm a prognostic ability for this biomarker. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Bronchial lavage P 16INK4A gene promoter methylation and lung cancer diagnosis: A meta-analysis.

PubMed

Yifan, D; Qun, L; Yingshuang, H; Xulin, L; Jianjun, W; Qian, M; Yuman, Yu; Zhaoyang, R

2015-12-01

To evaluate the diagnostic value of bronchial lavage P16INK4A promoter methylation and lung cancer. The databases of PubMed, Medline, China National Knowledge Infrastructure, and Wanfang were electronically searched by two reviewers to find the suitable studies related to the association between P16INK4A promoter methylation and lung cancer. The P16INK4A promoter methylation rate was extracted from each included individual study. The diagnostic sensitivity, specificity, and area under the receiver operating characteristic ROC curve of bronchial lavage P16INK4Aas a biomarker for diagnosis of lung cancer were pooled by stata 11.0 software (Stata Corporation, College Station, TX, USA). At last, 10 publications were included in this meta-analysis. Of the included 10 studies, five are published in English with relatively high quality and other five papers published in Chinese have relatively low quality. The pooled sensitivity and specificity of bronchial lavage P16INK4A promoter methylation for lung cancer diagnosis were 0.61 (95% confidence interval [CI]: 0.57-0.65) and 0.81 (95% CI: 0.78-0.85), respectively, with random effect model. The ROC curve were calculated and drawn according to Bayes' theorem by stata 11.0 software. The systematic area under the ROC was 0.72 (95% CI: 0.68-0.76), which indicated that the diagnostic value of bronchial lavage P16INK4A promoter methylation for lung cancer was relatively high. Moreover, no significant publication bias was existed in this meta-analysis (t = 0.69, P > 0.05). Bronchial lavage P16INK4A promoter methylation can be a potential biomarker for diagnosis of lung cancer.

The Regulatory Mechanisms of Tumor Suppressor P16INK4A and Relevance to Cancer†

PubMed Central

Li, Junan; Poi, Ming Jye; Tsai, Ming-Daw

2011-01-01

P16INK4A (also known as P16 and MTS1), a protein consisting exclusively of four ankyrin repeats, is recognized as a tumor suppressor mainly due to the prevalence of genetic inactivation of the p16INK4A (or CDKN2A) gene in virtually all types of human cancers. However, it has also been shown that elevated expression (up-regulation) of P16 is involved in cellular senescence, aging, and cancer progression, indicating that the regulation of P16 is critical for its function. Here, we discuss the regulatory mechanisms of P16 function at the DNA level, the transcription level, and the posttranscriptional level, as well as their implications in the structure-function relationship of P16 and in human cancers. PMID:21619050

The retinoblastoma protein/p16 INK4A pathway but not p53 is disrupted by human papillomavirus in penile squamous cell carcinoma.

PubMed

Stankiewicz, Elzbieta; Prowse, David M; Ktori, Elena; Cuzick, Jack; Ambroisine, Laurence; Zhang, Xiaoxi; Kudahetti, Sakunthala; Watkin, Nicholas; Corbishley, Catherine; Berney, Daniel M

2011-02-01

The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood. Human papillomavirus (HPV) may be involved in carcinogenesis, but few studies have compared cell-cycle protein expression in HPV positive and negative cancers. The aim was to determine the extent of HPV infection in different histological subtypes of PSCC and its impact on the expression of key cell-cycle proteins: p53, p21, p16(INK4A) and retinoblastoma (RB) protein. One hundred and forty-eight PSCC samples were examined immunohistochemically for RB, p16(INK4A) , p53 and p21 protein expression. One hundred and two cases were typed for HPV by PCR. HPV DNA was detected in 56% of tumours, with HPV16 present in 81%. Basaloid tumours were related strongly to HPV infection (10 of 13), while verrucous were not (three of 13). Fifty-nine per cent (38 of 64) of usual type SCCs had HPV infection. RB protein correlated negatively (P<0.0001) and p16(INK4A) (P<0.0001) and p21 (P=0.0002) correlated positively with HPV infection. p53 did not correlate with HPV infection. HPV infection is present in more than half of penile cancers and it is responsible for RB pathway disruption. However, no link between HPV and p53 immunodetection was found. Only basaloid and half of usual-type PSSCs correlate with HPV infection, confirming possible separate aetiologies for those tumours. © 2011 Blackwell Publishing Limited.

Deregulated expression of p16INK4a and p53 pathway members in benign and malignant myoepithelial tumours of the salivary glands.

PubMed

Vékony, H; Röser, K; Löning, T; Raaphorst, F M; Leemans, C R; Van der Waal, I; Bloemena, E

2008-12-01

Myoepithelial salivary gland tumours are uncommon and follow an unpredictable biological course. The aim was to examine their molecular background to acquire a better understanding of their clinical behaviour. Expression of protein (E2F1, p16(INK4a), p53, cyclin D1, Ki67 and Polycomb group proteins BMI-1, MEL-18 and EZH2) was investigated in 49 benign and 30 primary malignant myoepithelial tumours and five histologically benign recurrences by immunohistochemistry and the findings correlated with histopathological characteristics. Benign tumours showed a higher percentage of cells with expression of p16(INK4a) pathway members [p16(INK4a) and E2F1 (both P < 0.001), and cyclin D1, P = 0.002] compared with normal salivary gland. Furthermore, malignant tumours expressed p53 (P = 0.003) and EZH2 (P = 0.09) in a higher percentage. Recurrences displayed more p53 + tumour cells (P = 0.02) than benign primaries. Amongst the benign tumours, the clear cell type had the highest proliferation fraction (P = 0.05) and a higher percentage of EZH2 was detected in the plasmacytoid cell type (P = 0.002). This study is the first to demonstrate that deregulation of the p16(INK4a) senescence pathway is involved in the development of myoepithelial tumours. We propose that additional inactivation of p53 in malignant primaries and benign recurrences contributes to myoepithelial neoplastic transformation and aggressive tumour growth.

Associations of P16INK4a promoter hypermethylation with squamous intra-epithelial lesion, cervical cancer and their clinicopathological features: a meta-analysis

PubMed Central

Cui, Ning-hua; Zhang, Shuai; Wang, Chen; Zheng, Fang

2017-01-01

To assess the associations of P16INK4a methylation status with low-grade squamous intra-epithelial lesion (LSIL), high-grade squamous intra-epithelial lesion (HSIL), cervical cancer (CC) and their clinicopathological features, a meta-analysis with 29 eligible studies was conducted. Pooled odds ratios (ORs) with their 95% confidence intervals (CIs) were estimated to assess the strength of the associations. Heterogeneity, sensitivity of pooled results and publication bias were also evaluated. Overall, there was an increasing trend of P16INK4a hypermethylation rates among LSIL (21.4%), HSIL (30.9%) and CC (35.0%) specimens. P16INK4a hypermethylation was significantly associated with the increased risk of LSIL, HSIL and CC, with the pooled ORs of 3.26 (95% CI: 1.86-5.71), 5.80 (95% CI: 3.80-8.84) and 12.17 (95% CI: 5.86-25.27), respectively. A significant association was also found between P16INK4a hypermethylation and smoking habit (OR = 3.88, 95% CI: 2.13-7.08). Taken together, meta-analysis results support P16INK4a hypermethylation as an epigenetic marker for the progression of cervical carcinogenesis. PMID:27669738

Efficient immortalization of primary human cells by p16INK4a-specific short hairpin RNA or Bmi-1, combined with introduction of hTERT.

PubMed

Haga, Kei; Ohno, Shin-ichi; Yugawa, Takashi; Narisawa-Saito, Mako; Fujita, Masatoshi; Sakamoto, Michiie; Galloway, Denise A; Kiyono, Tohru

2007-02-01

Activation of telomerase is sufficient for immortalization of some types of human cells but additional factors may also be essential. It has been proposed that stress imposed by inadequate culture conditions induces senescence due to accumulation of p16(INK4a). Here, we present evidence that many human cell types undergo senescence by activation of the p16(INK4a)/Rb pathway, and that introduction of Bmi-1 can inhibit p16(INK4a) expression and extend the life span of human epithelial cells derived from skin, mammary gland and lung. Introduction of p16(INK4a)-specific short hairpin RNA, as well as Bmi-1, suppressed p16(INK4a) expression in human mammary epithelial cells without promoter methylation, and extended their life span. Subsequent introduction of hTERT, the telomerase catalytic subunit, into cells with low p16(INK4a) levels resulted in efficient immortalization of three cell types without crisis or growth arrest. The majority of the human mammary epithelial cells thus immortalized showed almost normal ploidy as judged by G-banding and spectral karyotyping analysis. Our data suggest that inhibition of p16(INK4a) and introduction of hTERT can immortalize many human cell types with little chromosomal instability.

PD-L1 expression is associated with p16INK4A expression in non-oropharyngeal head and neck squamous cell carcinoma

PubMed Central

Chen, San-Chi; Chang, Peter Mu-Hsin; Wang, Hsiao-Jung; Tai, Shyh-Kuan; Chu, Pen-Yuan; Yang, Muh-Hwa

2018-01-01

PD-L1 expression is critical in helping tumor cells evade the immune system. However, the level of PD-L1 expression in non-oropharyngeal head and neck squamous cell carcinoma (non-OPHNSCC) and its association with patient prognosis remains unclear. A retrospective clinicopathological analysis was performed on 106 patients with non-OPHNSCC diagnosed between 2007 and 2014. In the current study, tissue arrays from paraffin-embedded non-OPHNSCC samples obtained from patients were constructed, and PD-L1 and p16INK4A expression were determined using immunohistochemistry. Systemic inflammatory factors, including C-reactive protein, serum white blood cell, neutrophil, monocyte and lymphocyte counts were also analyzed. The current study demonstrated that PD-L1 was overexpressed in 32.1% (34/106) and p16INK4A in 20.8% (22/106) of patients. The expression of PD-L1 was associated with p16INK4A expression (P<0.01) but was not associated with levels of systemic inflammatory factors. Tumor stage was determined to be a significant prognostic value (stage I/II vs. III/IV, P=0.03), however, PD-L1, p16INK4A or other clinicopathological factors were not. The current study identified an association between PD-L1 and p16INK4A expression in non-OPHNSCC. This may facilitate the development of anti-PD1/PDL1 therapies to treat patients with head and neck cancer. PMID:29434933

Structural Basis of CDK4 Inhibition by p18INK4

DTIC Science & Technology

1999-05-01

have determined the crystal structure of p 18INK4c to 1.95 A resolution [4] and the atomic coordinates have been deposited in the PDB protein...p 18INK4c function. The results were published [4] (Attached) and the coordinates were deposited in the PDB Protein Structure Database (Accession...Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. ŘCurrent address: Institute of Molecular Agrobiology, National University of

p16INK4a immunostaining in cytological and histological specimens from the uterine cervix: a systematic review and meta-analysis

PubMed Central

Tsoumpou, I; Arbyn, M; Kyrgiou, M; Wentzensen, N; Koliopoulos, G; Martin-Hirsch, P; Paraskevaidis, E

2009-01-01

Background P16INK4a is a biomarker for transforming HPV infections that could act as an adjunct to current cytological and histological assessment of cervical smears and biopsies, allowing the identification of those women with ambiguous results that require referral to colposcopy and potentially treatment. Material and Methods We conducted a systematic review of all studies that evaluated the use of p16INK4a in cytological or histological specimens from the uterine cervix. We also estimated the mean proportion of samples that were positive for p16INK4a in cytology and histology, stratified by the grade of the lesion. Results Sixty-one studies were included. The proportion of cervical smears overexpressing p16INK4a increased with the severity of cytological abnormality. Among normal smears, only 12% (95% CI: 7–17%) were positive for the biomarker compared to 45% of ASCUS and LSIL (95% CI: 35–54% and 37– 57% respectively) and 89% of HSIL smears (95% CI: 84–95%). Similarly, in histology only 2% of normal biopsies (95% CI: 0.4– 30%) and 38% of CIN1 (95% CI: 23– 53%) showed diffuse staining for p16INK4a compared to 68% of CIN2 (95% CI: 44– 92%) and 82% of CIN3 (95% CI: 72– 92%). Conclusion Although there is good evidence that p16INK4a immunostaining correlates with the severity of cytological/histological abnormalities, the reproducibility is limited due to insufficiently standardized interpretation of the immunostaining. Therefore, a consensus needs to be reached regarding the evaluation of p16INK4a staining and the biomarker needs to be evaluated in various clinical settings addressing specific clinical questions. PMID:19261387

The role of histologic subtype, p16(INK4a) expression, and presence of human papillomavirus DNA in penile squamous cell carcinoma.

PubMed

Steinestel, Julie; Al Ghazal, Andreas; Arndt, Annette; Schnoeller, Thomas J; Schrader, Andres J; Moeller, Peter; Steinestel, Konrad

2015-04-03

Up to 50% of penile squamous cell carcinomas (pSCC) develop in the context of high-risk human papillomavirus (HR-HPV) infection. Most of these tumours have been reported to show basaloid differentiation and overexpression of tumour suppressor protein p16(INK4a). Whether HPV-triggered carcinogenesis in pSCC has an impact on tumour aggressiveness, however, is still subject to research. In tissue specimens from 58 patients with surgically treated pSCC between 1995 and 2012, we performed p16(INK4a) immunohistochemistry and DNA extraction followed by HPV subtyping using a PCR-based approach. The results were correlated with histopathological and clinical parameters. 90.4% of tumours were of conventional (keratinizing) subtype. HR-HPV DNA was detected in 29.3%, and a variety of p16(INK4a) staining patterns was observed in 58.6% of samples regardless of histologic subtype. Sensitivity of basaloid subtype to predict HR-HPV positivity was poor (11.8%). In contrast, sensitivity and specificity of p16(INK4a) staining to predict presence of HR-HPV DNA was 100% and 57%, respectively. By focussing on those samples with intense nuclear staining pattern for p16(INK4a), specificity could be improved to 83%. Both expression of p16(INK4a) and presence of HR-HPV DNA, but not histologic grade, were inversely associated with pSCC tumour invasion (p = 0.01, p = 0.03, and p = 0.71). However, none of these correlated with nodal involvement or distant metastasis. In contrast to pathological tumour stage, the HR-HPV status, histologic grade, and p16(INK4a) positivity failed to predict cancer-specific survival. Our results confirm intense nuclear positivity for p16(INK4a), rather than histologic subtype, as a good predictor for presence of HR-HPV DNA in pSCC. HR-HPV / p16(INK4a) positivity, independent of histological tumour grade, indicates a less aggressive local behaviour; however, its value as an independent prognostic indicator remains to be determined. Since local invasion

p15Ink4b is Key in Dendritic Cell Development | Center for Cancer Research

Cancer.gov

An important step in the initiation of leukemia is the ability of pre-leukemic and leukemic cells to evade the immune system. Dendritic cells are instrumental in maintaining the body’s immunity, and CCR scientists have shown for the first time that the tumor suppressor protein p15Ink4b regulates the differentiation and maturation of conventional dendritic cells.

Tumor suppressor p16 INK4a: Downregulation of galectin-3, an endogenous competitor of the pro-anoikis effector galectin-1, in a pancreatic carcinoma model.

PubMed

Sanchez-Ruderisch, Hugo; Fischer, Christian; Detjen, Katharina M; Welzel, Martina; Wimmel, Anja; Manning, Joachim C; André, Sabine; Gabius, Hans-Joachim

2010-09-01

The tumor suppressor p16(INK4a) has functions beyond cell-cycle control via cyclin-dependent kinases. A coordinated remodeling of N- and O-glycosylation, and an increase in the presentation of the endogenous lectin galectin-1 sensing these changes on the surface of p16(INK4a)-expressing pancreatic carcinoma cells (Capan-1), lead to potent pro-anoikis signals. We show that the p16(INK4a)-dependent impact on growth-regulatory lectins is not limited to galectin-1, but also concerns galectin-3. By monitoring its expression in relation to p16(INK4a) status, as well as running anoikis assays with galectin-3 and cell transfectants with up- or downregulated lectin expression, a negative correlation between anoikis and the presence of this lectin was established. Nuclear run-off and northern blotting experiments revealed an effect of the presence of p16(INK4a) on steady-state levels of galectin-3-specific mRNA that differed from decreasing the transcriptional rate. On the cell surface, galectin-3 interferes with galectin-1, which initiates signaling toward its pro-anoikis activity via caspase-8 activation. The detected opposite effects of p16(INK4a) at the levels of growth-regulatory galectins-1 and -3 shift the status markedly towards the galectin-1-dependent pro-anoikis activity. A previously undescribed orchestrated fine-tuning of this effector system by a tumor suppressor is discovered.

Case report of a p16INK4A-positive branchial cleft cyst.

PubMed

McLean, T; Iseli, C; Amott, D; Taylor, M

2015-06-01

To report the occurrence of a concurrent oropharyngeal papilloma and branchial cleft cyst linked by p16(INK4A) and human papillomavirus immunohistochemistry. A 42-year-old woman presented with a 1-month history of a left lateral neck mass. Contrast enhanced computed tomography showed a hypodense lesion 20 mm in diameter anteromedial to the left sternocleidomastoid muscle. Ultrasound-guided fine needle aspiration suggested a branchial cleft cyst. Panendoscopy was performed at the time of neck mass removal, and a papillomatous lesion was removed from the left hypopharynx. Histopathological analysis showed the neck lesion to be a branchial cyst containing lymphoid tissue, and the oral lesion to be a squamous papilloma. Immunohistochemical analysis showed both the branchial cleft cyst and papilloma to be positive for p16(INK4A) expression and human papillomavirus DNA. Histological and immunohistochemical analyses support the cystic transformation of lymph nodes, or the 'Inclusion Theory', as the aetiology of branchial apparatus anomalies, and raise the possibility that human papillomavirus infection may play a much larger role in disease of the head and neck than previously supposed.

An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress.

PubMed

Quereda, V; Porlan, E; Cañamero, M; Dubus, P; Malumbres, M

2016-03-01

Cell-cycle inhibitors of the Ink4 and Cip/Kip families are involved in cellular senescence and tumor suppression. These inhibitors are individually dispensable for the cell cycle and inactivation of specific family members results in increased proliferation and enhanced susceptibility to tumor development. We have now analyzed the consequences of eliminating a substantial part of the cell-cycle inhibitory activity in the cell by generating a mouse model, which combines the absence of both p21(Cip1) and p27(Kip1) proteins with the endogenous expression of a Cdk4 R24C mutant insensitive to Ink4 inhibitors. Pairwise combination of Cdk4 R24C, p21-null and p27-null alleles results in frequent hyperplasias and tumors, mainly in cells of endocrine origin such as pituitary cells and in mesenchymal tissues. Interestingly, complete abrogation of p21(Cip1) and p27(Kip1) in Cdk4 R24C mutant mice results in a different phenotype characterized by perinatal death accompanied by general hypoplasia in most tissues. This phenotype correlates with increased replicative stress in developing tissues such as the nervous system and subsequent apoptotic cell death. Partial inhibition of Cdk4/6 rescues replicative stress signaling as well as p53 induction in the absence of cell-cycle inhibitors. We conclude that one of the major physiological activities of cell-cycle inhibitors is to prevent replicative stress during development.

Expression of Ki-67 and P16 INK4a in chemically-induced perioral squamous cell carcinomas in mice.

PubMed

Alves, Ângela Valéria Farias; Ribeiro, Danielle Rodrigues; Lima, Sonia Oliveira; Reis, Francisco Prado; Soares, Andréa Ferreira; Gomes, Margarete Zanardo; Albuquerque, Ricardo Luiz Cavalcanti de

2016-01-01

to evaluate the influence of Ki-67 and P16INK4a proteins immunohistochemical expressions on the clinical and morphological parameters of perioral squamous cell carcinoma induced with 9,10-dimethyl-1,2-benzanthracene (DMBA) in mice. we topically induced the lesions in the oral commissure of ten Swiss mice for 20 weeks, determining the time to tumors onset and the average tumor volume up to 26 weeks. In histopathological analysis, the variables studied were histological malignancy grade and the immunohistochemical expression of Ki-67 and P16INK4a proteins. The correlation between variables was determined by application of the Spearman correlation test. the mean time to onset of perioral lesions was 21.1 ± 2.13 weeks; mean tumor volume was 555.91 ± 205.52 mm3. Of the induced tumors, 80% were classified as low score and 20% high score. There was diffuse positivity for Ki-67 in 100% of lesions - Proliferation Index (PI) of 50.1 ± 18.0. There was a strong direct correlation between Ki-67 immunoreactivity and tumor volume (R = 0.702) and a low correlation with the malignancy score (R = 0.486). The P16INK4a protein expression was heterogeneous, showing a weak correlation with tumor volume (R = 0.334). There was no correlation between the immunohistochemical expression of the two proteins studied. in an experimental model of DMBA-induced perioral carcinogenesis, tumor progression was associated with the tumor proliferative fraction (Ki-67 positive cells) and with tumor histological grading, but not with P16INK4a expression. avaliar a influência da expressão imuno-histoquímica das proteínas Ki-67 e p16INK4a sobre parâmetros clínico-morfológicos em carcinomas espinocelulares periorais quimicamente induzidos com 9,10-dimetil-1,2-benzantraceno (DMBA) em modelo murino. as lesões foram induzidas topicamente na comissura labial de dez camundongos Swiss durante 20 semanas, sendo determinado o momento de surgimento dos tumores e volume tumoral médio até 26 semanas. Na

Cigarette smoking and p16INK4α gene promoter hypermethylation in non-small cell lung carcinoma patients: a meta-analysis.

PubMed

Zhang, Bo; Zhu, Wei; Yang, Ping; Liu, Tao; Jiang, Mei; He, Zhi-Ni; Zhang, Shi-Xin; Chen, Wei-Qing; Chen, Wen

2011-01-01

Aberrant methylation of promoter DNA and transcriptional repression of specific tumor suppressor genes play an important role in carcinogenesis. Recently, many studies have investigated the association between cigarette smoking and p16(INK4α) gene hypermethylation in lung cancer, but could not reach a unanimous conclusion. Nineteen cross-sectional studies on the association between cigarette smoking and p16(INK4α) methylation in surgically resected tumor tissues from non-small cell lung carcinoma (NSCLC) patients were identified in PubMed database until June 2011. For each study, a 2×2 cross-table was extracted. In total, 2,037 smoker and 765 nonsmoker patients were pooled with a fixed-effects model weighting for the inverse of the variance. Overall, the frequency of p16(INK4α) hypermethylation was higher in NSCLC patients with smoking habits than that in non-smoking patients (OR = 2.25, 95% CI = 1.81-2.80). The positive association between cigarette smoking and p16(INK4α) hypermethylation was similar in adenocarcinoma and squamous-cell carcinoma. In the stratified analyses, the association was stronger in Asian patients and in the studies with larger sample sizes. Cigarette smoking is positively correlated to p16(INK4α) gene hypermethylation in NSCLC patients.

Regulation of T Cell Differentiation and Alloimmunity by the Cyclin-Dependent Kinase Inhibitor p18ink4c

PubMed Central

Rowell, Emily A.; Wang, Liqing; Chunder, Neelanjana; Hancock, Wayne W.; Wells, Andrew D.

2014-01-01

Cellular proliferation in response to mitogenic stimuli is negatively regulated by the Cip/Kip and the Ink4 families of cyclin-dependent kinase (CDK) inhibitors. Several of these proteins are elevated in anergic T cells, suggesting a potential role in the induction or maintenance of tolerance. Our previous studies showed that p27kip1 is required for the induction of T cell anergy and transplantation tolerance by costimulatory blockade, but a role for Ink4 proteins in these processes has not been established. Here we show that CD4+ T cells from mice genetically deficient for p18ink4c divide more rapidly than wild-type cells in response to antigenic, costimulatory and growth factor signals. However, this gain of proliferative function was accompanied by a moderate increase in the rate of cell death, and was accompanied by an overall defect in the generation of alloreactive IFNγ-producing effector cells. Consistent with this, p18ink4c-deficient T cells were unable to induce graft-vs-host disease in vivo, and p18ink4c deficiency cooperated with costimulatory blockade to significantly increase the survival of fully mismatched allografts in a cardiac transplantation model. While both p18ink4c and p27kip1 act to restrict T cell proliferation, p18ink4c exerts an opposite effect from p27kip1 on alloimmunity and organ transplant rejection, most likely by sustaining T cell survival and the development of effector function. Our studies point to additional important links between the cell cycle machinery and the processes of T cell differentiation, survival and tolerance. PMID:24614758

Association of antibody to E2 protein of human papillomavirus and p16INK4A with progression of HPV-infected cervical lesions.

PubMed

Chuerduangphui, Jureeporn; Pientong, Chamsai; Swangphon, Piyawut; Luanratanakorn, Sanguanchoke; Sangkomkamhang, Ussanee; Tungsiriwattana, Thumwadee; Kleebkaow, Pilaiwan; Burassakarn, Ati; Ekalaksananan, Tipaya

2018-05-09

Human papillomavirus (HPV) E2 and L1 proteins are expressed in cervical cells during the lytic stage of infection. Overexpression of p16 INK4A is a biomarker of HPV-associated cervical neoplasia. This study investigated antibodies to HPV16 E2, HPV16 L1, and p16 INK4A in sera from women with no squamous intraepithelial lesion (No-SIL) of the cervix, low-grade SIL, high-grade SIL, and cervical squamous cell carcinoma (SCC). HPV DNA was detected by polymerase chain reaction. Anti-E2, -L1, and -p16 INK4A antibodies in sera were determined by western blot. Among 116 samples, 69 (60%) were HPV DNA-positive. Percentages seropositive for anti-E2, -L1, and -p16 INK4A antibodies were 39.6, 22.4, and 23.3%, respectively. Anti-E2 antibody was significantly correlated with HPV DNA-positive cases. Eighty-seven women (75%) were regarded as infected with HPV, having at least one positive result from HPV DNA, L1, or E2 antibody. Antibody to p16 INK4A was associated with HPV infection (odds = 5.444, 95% CI 1.203-24.629, P = 0.028) and precancerous cervical lesions (odds = 5.132, 95% CI 1.604-16.415, P = 0.006). Interestingly, the concurrent detection of anti-E2 and -p16 INK4A antibodies was significantly associated with HPV infection (odds = 1.382, 95% CI 1.228-1.555, P = 0.044). These antibodies might be good candidate biomarkers for monitoring HPV-associated cervical lesion development to cancer.

[Sorting role of p16(INK4a)/Ki-67 double immunostaining in the cervical cytology specimens of ASCUS and LSIL cases].

PubMed

Yu, J; Zhu, H T; Zhao, J J; Su, J Z; Xia, Y D

2017-05-08

Objective: To investigate the sorting effect of p16(INK4a)/Ki-67 double immunostaining method in patients with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL) cytology results. Methods: Four-hundred and twenty cases collected during April 2014 to February 2015 of cervical cytology of ASCUS ( n =318) and LSIL ( n =102) were selected, and residual liquid-based cytology specimens were used for p16(INK4a)/Ki-67 double immunostaining. The sensitivity and specificity of the detection of cervical precancerous lesions and cervical cancer were calculated, and the results were compared with high risk HPV. Taking histological follow-up as the gold standard, the test was considered positive when at least one cell exhibited p16(INK4a)/Ki-67 co-staining, without requirement of adjunct morphologic interpretation of positive cells. Results: Further screening CIN2+ in cytology ASCUS and LSIL group , the sensitivity of p16(INK4a)/Ki-67 double immunostaining was slightly lower than high risk HPV (84.2% vs . 94.7%), while the specificity was higher (84.0% vs . 53.9%). For ASCUS patients, the sensitivity of p16(INK4a)/Ki-67 double immunostaining and high risk HPV was 82.6% and 91.3%, and the specificity was 88.8% and 63.7%, respectively. For LSIL patients, the sensitivity of p16(INK4a)/Ki-67 double immunostaining and high risk HPV was 86.7% and 100.0%, and the specificity was 67.8% and 20.7%, respectively. For patients younger and older than 30 years, specificity of p16(INK4a)/Ki-67 double immunostaining was both higher than that of high risk HPV (80.8% vs . 42.3%; 84.6% vs . 56.9%). Conclusions: p16(INK4a)/Ki-67 double immunostaining can effectively identify the high risk population in ASCUS or LSIL, with higher specificity than high risk HPV test. p16(INK4a)/Ki-67 double immunostaining may benefit patients younger than 30 years of age as a preliminary or potential cytology-combining screening tool.

Mangiferin induces islet regeneration in aged mice through regulating p16INK4a.

PubMed

Wang, Hailian; He, Xia; Lei, Tiantian; Liu, Yilong; Huai, Guoli; Sun, Minghan; Deng, Shaoping; Yang, Hongji; Tong, Rongsheng; Wang, Yi

2018-06-01

Previous studies by our group on mangiferin demonstrated that it exerts an anti‑hyperglycemic effect through the regulation of cell cycle proteins in 3‑month‑old, partially pancreatectomized (PPx) mice. However, β‑cell proliferation is known to become severely restricted with advanced age. Therefore, it is unknown whether mangiferin is able to reverse the diabetic condition and retain β‑cell regeneration capability in aged mice. In the present study, 12‑month‑old C57BL/6J mice that had undergone PPx were subjected to mangiferin treatment (90 mg/kg) for 28 days. Mangiferin‑treated aged mice exhibited decreased blood glucose levels and increased glucose tolerance, which was accompanied with higher serum insulin levels when compared with those in untreated PPx control mice. In addition, islet hyperplasia, elevated β‑cell proliferation and reduced β‑cell apoptosis were also identified in the mice that received mangiferin treatment. Further studies on the mRNA transcript and protein expression levels indicated comparatively increased levels of cyclins D1 and D2 and cyclin‑dependent kinase 4 in mangiferin‑treated mice, while the levels of p27Kip1 and p16INK4a were decreased relative to those in the untreated PPx controls. Of note, mangiferin treatment improved the proliferation rate of islet β‑cells in adult mice overexpressing p16INK4a, suggesting that mangiferin induced β‑cell proliferation via the regulation of p16INK4a. In addition, the mRNA transcription levels of critical genes associated with insulin secretion, including pancreatic and duodenal homeobox 1, glucose transporter 2 and glucokinase, were observed to be upregulated after mangiferin treatment. Taken together, it was indicated that mangiferin treatment significantly induced β‑cell proliferation and inhibited β‑cell apoptosis by regulating cell cycle checkpoint proteins. Furthermore, mangiferin was also demonstrated to regulate genes associated with insulin

Mangiferin induces islet regeneration in aged mice through regulating p16INK4a

PubMed Central

Liu, Yilong; Huai, Guoli; Sun, Minghan; Deng, Shaoping; Yang, Hongji; Tong, Rongsheng; Wang, Yi

2018-01-01

Previous studies by our group on mangiferin demonstrated that it exerts an antihyperglycemic effect through the regulation of cell cycle proteins in 3-month-old, partially pancreatectomized (PPx) mice. However, β-cell proliferation is known to become severely restricted with advanced age. Therefore, it is unknown whether mangiferin is able to reverse the diabetic condition and retain β-cell regeneration capability in aged mice. In the present study, 12-month-old C57BL/6J mice that had undergone PPx were subjected to mangiferin treatment (90 mg/kg) for 28 days. Mangiferin-treated aged mice exhibited decreased blood glucose levels and increased glucose tolerance, which was accompanied with higher serum insulin levels when compared with those in untreated PPx control mice. In addition, islet hyperplasia, elevated β-cell proliferation and reduced β-cell apoptosis were also identified in the mice that received mangiferin treatment. Further studies on the mRNA transcript and protein expression levels indicated comparatively increased levels of cyclins D1 and D2 and cyclin-dependent kinase 4 in mangiferin-treated mice, while the levels of p27Kip1 and p16INK4a were decreased relative to those in the untreated PPx controls. Of note, mangiferin treatment improved the proliferation rate of islet β-cells in adult mice overexpressing p16INK4a, suggesting that mangiferin induced β-cell proliferation via the regulation of p16INK4a. In addition, the mRNA transcription levels of critical genes associated with insulin secretion, including pancreatic and duodenal homeobox 1, glucose transporter 2 and glucokinase, were observed to be upregulated after mangiferin treatment. Taken together, it was indicated that mangiferin treatment significantly induced β-cell proliferation and inhibited β-cell apoptosis by regulating cell cycle checkpoint proteins. Furthermore, mangiferin was also demonstrated to regulate genes associated with insulin secretion. Collectively these, results

P16INK4A immunohistochemistry for detection of human papilloma virus-associated penile squamous cell carcinoma is superior to in-situ hybridization.

PubMed

Aumayr, K; Susani, M; Horvat, R; Wrba, F; Mazal, P; Klatte, T; Koller, A; Neudert, B; Haitel, A

2013-01-01

We evaluated p16INK4A as a reliable option to detect human papilloma virus (HPV) DNA in penile tumor specimens. Formalin-fixed paraffin embedded samples of 26 patients with penile cancer and another 18 cases with non-tumorigenic lesions were stained by three different widely used commercially available chromogenic in-situ hybridization assays high-risk HPV CISH Y1443 (Genpoint, DAKO), pan HPV CISH Y1404 (Genpoint, DAKO), INFORM HPV III (Ventana, Tucson, Arizona) and p16INK4A immunohistochemistry, then compared to the known gold standard polymerase chain reaction detecting HPV 16, 18, 31, and 33. Immunoreactivity for p16INK4A was evaluated by using a 4-tiered (0, 1, 2, and 3) pattern based system. 19 cases were positive for p16INK4A, 13 of which showed a continuous transepithelial staining (pattern 3). Pan HPV ISH showed positivity in 9 cases, high-risk HPV ISH in 7 cases and INFORM HPVIII ISH in 7 cases. p16INK4A IHC pattern 3 versus pattern 0, 1 and 2 exhibited a specificity and positive predictive value of 100 percent, with a sensitivity and negative predictive value of 72 and 62 percent, respectively, which was much better than all HPV in-situ hybridization methods referred to polymerase chain reaction. p16INK4A seems to be a superior marker for the detection of HPV-associated penile squamous cell carcinoma compared to CISH tests, but is not recommend for the detection of non-tumorigenic lesions, where PCR should be used for the initial assessment.

P16INK4a Positive Cells in Human Skin Are Indicative of Local Elastic Fiber Morphology, Facial Wrinkling, and Perceived Age

PubMed Central

Waaijer, Mariëtte E. C.; Gunn, David A.; Adams, Peter D.; Pawlikowski, Jeff S.; Griffiths, Christopher E. M.; van Heemst, Diana; Slagboom, P. Eline; Westendorp, Rudi G. J.; Maier, Andrea B.

2016-01-01

Senescent cells are more prevalent in aged human skin compared to young, but evidence that senescent cells are linked to other biomarkers of aging is scarce. We counted cells positive for the tumor suppressor and senescence associated protein p16INK4a in sun-protected upper-inner arm skin biopsies from 178 participants (aged 45–81 years) of the Leiden Longevity Study. Local elastic fiber morphology, facial wrinkles, and perceived facial age were compared to tertiles of p16INK4a counts, while adjusting for chronological age and other potential confounders. The numbers of epidermal and dermal p16INK4a positive cells were significantly associated with age-associated elastic fiber morphologic characteristics, such as longer and a greater number of elastic fibers. The p16INK4a positive epidermal cells (identified as primarily melanocytes) were also significantly associated with more facial wrinkles and a higher perceived age. Participants in the lowest tertile of epidermal p16INK4a counts looked 3 years younger than those in the highest tertile, independently of chronological age and elastic fiber morphology. In conclusion, p16INK4a positive cell numbers in sun-protected human arm skin are indicative of both local elastic fiber morphology and the extent of aging visible in the face. PMID:26286607

Human papillomavirus-associated increase in p16INK4A expression in penile lichen sclerosus and squamous cell carcinoma.

PubMed

Prowse, D M; Ktori, E N; Chandrasekaran, D; Prapa, A; Baithun, S

2008-02-01

Human papillomaviruses (HPVs) are sexually transmitted human carcinogens that may play a role in the oncogenesis of penile cancer. To investigate the role of HPV infection and expression of the tumour suppressor protein p16INK4A in the pathogenesis of penile cancer. By means of polymerase chain reaction amplification and reverse hybridization line probe assay to detect HPV infection, and immunohistochemical staining for p16INK4A and Ki67, we analysed 26 penile squamous cell carcinomas (SCCs) and 20 independent penile lichen sclerosus (LS) lesions from 46 patients. HPV DNA was found in 54% of penile SCCs and 33% of penile LS cases in single and multiple infections. High-risk HPV 16 was the predominant HPV type detected. No relationship between Ki67 expression and HPV infection was observed. Strong immunostaining for p16INK4A correlated with HPV 16/18 infection in both penile LS and penile SCC. In our penile SCC series the cancer margins were also associated with penile LS in 13 of 26 lesions, and HPV was detected in seven of the 13 SCC cases associated with LS and in six of the 11 SCC lesions not involving LS. Our study shows a high prevalence of HPV 16 and p16INK4A expression in penile lesions, consistent with an active role for HPV in interfering with the retinoblastoma pathway. High-risk HPV infection could be involved in the tumorigenic process in 50% of penile cancers, and the use of prophylactic HPV vaccines has the potential to prevent these cancers.

Culturing on Wharton's jelly extract delays mesenchymal stem cell senescence through p53 and p16INK4a/pRb pathways.

PubMed

Hao, Haojie; Chen, Guanghui; Liu, Jiejie; Ti, Dongdong; Zhao, Yali; Xu, Shenjun; Fu, Xiaobing; Han, Weidong

2013-01-01

Mesenchymal stem cells (MSCs) hold great therapeutic potential. However, MSCs undergo replication senescence during the in vitro expansion process. Wharton's jelly from the human umbilical cord harbors a large number of MSCs. In this study, we hypothesized that Wharton's jelly would be beneficial for in vitro expansion of MSCs. Wharton's jelly extract (WJEs), which is mainly composed of extracellular matrix and cytokines, was prepared as coating substrate. Human MSCs were isolated and cultured on WJE-coated plates. Although the proliferation capacity of cells was not augmented by WJE in early phase culture, adynamic growth in late-phase culture was clearly reduced, suggesting that the replicative senescence of MSCs was efficiently slowed by WJE. This was confirmed by β-galactosidase staining and telomere length measurements of MSCs in late-phase culture. In addition, the decreased differentiation ability of MSCs after long-term culture was largely ameliorated by WJE. Reactive oxygen species (ROS), p53, and p16INK4a/pRb expression increased with passaging. Analysis at the molecular level revealed that WJE-based culture efficiently suppressed the enhancement of intracellular ROS, p53, and p16INK4a/pRb in MSCs. These data demonstrated that WJE provided an ideal microenvironment for MSCs culture expansion in vitro preserved MSC properties by delaying MSCs senescence, and allowed large numbers of MSCs to be obtained for basic research and clinical therapies.

Extremely stringent activation of p16INK4a prevents immortalization of uterine cervical epithelial cells without human papillomavirus oncogene expression

PubMed Central

Hang, Su; Tiwari, Agnes F.Y.; Ngan, Hextan Y.S.; Yip, Yim-Ling; Cheung, Annie L.M.; Tsao, Sai Wah; Deng, Wen

2016-01-01

Cervical epithelial cell immortalization with defined genetic factors without viral oncogenes has never been reported. Here we report that HPV-negative cervical epithelial cells failed to be immortalized by telomerase activation or the combination of p53 knockdown and telomerase activation. Under those conditions, p16INK4a expression was always elevated during the late stage of limited cell lifespan, suggesting that cervical epithelial cells possess an intrinsic property of uniquely stringent activation of p16INK4a, which may offer an explanation for the rarity of HPV-negative cervical cancer. Combining p16INK4a knockdown with telomerase activation resulted in efficient immortalization of HPV-negative cervical epithelial cells under ordinary culture conditions. Compared with the HPV16-E6E7-immortalized cell lines derived from the same primary cell sources, the novel HPV-negative immortalized cell lines had lower degrees of chromosomal instability, maintained more sensitive p53/p21 response to DNA damage, exhibited more stringent G2 checkpoint function, and were more resistant to replication-stress-induced genomic instability. The newly immortalized HPV-negative cervical epithelial cell lines were non-tumorigenic in nude mice. The cell lines can be used not only as much-needed HPV-negative non-malignant cell models but also as starting models that can be genetically manipulated in a stepwise fashion to investigate the roles of defined genetic alterations in the development of HPV-negative cervical cancer. PMID:27344169

Identification and genotyping of human papillomavirus in a Spanish cohort of penile squamous cell carcinomas: correlation with pathologic subtypes, p16(INK4a) expression, and prognosis.

PubMed

Ferrándiz-Pulido, Carla; Masferrer, Emili; de Torres, Ines; Lloveras, Belen; Hernandez-Losa, Javier; Mojal, Sergio; Salvador, Carlos; Morote, Juan; Ramon y Cajal, Santiago; Pujol, Ramon M; Garcia-Patos, Vicente; Toll, Agustin

2013-01-01

Penile squamous cell carcinoma (PSCC) is a tumor with a high metastatic potential. In PSCC the attributable fraction to human papillomavirus (HPV) is not well established. We sought to provide novel data about the prevalence of HPV in a large series of penile intraepithelial neoplasia (PeIN) and invasive PSCC, correlating the results with the histologic subtype, p16(INK4a) immunostaining, and prognosis. A total of 82 PSCC were included in the study, 69 invasive and 13 PeIN. HPV detection was performed by polymerase chain reaction with SPF-10 broad-spectrum primers followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe assay. P16(INK4a) immunohistochemical expression on tissue microarrays was also analyzed. HPV DNA was identified in 31 of 77 (40.2%) PSCC (22 of 67 invasive and 9 of 10 PeIN). In 25 of 31 (80.6%) cases HPV-16 was identified. HPV detection was significantly associated with some histologic subtypes: most basaloid and warty tumors were high-risk HPV (hrHPV) positive, whereas only 15% of usual PSCC were hr-HPV positive. All hrHPV-positive PSCC had an adjacent undifferentiated PeIN. Strong p16(INK4a) immunostaining correlated with hrHPV infection. Most undifferentiated PeIN showed p16(INK4a) immunohistochemical overexpression. Both hrHPV-positive and p16(INK4a)-positive tumors showed a better overall survival without reaching statistical significance. This was a retrospective study. Our results suggest that most hrHPV-positive PSCC develop from undifferentiated hrHPV-positive PeIN. P16(INK4a) immunostaining may be useful in identifying both etiologically related hrHPV-positive tumors and those with better outcome. The routine use of p16(INK4a) staining should be incorporated in histologic evaluation of PSCC. Copyright © 2012 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

The induction of H3K9 methylation by PIWIL4 at the p16{sup Ink4a} locus

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

Sugimoto, Keiki; Kage, Hidenori; Aki, Naomi

The field of epigenetics has made progress by the identification of the small RNA-mediated epigenetic modification. However, little is known about the key proteins. Here, we report that the human PIWI-like family is a candidate protein that is involved in the pathway responsible for chromatin remodeling. The PIWI-like family proteins, expressed as the Flag-fusion proteins, formed a bulky body and localized to the nuclear periphery. Transient transfection of PIWI-like 4 (PIWIL4), only member of the PIWI-like family that was ubiquitously expressed in human tissues, induced histone H3 lysine 9 methylation at the p16{sup Ink4a} (CDKN2A) locus. The elevated level ofmore » histone methylation resulted in the downregulation of the p16{sup Ink4a} gene. These results suggest PIWIL4 plays important roles in the chromatin-modifying pathway in human somatic cells.« less

Germline CDKN2A/P16INK4A mutations contribute to genetic determinism of sarcoma.

PubMed

Jouenne, Fanélie; Chauvot de Beauchene, Isaure; Bollaert, Emeline; Avril, Marie-Françoise; Caron, Olivier; Ingster, Olivier; Lecesne, Axel; Benusiglio, Patrick; Terrier, Philippe; Caumette, Vincent; Pissaloux, Daniel; de la Fouchardière, Arnaud; Cabaret, Odile; N'Diaye, Birama; Velghe, Amélie; Bougeard, Gaelle; Mann, Graham J; Koscielny, Serge; Barrett, Jennifer H; Harland, Mark; Newton-Bishop, Julia; Gruis, Nelleke; Van Doorn, Remco; Gauthier-Villars, Marion; Pierron, Gaelle; Stoppa-Lyonnet, Dominique; Coupier, Isabelle; Guimbaud, Rosine; Delnatte, Capucine; Scoazec, Jean-Yves; Eggermont, Alexander M; Feunteun, Jean; Tchertanov, Luba; Demoulin, Jean-Baptiste; Frebourg, Thierry; Bressac-de Paillerets, Brigitte

2017-09-01

Sarcomas are rare mesenchymal malignancies whose pathogenesis is poorly understood; both environmental and genetic risk factors could contribute to their aetiology. We performed whole-exome sequencing (WES) in a familial aggregation of three individuals affected with soft-tissue sarcoma (STS) without TP53 mutation (Li-Fraumeni-like, LFL) and found a shared pathogenic mutation in CDKN2A tumour suppressor gene. We searched for individuals with sarcoma among 474 melanoma-prone families with a CDKN2A -/+ genotype and for CDKN2A mutations in 190 TP53 -negative LFL families where the index case was a sarcoma. Including the initial family, eight independent sarcoma cases carried a germline mutation in the CDKN2A /p16 INK4A gene. In five out of seven formalin-fixed paraffin-embedded sarcomas, heterozygosity was lost at germline CDKN2A mutations sites demonstrating complete loss of function. As sarcomas are rare in CDKN2A /p16 INK4A carriers, we searched in constitutional WES of nine carriers for potential modifying rare variants and identified three in platelet-derived growth factor receptor ( PDGFRA ) gene. Molecular modelling showed that two never-described variants could impact the PDGFRA extracellular domain structure. Germline mutations in CDKN2A /P16 INK4A , a gene known to predispose to hereditary melanoma, pancreatic cancer and tobacco-related cancers, account also for a subset of hereditary sarcoma. In addition, we identified PDGFRA as a candidate modifier gene. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Up-regulation of Survivin during Immortalization of Human Myofibroblasts Is Linked to Repression of Tumor Suppressor p16INK4a Protein and Confers Resistance to Oxidative Stress*

PubMed Central

Kan, Chin-Yi; Petti, Carlotta; Bracken, Lauryn; Maritz, Michelle; Xu, Ning; O'Brien, Rosemary; Yang, Chen; Liu, Tao; Yuan, Jun; Lock, Richard B.; MacKenzie, Karen L.

2013-01-01

Survivin is an essential component of the chromosomal passenger complex and a member of the inhibitor of apoptosis family. It is expressed at high levels in a large variety of malignancies, where it has been implicated in drug resistance. It was also shown previously that survivin is up-regulated during telomerase-mediated immortalization, which occurs at a relatively early stage during carcinogenesis. This study shows that up-regulation of survivin during immortalization of human myofibroblasts is an indirect consequence of the repression of p16INK4a. Survivin and p16INK4a were functionally linked by assays that showed that either the up-regulation of survivin or repression of p16INK4a rendered telomerase-transduced MRC-5 myofibroblasts resistant to oxidative stress. Conversely, siRNA-mediated down-regulation of survivin activated caspases and enhanced the sensitivity of immortal MRC-5 cells to oxidative stress. The E2F1 transcription factor, which is negatively regulated by the pRB/p16INK4a tumor suppressor pathway, was implicated in the up-regulation of survivin. Using the ChIP assay, it was shown that E2F1 directly interacted with the survivin gene (BIRC5) promoter in cells that spontaneously silenced p16INK4a during telomerase-mediated immortalization. E2F1 binding to the BIRC5 was also enhanced in telomerase-transduced cells subjected to shRNA-mediated repression of p16INK4a. Together, these data show that repression of p16INK4a contributes to the up-regulation of survivin and thereby provides a survival advantage to cells exposed to oxidative stress during immortalization. The up-regulation of survivin during immortalization likely contributes to the vulnerability of immortal cells to transformation by oncogenes that alter intracellular redox state. PMID:23449974

p16INK4A expression as biomarker for HPV 16-related vulvar neoplasias.

PubMed

Riethdorf, Sabine; Neffen, Eduardo F; Cviko, Aida; Löning, Thomas; Crum, Christopher P; Riethdorf, Lutz

2004-12-01

Up-regulation of p16INK4A is associated with high-risk human papillomavirus (HPV) in preinvasive and invasive cervical neoplasia. However, its expression in vulvar carcinomas, which have a diverse pathogenesis, has not been extensively studied. One hundred seventy-seven vulvar intraepithelial neoplasms (VIN), squamous cell carcinomas (SCC), and benign squamous epithelia were analyzed for p16 expression. RNA/RNA in situ hybridization was used to detect HPV 16 E6/E7 transcripts in 112. Ninety-five percent of VIN 3 and basaloid or warty SCCs (76/80) and 4% of keratinizing SCC (2/48) were moderately to strongly immunopositive for p16, which localized to nucleus and cytoplasm; 52/58 analyzed (90%) contained HPV 16 transcripts. The positive predictive value (PPV) of moderate to strong diffuse p16 immunostaining and HPV positivity for the diagnosis of VIN 3 and of basaloid or warty SCC was 97% and 95%, respectively. Conversely, 94% of keratinizing SCC contained heterogeneous staining, and when present, it was strictly cytoplasmic and frequently localized to the cells at the epithelial-stromal interface. Benign squamous epithelia were p16 negative, with the exception of lichen sclerosus, which contained focal and heterogeneously p16 positive in 42%. As in the cervix, intense diffuse p16 expression supports an HPV-related neoplastic process in vulvar neoplasia, irrespective of the level of differentiation. Up-regulation of p16 at the epithelial-stromal interface in HPV negative keratinizing SCCs is consistent with an HPV-independent response to alterations associated with invasion. These disparate patterns of p16 expression underscore 2 different mechanisms for p16 expression in HPV-related and HPV-unrelated vulvar carcinomas.

p16(INK4a) /Ki-67 dual labelling as a marker for the presence of high-grade cancer cells or disease progression in urinary cytopathology.

PubMed

Piaton, E; Advenier, A S; Carré, C; Decaussin-Petrucci, M; Mege-Lechevallier, F; Ruffion, A

2013-10-01

Overexpression of p16(INK4a) independent of the presence of E6-E7 oncoproteins of high-risk papillomaviruses has been identified in bladder carcinoma in situ lesions with or without concurrent papillary or invasive high-grade (HG) urothelial carcinoma. As p16(INK4a) and Ki-67 co-expression clearly indicates deregulation of the cell cycle, the aim of this study was to investigate the frequency of p16(INK4a) /Ki-67 dual labelling in urinary cytology samples. Immunolabelling was performed in demounted, destained Papanicolaou slides after ThinPrep(®) processing. A total of 84 urinary cytology samples (18 negative, 10 low grade, 19 atypical urothelial cells and 37 high grade) were analysed for p16(INK4a) /Ki-67 co-expression. We assessed underlying urothelial malignancy with cystoscopy, histopathology and follow-up data in every case. Compared with raw histopathological results, p16 (INK4a) /Ki-67 dual labelling was observed in 48 out of 55 (87.3%) HG lesions and in 11 out of 29 (37.9%) negative, papillary urothelial neoplasia of low malignant potential or low-grade carcinomas (P = 0.05). All cases with high-grade/malignant cytology were dual labelled. Sixteen out of 17 (94.1%) carcinoma in situ cases and eight out of 14 (57.1%) cases with atypical urothelial cells matching with HG lesions were dual labelled. Extended follow-up allowed three cases of progression to be diagnosed in dual-labelled cases with negative/low-grade cytology results after a 9- to 11-months delay. The data show that p16(INK4a) /Ki-67 co-expression allows most HG cancer cells to be detected initially and in the follow-up period. Additional studies are needed in order to determine whether dual labelling can be used as a triage tool for atypical urothelial cells in the urine. © 2012 John Wiley & Sons Ltd.

E2F1 induces p19INK4d, a protein involved in the DNA damage response, following UV irradiation.

PubMed

Carcagno, Abel L; Giono, Luciana E; Marazita, Mariela C; Castillo, Daniela S; Pregi, Nicolás; Cánepa, Eduardo T

2012-07-01

Central to the maintenance of genomic integrity is the cellular DNA damage response. Depending on the type of genotoxic stress and through the activation of multiple signaling cascades, it can lead to cell cycle arrest, DNA repair, senescence, and apoptosis. p19INK4d, a member of the INK4 family of CDK inhibitors, plays a dual role in the DNA damage response, inhibiting cell proliferation and promoting DNA repair. Consistently, p19INK4d has been reported to become upregulated in response to UV irradiation and a great variety of genotoxic agents. Here, this induction is shown to result from a transcriptional stimulatory mechanism that can occur at every phase of the cell cycle except during mitosis. Moreover, evidence is presented that demonstrates that E2F1 is involved in the induction of p19INK4d following UV treatment, as it is prevented by E2F1 protein ablation and DNA-binding inhibition. Specific inhibition of this regulation using triplex-forming oligonucleotides that target the E2F response elements present in the p19INK4d promoter also block p19INK4d upregulation and sensitize cells to DNA damage. These results constitute the first description of a mechanism for the induction of p19INK4d in response to UV irradiation and demonstrate the physiological relevance of this regulation following DNA damage.

Microsatellite DNA assays reveal an allelic imbalance in p16(Ink4), GALT, p53, and APOA2 loci in patients with endometriosis.

PubMed

Goumenou, A G; Arvanitis, D A; Matalliotakis, I M; Koumantakis, E E; Spandidos, D A

2001-01-01

To detect allelic imbalance on specific genetic loci occurring in endometriosis. Microsatellite analysis. Paraffin-embedded tissues histologically confirmed as endometriotic or normal endometrium. Premenopausal women undergoing laparoscopy for suspected endometriosis. Laparoscopic excision of specimens. Allelic imbalance and alterations of intensity of microsatellite alleles. Five of 17 microsatellite DNA markers (29.4%) showed allelic imbalance. Eight samples (36.4%) showed allelic imbalance in at least one locus. Loci 9p21, 1q21, and 17p13.1 exhibited imbalance in 27.3%, 4.5%, and 4.5%, respectively. A 3-fold increase of the fractional allelic loss was observed from disease stage II to III and IV, whereas only 1.3-fold was found between patients of 41-50 and 20-40 years. We found that loss of heterozygosity on p16(Ink4), GALT, and p53, as well as on APOA2, a region frequently lost in ovarian cancer, occurs in endometriosis, even in stage II of the disease. The occurrence of such genomic alterations may represent important events in the development of endometriosis. The 9p21 locus may contain a gene associated with the pathogenesis of the disease, and therefore its loss may be a prognostic marker of the disease.

Correlation of P16 (Ink4a) and CK17 to HPV (16E6+18E6) in Premalignant and Malignant Lesions of Uterine Cervix: A Clinicopathologic Study

PubMed Central

Chaloob, Mohammed K.; Hussein, Alaa G.; Qasim, Ban J.

2016-01-01

Background: This research was accomplished to evaluate the IHC expression of p16 (ink4a) and CK17 in low grade cervical intraepithelial lesions (LSIL), high grade cervical intraepithelial lesions (HSIL) and invasive cervical carcinomas and to assess their correlation to HPV (16E6+18E6). Methods: The study included (127) formalin-fixed paraffin-embedded cervical biopsies; of which 22 cases were chronic cervicitis, 24 cases were LSIL, 28 cases were HSIL and 53 cases were invasive cervical carcinomas. Sections were immunohistochemically stained for p16 (ink4a), CK17 and HPV (16E6+18E6). Results: The study established a highly significant increase in IHC of expression of p16 (ink4a), CK17 and HPV (16E6+18E6) from LSIL through HSIL to invasive carcinomas (P-value˂0.001). There was non-significant association between IHC expression of all makers with age of patients; types, grade and stage of cervical carcinomas (P-value˃0.05). HPV (16E6+18E6) revealed a significantly positive correlation with p16 (ink4a) (P-value˂0.05) and a non- significant correlation with CK17 (P-value˃0.05); in LSIL, HSIL and invasive carcinoma cases. Conclusion: p16 (ink4a) expression directly reflects infection with high risk HPV in cervical lesions and can add a significant diagnostic accuracy in the evaluation of CIN. CK 17 is a good marker of malignant transformation, with increasing in its expression according to the severity of cervical lesions; however, it is not related to HPV infection. Both markers are not related to prognostic variables of patients with cervical carcinoma. PMID:28855930

Clinical Significance of p53 and p16(ink4a) Status in a Contemporary North American Penile Carcinoma Cohort.

PubMed

Zargar-Shoshtari, Kamran; Spiess, Philippe E; Berglund, Anders E; Sharma, Pranav; Powsang, Julio M; Giuliano, Anna; Magliocco, Anthony M; Dhillon, Jasreman

2016-08-01

Because of the low incidence of penile carcinoma (PC), the value of p16(ink4a), p53, and human papilloma virus (HPV) infection status in clinical practice remains unclear. Herein, we report our experience with potential clinical utility of these markers in men with PC treated at our institution. Tissue microarrays of 57 cases of invasive penile squamous cell carcinomas were immunohistochemically stained for p16 and p53. HPV in situ hybridization (ISH) for high-risk subtypes was also performed. Association between marker status, nodal disease, overall (OS) and cancer-specific survival (CSS) were assessed. p16 and HPV ISH were positive in 23 (40%) and 24 (42%) of the cohort, respectively. The proportion of warty, basaloid, or mixed warty basaloid tumor subtypes were significantly greater in the p16-positive patients (48% vs. 3%; P < .01). p53 expression was negative in 31 (54%) cases. Only in p16-negative patients, positive p53 status was associated with pN+ disease (odds ratio, 4.4 [95% confidence interval (CI), 1.04-18.6]). In Kaplan-Meier analysis, the unadjusted estimated OS was insignificantly longer in p16-positive patients (median OS, 75 vs. 27 months; P = .27) and median CSS was not reached (P = .16). In a multivariable Cox proportional hazard model, when controlling for pathological nodal status and adjuvant chemotherapy, p16 status was a significant predictor for improved CSS (hazard ratio, 0.36 [95% CI, 0.13-0.99]). The worst CSS was seen in pN+ patients with double negative p16 and p53 expression (8 vs. 34 months; P = .01). In this current cohort, p53 and p16 status showed clinical utility in predicting nodal disease as well as survival. Copyright © 2015 Elsevier Inc. All rights reserved.

Performance of in silico tools for the evaluation of p16INK4a (CDKN2A) variants in CAGI.

PubMed

Carraro, Marco; Minervini, Giovanni; Giollo, Manuel; Bromberg, Yana; Capriotti, Emidio; Casadio, Rita; Dunbrack, Roland; Elefanti, Lisa; Fariselli, Pietro; Ferrari, Carlo; Gough, Julian; Katsonis, Panagiotis; Leonardi, Emanuela; Lichtarge, Olivier; Menin, Chiara; Martelli, Pier Luigi; Niroula, Abhishek; Pal, Lipika R; Repo, Susanna; Scaini, Maria Chiara; Vihinen, Mauno; Wei, Qiong; Xu, Qifang; Yang, Yuedong; Yin, Yizhou; Zaucha, Jan; Zhao, Huiying; Zhou, Yaoqi; Brenner, Steven E; Moult, John; Tosatto, Silvio C E

2017-09-01

Correct phenotypic interpretation of variants of unknown significance for cancer-associated genes is a diagnostic challenge as genetic screenings gain in popularity in the next-generation sequencing era. The Critical Assessment of Genome Interpretation (CAGI) experiment aims to test and define the state of the art of genotype-phenotype interpretation. Here, we present the assessment of the CAGI p16INK4a challenge. Participants were asked to predict the effect on cellular proliferation of 10 variants for the p16INK4a tumor suppressor, a cyclin-dependent kinase inhibitor encoded by the CDKN2A gene. Twenty-two pathogenicity predictors were assessed with a variety of accuracy measures for reliability in a medical context. Different assessment measures were combined in an overall ranking to provide more robust results. The R scripts used for assessment are publicly available from a GitHub repository for future use in similar assessment exercises. Despite a limited test-set size, our findings show a variety of results, with some methods performing significantly better. Methods combining different strategies frequently outperform simpler approaches. The best predictor, Yang&Zhou lab, uses a machine learning method combining an empirical energy function measuring protein stability with an evolutionary conservation term. The p16INK4a challenge highlights how subtle structural effects can neutralize otherwise deleterious variants. © 2017 Wiley Periodicals, Inc.

Aging of mice is associated with p16(Ink4a)- and β-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells.

PubMed

Hall, Brandon M; Balan, Vitaly; Gleiberman, Anatoli S; Strom, Evguenia; Krasnov, Peter; Virtuoso, Lauren P; Rydkina, Elena; Vujcic, Slavoljub; Balan, Karina; Gitlin, Ilya; Leonova, Katerina; Polinsky, Alexander; Chernova, Olga B; Gudkov, Andrei V

2016-07-01

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and β-galactosidase activity at pH6.0 (β-gal(pH6)), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/β-gal(pH6)-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/β-gal(pH6)-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/β-gal(pH6)-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/β-gal(pH6)-positive cells and reconsideration of potential cellular target for anti-aging treatment.

Feedback Circuit among INK4 Tumor Suppressors Constrains Human Glioblastoma Development

PubMed Central

Wiedemeyer, Ruprecht; Brennan, Cameron; Heffernan, Timothy P.; Xiao, Yonghong; Mahoney, John; Protopopov, Alexei; Zheng, Hongwu; Bignell, Graham; Furnari, Frank; Cavenee, Webster K.; Hahn, William C.; Ichimura, Koichi; Collins, V. Peter; Chu, Gerald C.; Stratton, Michael R.; Ligon, Keith L.; Futreal, P. Andrew; Chin, Lynda

2008-01-01

Summary We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18INK4C and p16INK4A codeletion. Functional reconstitution of p18INK4C in GBM cells null for both p16INK4A and p18INK4C resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18INK4C in p16INK4A-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16INK4A in primary astrocytes induced a concomitant increase in p18INK4C. Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18INK4C in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation. PMID:18394558

Comparative modeling and docking studies of p16ink4/cyclin D1/Rb pathway genes in lung cancer revealed functionally interactive residue of RB1 and its functional partner E2F1.

PubMed

Naqsh e Zahra, Syeda; Khattak, Naureen Aslam; Mir, Asif

2013-01-01

Lung cancer is the major cause of mortality worldwide. Major signalling pathways that could play significant role in lung cancer therapy include (1) Growth promoting pathways (Epidermal Growth Factor Receptor/Ras/ PhosphatidylInositol 3-Kinase) (2) Growth inhibitory pathways (p53/Rb/P14ARF, STK11) (3) Apoptotic pathways (Bcl-2/Bax/Fas/FasL). Insilico strategy was implemented to solve the mystery behind selected lung cancer pathway by applying comparative modeling and molecular docking studies. YASARA [v 12.4.1] was utilized to predict structural models of P16-INK4 and RB1 genes using template 4ELJ-A and 1MX6-B respectively. WHAT CHECK evaluation tool demonstrated overall quality of predicted P16-INK4 and RB1 with Z-score of -0.132 and -0.007 respectively which showed a strong indication of reliable structure prediction. Protein-protein interactions were explored by utilizing STRING server, illustrated that CDK4 and E2F1 showed strong interaction with P16-INK4 and RB1 based on confidence score of 0.999 and 0.999 respectively. In order to facilitate a comprehensive understanding of the complex interactions between candidate genes with their functional interactors, GRAMM-X server was used. Protein-protein docking investigation of P16-INK4 revealed four ionic bonds illustrating Arg47, Arg80,Cys72 and Met1 residues as actively participating in interactions with CDK4 while docking results of RB1 showed four hydrogen bonds involving Glu864, Ser567, Asp36 and Arg861 residues which interact strongly with its respective functional interactor E2F1. This research may provide a basis for understanding biological insights of P16-INK4 and RB1 proteins which will be helpful in future to design a suitable drug to inhibit the disease pathogenesis as we have determined the interacting amino acids which can be targeted in order to design a ligand in-vitro to propose a drug for clinical trials. Protein -protein docking of candidate genes and their important interacting residues likely

p16(INK4A) mediates age-related changes in mesenchymal stem cells derived from human dental pulp through the DNA damage and stress response.

PubMed

Feng, Xingmei; Xing, Jing; Feng, Guijuan; Huang, Dan; Lu, Xiaohui; Liu, Suzhe; Tan, Wei; Li, Liren; Gu, Zhifeng

2014-01-01

Mesenchymal stem cells derived from human dental pulp (DP-MSCs) are characterized by self-renewal and multi-lineage differentiation, which play important roles in regenerative medicine. Autologous transfers, as non-immunogenic, constitute the safest approach in cellular transplantations. However, their use may be limited by age-related changes. In the study, we compared DP-MSCs isolated from human in five age groups: 5-12 y, 12-20 y, 20-35 y, 35-50 y, and >50 y. We tested the effect of age on proliferation, differentiation, senescence-associated β-galactosidase (SA-β-gal), cell cycle and programmed cell death. DP-MSCs showed characteristics of senescence as a function of age. Meanwhile, the expression of p16(INK4A) and γ-H2A.X significantly increased with age, whereas heat shock protein 60 (HSP60) was decreased in the senescent DP-MSCs. Reactive oxygen species (ROS) staining showed the number of ROS-stained cells and the DCFH fluorescent level were higher in the aged group. Further we examined the senescence of DP-MSCs after modulating p16(INK4A) signaling. The results indicated the dysfunction of DP-MSCs was reversed by p16(INK4A) siRNA. In summary, our study indicated p16(INK4A) pathway may play a critical role in DP-MSCs age-related changes and the DNA damage response (DDR) and stress response may be the main mediators of DP-MSCs senescence induced by excessive activation of p16(INK4A) signaling. Copyright © 2014. Published by Elsevier Ireland Ltd.

Performance of p16INK4a ELISA as a primary cervical cancer screening test among a large cohort of HIV-infected women in western Kenya: a 2-year cross-sectional study.

PubMed

Wu, Tara J; Smith-McCune, Karen; Reuschenbach, Miriam; von Knebel Doeberitz, Magnus; Maloba, May; Huchko, Megan J

2016-09-13

A biomarker with increased specificity for cervical dysplasia compared with human papillomavirus (HPV) testing would be an attractive option for cervical cancer screening among HIV-infected women in resource-limited settings. p16(INK4a) has been explored as a biomarker for screening in general populations. A 2-year cross-sectional study. 2 large HIV primary care clinics in western Kenya. 1054 HIV-infected women in western Kenya undergoing cervical cancer screening as part of routine HIV care from October 2010 to November 2012. Participants underwent p16(INK4a) specimen collection and colposcopy. Lesions with unsatisfactory colposcopy or suspicious for cervical intraepithelial neoplasia 2+ (CIN2+; including CIN2/3 or invasive cervical cancer) were biopsied. Following biopsy, disease status was determined by histopathological diagnosis. We measured the sensitivity, specificity and predictive values of p16(INK4a) ELISA for CIN2+ detection among HIV-infected women and compared them to the test characteristics of current screening methods used in general as well as HIV-infected populations. Average p16(INK4a) concentration in cervical samples was 37.4 U/mL. After colposcopically directed biopsy, 127 (12%) women were determined to have CIN2+. Receiver operating characteristic analysis showed an area under the curve of 0.664 for p16(INK4a) to detect biopsy-proven CIN2+. At a p16(INK4a) cut-off level of 9 U/mL, sensitivity, specificity, positive and negative predictive values were 89.0%, 22.9%, 13.6% and 93.8%, respectively. The overall p16(INK4a) positivity at a cut-off level of 9 U/mL was 828 (78.6%) women. There were 325 (30.8%) cases of correct p16(INK4a) prediction to detect or rule out CIN2+, and 729 (69.2%) cases of incorrect p16(INK4a) prediction. p16(INK4a) ELISA did not perform well as a screening test for CIN2+ detection among HIV-infected women due to low specificity. Our study contributes to the ongoing search for a more specific alternative to HPV

Performance of p16INK4a ELISA as a primary cervical cancer screening test among a large cohort of HIV-infected women in western Kenya: a 2-year cross-sectional study

PubMed Central

Wu, Tara J; Smith-McCune, Karen; Reuschenbach, Miriam; von Knebel Doeberitz, Magnus; Maloba, May; Huchko, Megan J

2016-01-01

Objective A biomarker with increased specificity for cervical dysplasia compared with human papillomavirus (HPV) testing would be an attractive option for cervical cancer screening among HIV-infected women in resource-limited settings. p16INK4a has been explored as a biomarker for screening in general populations. Design A 2-year cross-sectional study. Setting 2 large HIV primary care clinics in western Kenya. Participants 1054 HIV-infected women in western Kenya undergoing cervical cancer screening as part of routine HIV care from October 2010 to November 2012. Interventions Participants underwent p16INK4a specimen collection and colposcopy. Lesions with unsatisfactory colposcopy or suspicious for cervical intraepithelial neoplasia 2+ (CIN2+; including CIN2/3 or invasive cervical cancer) were biopsied. Following biopsy, disease status was determined by histopathological diagnosis. Primary and secondary outcome measures We measured the sensitivity, specificity and predictive values of p16INK4a ELISA for CIN2+ detection among HIV-infected women and compared them to the test characteristics of current screening methods used in general as well as HIV-infected populations. Results Average p16INK4a concentration in cervical samples was 37.4 U/mL. After colposcopically directed biopsy, 127 (12%) women were determined to have CIN2+. Receiver operating characteristic analysis showed an area under the curve of 0.664 for p16INK4a to detect biopsy-proven CIN2+. At a p16INK4a cut-off level of 9 U/mL, sensitivity, specificity, positive and negative predictive values were 89.0%, 22.9%, 13.6% and 93.8%, respectively. The overall p16INK4a positivity at a cut-off level of 9 U/mL was 828 (78.6%) women. There were 325 (30.8%) cases of correct p16INK4a prediction to detect or rule out CIN2+, and 729 (69.2%) cases of incorrect p16INK4a prediction. Conclusions p16INK4a ELISA did not perform well as a screening test for CIN2+ detection among HIV-infected women due to low

Nuclear translocation of p19INK4d in response to oxidative DNA damage promotes chromatin relaxation.

PubMed

Sonzogni, Silvina V; Ogara, María F; Castillo, Daniela S; Sirkin, Pablo F; Radicella, J Pablo; Cánepa, Eduardo T

2015-01-01

DNA is continuously exposed to damaging agents that can lead to changes in the genetic information with adverse consequences. Nonetheless, eukaryotic cells have mechanisms such as the DNA damage response (DDR) to prevent genomic instability. The DNA of eukaryotic cells is packaged into nucleosomes, which fold the genome into highly condensed chromatin, but relatively little is known about the role of chromatin accessibility in DNA repair. p19INK4d, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle regulation and cellular DDR. Extensive data indicate that p19INK4d is a critical factor in the maintenance of genomic integrity and cell survival. p19INK4d is upregulated by various genotoxics, improving the repair efficiency for a variety of DNA lesions. The evidence of p19INK4d translocation into the nucleus and its low sequence specificity in its interaction with DNA prompted us to hypothesize that p19INK4d plays a role at an early stage of cellular DDR. In the present study, we demonstrate that upon oxidative DNA damage, p19INK4d strongly binds to and relaxes chromatin. Furthermore, in vitro accessibility assays show that DNA is more accessible to a restriction enzyme when a chromatinized plasmid is incubated in the presence of a protein extract with high levels of p19INK4d. Nuclear protein extracts from cells overexpressing p19INK4d are better able to repair a chromatinized and damaged plasmid. These observations support the notion that p19INK4d would act as a chromatin accessibility factor that allows the access of the repair machinery to the DNA damage site.

Induction of the Tumor-Suppressor p16INK4a within Regenerative Epithelial Crypts in Ulcerative Colitis1

PubMed Central

Furth, Emma E; Gustafson, Karen S; Dai, Charlotte Y; Gibson, Steven L; Menard-Katcher, Paul; Chen, Tina; Koh, Jim; Enders, Greg H

2006-01-01

Abstract p16INK4a is a major tumor-suppressor protein, but its regulation and settings of fuction remain poorly understood. To explore the notion that p16 is induced in vivo in response to replicative stress, we examined p16 expression in tissues from human ulcerative colitis (UC; n = 25) and normal controls (n = 20). p16 was expressed strongly in UC-associated neoplasms (n = 17), as seen previously in sporadic colonic neoplasms. In non-neoplastic UC epithelium, p16 was expressed in 33% of crypts (the proliferative compartment) compared to < 1% of normal controls. p16 expression did not correlate with degree of inflammation but did correlate with the degree of crypt architecture distortion (P = .002)—a reflection of epithelial regeneration. In coimmunofluorescence studies with Ki67, p16 expression was associated with cell cycle arrest (P < .001). Both UC and normal crypts displayed evidence for the activation of the DNA damage checkpoint pathway, and p16 was induced in primary cultures of normal epithelial cells by ionizing irradiation (IR). However, induction by IR displayed delayed kinetics, implying that p16 is not an immediate target of the checkpoint pathway. These findings support a model in which p16 is induced as an “emergency brake” in cells experiencing sustained replicative stress. PMID:16820088

Performance of p16INK4a/Ki-67 immunocytochemistry for identifying CIN2+ in atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion specimens: a Japanese Gynecologic Oncology Group study.

PubMed

Fujii, Takuma; Saito, Miyuki; Hasegawa, Toshihiko; Iwata, Takashi; Kuramoto, Hiroyuki; Kubushiro, Kaneyuki; Ohmura, Mineo; Ochiai, Kazunori; Arai, Hiroharu; Sakamoto, Masaru; Motoyama, Teiichi; Aoki, Daisuke

2015-02-01

p16(INK4a) immunohistochemistry has revealed a high rate of positivity in cervical intraepithelial neoplasia grade 2 (CIN2) and more severe conditions (CIN2+). The Lower Anogenital Squamous Terminology Standardization project proposed p16(INK4a) immunohistochemistry as an ancillary test for CIN. Immunocytochemistry involving dual staining for p16(INK4a) and Ki-67 in the triage of atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) is reported to be useful in the identification of CIN2+. However, it is unclear whether p16(INK4a)/Ki-67 immunocytochemistry is of practical relevance for the triage of ASCUS and LSIL in the Japanese screening system. From 427 women fulfilling the eligibility criteria, 188 ASCUS and 239 LSIL specimens were analyzed. The accuracy of p16(INK4a)/Ki-67 immunocytochemistry and genotyping of high-risk human papillomaviruses (HPVs) in detecting CIN2+ were compared. p16(INK4a)/Ki-67 immunocytochemistry was positive in 33.5 % (63/188) of ASCUS, and 36.8 % (88/239) of LSIL specimens. The sensitivity and specificity of p16(INK4a)/Ki-67 immunocytochemistry was 87.3 % (95 % confidence interval 78.0-93.8 %) and 76.4 % (71.6-80.8 %), respectively. The positive and negative predictive values were 45.7 % (37.6-54.0 %) and 96.4 % (93.4-98.3 %), respectively; positive and negative likelihood ratios were 3.71 and 0.17, respectively. Using the McNemar test, p16(INK4a)/Ki-67 immunocytochemistry showed equivalent sensitivity but higher specificity than the HPV genotyping test Compared with high-risk HPV genotyping, p16(INK4a)/Ki-67 immunocytochemistry was a more accurate triage test for identifying CIN2+ in ASCUS and LSIL specimens.

P16INK4a MEDIATED SUPPRESSION OF TELOMERASE IN NORMAL AND MALIGNANT HUMAN BREAST CELLS

PubMed Central

Bazarov, Alexey V.; van Sluis, Marjolein; Hines, Curtis; Bassett, Ekaterina; Beliveau, Alain; Campeau, Eric; Mukhopadhyay, Rituparna; Lee, Won Jae; Melodyev, Sonya; Zaslavsky, Yuri; Lee, Leonard; Rodier, Francis; Chicas, Agustin; Lowe, Scott W.; Benhattar, Jean; Ren, Bing; Campisi, Judith; Yaswen, Paul

2010-01-01

Summary The cyclin-dependent kinase inhibitor p16INK4a (CDKN2A) is an important tumor-suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti-oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short-term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self-renewal. PMID:20569236

Aging of mice is associated with p16(Ink4a)- and β-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells

PubMed Central

Hall, Brandon M.; Balan, Vitaly; Gleiberman, Anatoli S.; Strom, Evguenia; Krasnov, Peter; Virtuoso, Lauren P.; Rydkina, Elena; Vujcic, Slavoljub; Balan, Karina; Gitlin, Ilya; Leonova, Katerina; Polinsky, Alexander; Chernova, Olga B.; Gudkov, Andrei V.

2016-01-01

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and β-galactosidase activity at pH6.0 (β-galpH6), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/β-galpH6-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/β-galpH6-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/β-galpH6-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/β-galpH6-positive cells and reconsideration of potential cellular target for anti-aging treatment. PMID:27391570

INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions.

PubMed

Cánepa, Eduardo T; Scassa, María E; Ceruti, Julieta M; Marazita, Mariela C; Carcagno, Abel L; Sirkin, Pablo F; Ogara, María F

2007-07-01

The cyclin D-Cdk4-6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The members of INK4 family, comprising p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d), block the progression of the cell cycle by binding to either Cdk4 or Cdk6 and inhibiting the action of cyclin D. These INK4 proteins share a similar structure dominated by several ankyrin repeats. Although they appear to be structurally redundant and equally potent as inhibitors, the INK4 family members are differentially expressed during mouse development. The striking diversity in the pattern of expression of INK4 genes suggested that this family of cell cycle inhibitors might have cell lineage-specific or tissue-specific functions. The INK4 proteins are commonly lost or inactivated by mutations in diverse types of cancer, and they represent established or candidate tumor suppressors. Apart from their capacity to arrest cells in the G1-phase of the cell cycle they have been shown to participate in an increasing number of cellular processes. Given their emerging roles in fundamental physiological as well as pathological processes, it is interesting to explore the diverse roles for the individual INK4 family members in different functions other than cell cycle regulation. Extensive studies, over the past few years, uncover the involvement of INK4 proteins in senescence, apoptosis, DNA repair, and multistep oncogenesis. We will focus the discussion here on these unexpected issues.

Value of p16(INK)⁴(a) in the pathology of invasive penile squamous cell carcinomas: A report of 202 cases.

PubMed

Cubilla, Antonio L; Lloveras, Belén; Alejo, Maria; Clavero, Omar; Chaux, Alcides; Kasamatsu, Elena; Monfulleda, Núria; Tous, Sara; Alemany, Laia; Klaustermeier, Joellen; Muñoz, Nubia; Quint, Wim; de Sanjose, Silvia; Bosch, Francisco Xavier

2011-02-01

One third to one half of penile squamous cell carcinomas (SCCs) are related to human papillomavirus (HPV) infection. Viral detection is usually carried out by polymerase chain reaction (PCR) or other molecular methods. In this study, we evaluated p16(INK)⁴(a) immunohistochemical expression, which is simpler and less costly, as a potential marker of high-risk HPV (HR-HPV) infection in penile SCC. We pathologically classified 202 invasive penile carcinomas and performed HPV genotyping by short PCR fragment (SPF)₁₀ PCR and p16(INK)⁴(a) immunohistochemistry. We also evaluated HPV and p16(INK)⁴(a) according to the histologic subtypes of penile SCC. Tumors depicting continuous p16(INK)⁴(a) immunostain in all neoplastic cells were considered positive. HPV and p16(INK)⁴(a) status were compared using classifier performances and concordance indexes. Evidence of HPV (low-risk and high-risk genotypes) was found in 63 cases (31%) by PCR. Fifty-three p16(INK)⁴(a)-positive cases were identified (26%). Overexpression of p16(INK)⁴(a) had a sensitivity of 67% and a specificity of 91% for defining the HPV status. Concordance indexes between p16(INK)⁴(a) and HPV status were high (≥78%) in general cases and in all histologic subtypes of penile SCC. The stain was useful in the differential diagnosis of basaloid and low-grade warty carcinomas. Low-risk HPV genotypes were found in 5 tumors, 4 of which were p16(INK)⁴(a) negative. Basaloid and nonbasaloid high-grade (grade 3) SCCs were more likely to be HR-HPV positive when compared with grades 1 to 2 tumors (P<0.000001 and 0.0417, respectively). p16(INK)⁴(a) overexpression was found to be a reliable marker for HR-HPV and a helpful tool in the differential diagnosis of low-grade verruciform and high-grade solid penile tumors. SCC variants depicting basaloid features were more likely to be HPV and p16(INK)⁴(a) positive than low-grade, keratinizing lesions. We also observed a tendency toward HPV positivity in high

p16(INK4a) -mediated suppression of telomerase in normal and malignant human breast cells.

PubMed

Bazarov, Alexey V; Van Sluis, Marjolein; Hines, William C; Bassett, Ekaterina; Beliveau, Alain; Campeau, Eric; Mukhopadhyay, Rituparna; Lee, Won Jae; Melodyev, Sonya; Zaslavsky, Yuri; Lee, Leonard; Rodier, Francis; Chicas, Agustin; Lowe, Scott W; Benhattar, Jean; Ren, Bing; Campisi, Judith; Yaswen, Paul

2010-10-01

The cyclin-dependent kinase inhibitor p16(INK4a) (CDKN2A) is an important tumor suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti-oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short-term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self-renewal. © 2010 The Authors Aging Cell © 2010 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

Induction of the tumor-suppressor p16(INK4a) within regenerative epithelial crypts in ulcerative colitis.

PubMed

Furth, Emma E; Gustafson, Karen S; Dai, Charlotte Y; Gibson, Steven L; Menard-Katcher, Paul; Chen, Tina; Koh, Jim; Enders, Greg H

2006-06-01

p16(INK4a) is a major tumor-suppressor protein, but its regulation and settings of fuction remain poorly understood. To explore the notion that p16 is induced in vivo in response to replicative stress, we examined p16 expression in tissues from human ulcerative colitis (UC; n = 25) and normal controls (n = 20). p16 was expressed strongly in UC-associated neoplasms (n = 17), as seen previously in sporadic colonic neoplasms. In non-neoplastic UC epithelium, p16 was expressed in 33% of crypts (the proliferative compartment) compared to < 1% of normal controls. p16 expression did not correlate with degree of inflammation but did correlate with the degree of crypt architecture distortion (P = .002)-a reflection of epithelial regeneration. In coimmunofluorescence studies with Ki67, p16 expression was associated with cell cycle arrest (P < .001). Both UC and normal crypts displayed evidence for the activation of the DNA damage checkpoint pathway, and p16 was induced in primary cultures of normal epithelial cells by ionizing irradiation (IR). However, induction by IR displayed delayed kinetics, implying that p16 is not an immediate target of the checkpoint pathway. These findings support a model in which p16 is induced as an "emergency brake" in cells experiencing sustained replicative stress.

Double positivity for HPV-DNA/p16ink4a is the biomarker with strongest diagnostic accuracy and prognostic value for human papillomavirus related oropharyngeal cancer patients.

PubMed

Mena, Marisa; Taberna, Miren; Tous, Sara; Marquez, Sandra; Clavero, Omar; Quiros, Beatriz; Lloveras, Belen; Alejo, Maria; Leon, Xavier; Quer, Miquel; Bagué, Silvia; Mesia, Ricard; Nogués, Julio; Gomà, Montserrat; Aguila, Anton; Bonfill, Teresa; Blazquez, Carmen; Guix, Marta; Hijano, Rafael; Torres, Montserrat; Holzinger, Dana; Pawlita, Michael; Pavon, Miguel Angel; Bravo, Ignacio G; de Sanjosé, Silvia; Bosch, Francesc Xavier; Alemany, Laia

2018-03-01

The etiologic role of human papillomaviruses (HPV) in oropharyngeal cancer (OPC) is well established. Nevertheless, information on survival differences by anatomic sub-site or treatment remains scarce, and it is still unclear the HPV-relatedness definition with best diagnostic accuracy and prognostic value. We conducted a retrospective cohort study of all patients diagnosed with a primary OPC in four Catalonian hospitals from 1990 to 2013. Formalin-fixed, paraffin-embedded cancer tissues were subjected to histopathological evaluation, DNA quality control, HPV-DNA detection, and p16 INK4a /pRb/p53/Cyclin-D1 immunohistochemistry. HPV-DNA positive and a random sample of HPV-DNA negative cases were subjected to HPV-E6*I mRNA detection. Demographic, tobacco/alcohol use, clinical and follow-up data were collected. Multivariate models were used to evaluate factors associated with HPV positivity as defined by four different HPV-relatedness definitions. Proportional-hazards models were used to compare the risk of death and recurrence among HPV-related and non-related OPC. 788 patients yielded a valid HPV-DNA result. The percentage of positive cases was 10.9%, 10.2%, 8.5% and 7.4% for p16 INK4a , HPV-DNA, HPV-DNA/HPV-E6*I mRNA, and HPV-DNA/p16 INK4a , respectively. Being non-smoker or non-drinker was consistently associated across HPV-relatedness definitions with HPV positivity. A suggestion of survival differences between anatomic sub-sites and treatments was observed. Double positivity for HPV-DNA/p16 INK4a showed strongest diagnostic accuracy and prognostic value. Double positivity for HPV-DNA/p16 INK4a , a test that can be easily implemented in the clinical practice, has optimal diagnostic accuracy and prognostic value. Our results have strong clinical implications for patients' classification and handling and also suggest that not all the HPV-related OPC behave similarly. Copyright © 2018 Elsevier Ltd. All rights reserved.

E2F1-mediated upregulation of p19INK4d determines its periodic expression during cell cycle and regulates cellular proliferation.

PubMed

Carcagno, Abel L; Marazita, Mariela C; Ogara, María F; Ceruti, Julieta M; Sonzogni, Silvina V; Scassa, María E; Giono, Luciana E; Cánepa, Eduardo T

2011-01-01

A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity.

E2F1-Mediated Upregulation of p19INK4d Determines Its Periodic Expression during Cell Cycle and Regulates Cellular Proliferation

PubMed Central

Carcagno, Abel L.; Marazita, Mariela C.; Ogara, María F.; Ceruti, Julieta M.; Sonzogni, Silvina V.; Scassa, María E.; Giono, Luciana E.; Cánepa, Eduardo T.

2011-01-01

Background A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. Methodology/Principal Findings In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. Conclusions/Significance The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an

p16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells

PubMed Central

Cen, Ling; Carlson, Brett L.; Schroeder, Mark A.; Ostrem, Jamie L.; Kitange, Gaspar J.; Mladek, Ann C.; Fink, Stephanie R.; Decker, Paul A.; Wu, Wenting; Kim, Jung-Sik; Waldman, Todd; Jenkins, Robert B.; Sarkaria, Jann N.

2012-01-01

Deregulation of the p16INK4a-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16INK4a-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16INK4a-Cdk4/6-Rb pathway. In vitro evaluation using short-term explant cultures from selected GBM xenograft lines showed that PD0332991 effectively arrested cell cycle in G1-phase and inhibited cell proliferation dose-dependently in lines deleted for CDKN2A/B-p16INK4a and either single-copy deletion of CDK4 (GBM22), high-level CDK6 amplification (GBM34), or deletion of CDKN2C/p18INK4c (GBM43). In contrast, 2 GBM lines with p16INK4a expression and either CDK4 amplification (GBM5) or RB mutation (GBM28) were completely resistant to PD0332991. Additional xenograft lines were screened, and GBM63 was identified to have p16INK4a expression and CDK4 amplification. Similar to the results with GBM5, GBM63 was resistant to PD0332991 treatment. In an orthotopic survival model, treatment of GBM6 xenografts (CDKN2A/B-deleted and CDK4 wild-type) with PD0332991 significantly suppressed tumor cell proliferation and prolonged survival. Collectively, these data support the concept that GBM tumors lacking p16INK4a expression and with nonamplified CDK4 and wild-type RB status may be more susceptible to Cdk4/6 inhibition using PD0332991. PMID:22711607

Simulation of Different Truncated p16INK4a Forms and In Silico Study of Interaction with Cdk4

PubMed Central

Fahham, Najmeh; Ghahremani, Mohammad Hossein; Sardari, Soroush; Vaziri, Behrouz; Ostad, Seyed Nasser

2008-01-01

Protein-protein interactions studies can greatly increase the amount of structural and functional information pertaining to biologically active molecules and processes. The information obtained from such studies can lead to design and application of new modification in order to obtain a desired bioactivity. Many application packages and servers performing docking, such as HEX, DOT, AUTODOCK, and ZDOCK are now available for predicting the lowest free energy state of a protein complex. In this study, we have focused on cyclin-dependent kinase 4 (Cdk4), a key molecule in the regulation of cell cycle progression at the G1-S phase restriction point and p16INK4a, a tumor suppressor which inhibits Cdk4 activity. Truncated structures were created to find the more critical regions of p16 for interaction. The tertiary structures were determined by ProSAL, GENO3D Web Server. We evaluated their interactions with Cdk4 using two docking systems, HEX 4.5 and DOT 1. Calculations were performed on a high-speed computer. Minimizations and visualizations were carried out by PdbViewer 3.7. Considering shape and shape/electrostatic total energy, structures containing ANK II, III and IV motifs that lack the N-terminal region of the full length p16 molecule showed the best fit complexes among the p16 truncated forms. The free energies were compatible with that of p16 full length original form, the full length. It seems that the N-terminal of the molecule is not crucial for the interaction since the truncated structure containing only this region did not show a good total energy. PMID:19352455

Methylation of p16(INK4a) promoters occurs in vivo in histologically normal human mammary epithelia

NASA Technical Reports Server (NTRS)

Holst, Charles R.; Nuovo, Gerard J.; Esteller, Manel; Chew, Karen; Baylin, Stephen B.; Herman, James G.; Tlsty, Thea D.

2003-01-01

Cultures of human mammary epithelial cells (HMECs) contain a subpopulation of variant cells with the capacity to propagate beyond an in vitro proliferation barrier. These variant HMECs, which contain hypermethylated and silenced p16(INK4a) (p16) promoters, eventually accumulate multiple chromosomal changes, many of which are similar to those detected in premalignant and malignant lesions of breast cancer. To determine the origin of these variant HMECs in culture, we used Luria-Delbruck fluctuation analysis and found that variant HMECs exist within the population before the proliferation barrier, thereby raising the possibility that variant HMECs exist in vivo before cultivation. To test this hypothesis, we examined mammary tissue from normal women for evidence of p16 promoter hypermethylation. Here we show that epithelial cells with methylation of p16 promoter sequences occur in focal patches of histologically normal mammary tissue of a substantial fraction of healthy, cancer-free women.

[The mechanisms of p21WAF1/Cip-1 expression in MOLT-4 cell line induced by TSA].

PubMed

Song, Yi; Liu, Mei-Ju; Zhao, Guo-Wei; Qian, Jun-Jie; Dong, Yan; Liu, Hua; Sun, Guo-Jing; Mei, Zhu-Zhong; Liu, Bin; Tian, Bao-Lei; Sun, Zhi-Xian

2005-04-01

To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.

Transcriptional upregulation of p19INK4d upon diverse genotoxic stress is critical for optimal DNA damage response.

PubMed

Ceruti, Julieta M; Scassa, María E; Marazita, Mariela C; Carcagno, Abel C; Sirkin, Pablo F; Cánepa, Eduardo T

2009-06-01

p19INK4d promotes survival of several cell lines after UV irradiation due to enhanced DNA repair, independently of CDK4 inhibition. To further understand the action of p19INK4d in the cellular response to DNA damage, we aimed to elucidate whether this novel regulator plays a role only in mechanisms triggered by UV or participates in diverse mechanisms initiated by different genotoxics. We found that p19INK4d is induced in cells injured with cisplatin or beta-amyloid peptide as robustly as with UV. The mentioned genotoxics transcriptionally activate p19INK4d expression as demonstrated by run-on assay without influencing its mRNA stability and with partial requirement of protein synthesis. It is not currently known whether DNA damage-inducible genes are turned on by the DNA damage itself or by the consequences of that damage. Experiments carried out in cells transfected with distinct damaged DNA structures revealed that the damage itself is not responsible for the observed up-regulation. It is also not known whether the increased expression of DNA-damage-inducible genes is related to immediate protective responses such as DNA repair or to more delayed responses such as cell cycle arrest or apoptosis. We found that ectopic expression of p19INK4d improves DNA repair ability and protects neuroblastoma cells from apoptosis caused by cisplatin or beta-amyloid peptide. Using clonal cell lines where p19INK4d levels can be modified at will, we show that p19INK4d expression correlates with increased survival and clonogenicity. The results presented here, prompted us to suggest that p19INK4d displays an important role in an early stage of cellular DNA damage response.

hERG1/Kv11.1 activation stimulates transcription of p21waf/cip in breast cancer cells via a calcineurin-dependent mechanism.

PubMed

Perez-Neut, Mathew; Rao, Vidhya R; Gentile, Saverio

2016-09-13

The function of Kv11.1 is emerging in breast cancer biology, as a growing body of evidence indicates that the hERG1/Kv11.1 potassium channel is aberrantly expressed in several cancer types including breast cancers.The biological effects of Kv11.1 channel blockers and their associated side effects are very well known but the potential use of Kv11.1 activators as an anticancer strategy are still unexplored. In our previous work, we have established that stimulation of the Kv11.1 potassium channel activates a senescent-like program that is characterized by a significant increase in tumor suppressor protein levels, such as p21waf/cip and p16INK4A. In this study we investigated the mechanism linking Kv11.1 stimulation to augmentation of p21waf/cip protein level. We have demonstrated that the Kv11.1 channel activator NS1643 activates a calcineurin-dependent transcription of p21waf/cip and that this event is fundamental for the inhibitory effect of NS1643 on cell proliferation. Our results reveal a novel mechanism by which stimulation of Kv11.1 channel leads to transcription of a potent tumor suppressor and suggest a potential therapeutic use for Kv11.1 channel activators.

hERG1/Kv11.1 activation stimulates transcription of p21waf/cip in breast cancer cells via a calcineurin-dependent mechanism

PubMed Central

Perez-Neut, Mathew; Rao, Vidhya R.; Gentile, Saverio

2016-01-01

The function of Kv11.1 is emerging in breast cancer biology, as a growing body of evidence indicates that the hERG1/Kv11.1 potassium channel is aberrantly expressed in several cancer types including breast cancers. The biological effects of Kv11.1 channel blockers and their associated side effects are very well known but the potential use of Kv11.1 activators as an anticancer strategy are still unexplored. In our previous work, we have established that stimulation of the Kv11.1 potassium channel activates a senescent-like program that is characterized by a significant increase in tumor suppressor protein levels, such as p21waf/cip and p16INK4A. In this study we investigated the mechanism linking Kv11.1 stimulation to augmentation of p21waf/cip protein level. We have demonstrated that the Kv11.1 channel activator NS1643 activates a calcineurin-dependent transcription of p21waf/cip and that this event is fundamental for the inhibitory effect of NS1643 on cell proliferation. Our results reveal a novel mechanism by which stimulation of Kv11.1 channel leads to transcription of a potent tumor suppressor and suggest a potential therapeutic use for Kv11.1 channel activators. PMID:25945833

Chromatin relaxation-mediated induction of p19INK4d increases the ability of cells to repair damaged DNA.

PubMed

Ogara, María F; Sirkin, Pablo F; Carcagno, Abel L; Marazita, Mariela C; Sonzogni, Silvina V; Ceruti, Julieta M; Cánepa, Eduardo T

2013-01-01

The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies.

Chromatin Relaxation-Mediated Induction of p19INK4d Increases the Ability of Cells to Repair Damaged DNA

PubMed Central

Carcagno, Abel L.; Marazita, Mariela C.; Sonzogni, Silvina V.; Ceruti, Julieta M.; Cánepa, Eduardo T.

2013-01-01

The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies. PMID:23593412

Combining routine morphology, p16(INK4a) immunohistochemistry, and in situ hybridization for the detection of human papillomavirus infection in penile carcinomas: a tissue microarray study using classifier performance analyses.

PubMed

Chaux, Alcides; Cubilla, Antonio L; Haffner, Michael C; Lecksell, Kristen L; Sharma, Rajni; Burnett, Arthur L; Netto, George J

2014-02-01

Infection by high-risk human papillomavirus (HR-HPV) plays an important role in the pathogenesis of penile cancer in approximately 50% of the patients. The gold standard for human papillomavirus (HPV) detection is the polymerase chain reaction (PCR) assay. However, technical requirements and associated costs preclude the worldwide use of PCR assays on a routine basis. Herein, we evaluated the predictive abilities of tumor morphology, immunohistochemistry for p16(INK4a) expression, and in situ hybridization (ISH) for HR-HPV detection in defining HPV status, as established by PCR. Tissue samples from 48 patients with HPV-positive penile squamous cell carcinoma (SCC) were included in 4 tissue microarrays (TMA). Sensitivities and specificities were as follows: tumor morphology, 70% and 68%; p16(INK4a) immunohistochemistry, 65% and 90%; HR-HPV ISH, 47% and 100%. Regarding combinations of the predictors, the best performance was seen when HR-HPV ISH and p16(INK4a) immunohistochemistry were combined, regardless of the tumor morphology: sensitivity, 88%; specificity, 64%; area under the receiver-operating characteristic (AUC) curve, 0.83. Combinations of tumor morphology with p16(INK4a) immunohistochemistry or with HR-HPV ISH performed similarly well. In penile SCC, both p16(INK4a) immunohistochemistry and ISH for HR-HPV increase the predictive ability of routine morphology in defining HPV status. These tests can be interpreted differentially, depending on the necessity of a higher sensitivity or a higher specificity. For research/screening studies, we recommend combining tumor morphology, p16(INK4a) immunohistochemistry, and HR-HPV ISH. To increase sensitivity, positivity in any of these predictors should be considered as indicative of HPV infection. For routine diagnosis of clinical cases, criteria should be more stringent, and, to achieve the highest specificity in classifying a case as HPV-related, all predictors should be consistently positive. The data generated in

Cell cycle inhibitor, p19INK4d, promotes cell survival and decreases chromosomal aberrations after genotoxic insult due to enhanced DNA repair.

PubMed

Scassa, María E; Marazita, Mariela C; Ceruti, Julieta M; Carcagno, Abel L; Sirkin, Pablo F; González-Cid, Marcela; Pignataro, Omar P; Cánepa, Eduardo T

2007-05-01

Genome integrity and cell proliferation and survival are regulated by an intricate network of pathways that includes cell cycle checkpoints, DNA repair and recombination, and programmed cell death. It makes sense that there should be a coordinated regulation of these different processes, but the components of such mechanisms remain unknown. In this report, we demonstrate that p19INK4d expression enhances cell survival under genotoxic conditions. By using p19INK4d-overexpressing clones, we demonstrated that p19INK4d expression correlates with the cellular resistance to UV treatment with increased DNA repair activity against UV-induced lesions. On the contrary, cells transfected with p19INK4d antisense cDNA show reduced ability to repair DNA damage and increased sensitivity to genotoxic insult when compared with their p19INK4d-overexpressing counterparts. Consistent with these findings, our studies also show that p19INK4d-overexpressing cells present not only a minor accumulation of UV-induced chromosomal aberrations but a lower frequency of spontaneous chromosome abnormalities than p19INK4d-antisense cells. Lastly, we suggest that p19INK4d effects are dissociated from its role as CDK4/6 inhibitor. The results presented herein support a crucial role for p19INK4d in regulating genomic stability and overall cell viability under conditions of genotoxic stress. We propose that p19INK4d would belong to a protein network that would integrate DNA repair, apoptotic and checkpoint mechanisms in order to maintain the genomic integrity.

Regulation of proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19INK4d.

PubMed

Kero, Darko; Vukojevic, Katarina; Stazic, Petra; Sundov, Danijela; Mardesic Brakus, Snjezana; Saraga-Babic, Mirna

2017-10-02

Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19 INK4d . p19 INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19 INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19 INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19 INK4d throughout the investigated period indicates that p19 INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19 INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.

Human Keratinocytes That Express hTERT and Also Bypass a p16INK4a-Enforced Mechanism That Limits Life Span Become Immortal yet Retain Normal Growth and Differentiation Characteristics

PubMed Central

Dickson, Mark A.; Hahn, William C.; Ino, Yasushi; Ronfard, Vincent; Wu, Jenny Y.; Weinberg, Robert A.; Louis, David N.; Li, Frederick P.; Rheinwald, James G.

2000-01-01

Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16INK4a cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16INK4a-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT+ keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16INK4a expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16INK4a-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes

HFE polymorphisms influence the response to chemotherapeutic agents via induction of p16INK4A.

PubMed

Lee, Sang Y; Liu, Siying; Mitchell, Ryan M; Slagle-Webb, Becky; Hong, Young-Soo; Sheehan, Jonas M; Connor, James R

2011-11-01

HFE is a protein that impacts cellular iron uptake. HFE gene variants are identified as risk factors or modifiers for multiple diseases. Using HFE stably transfected human neuroblastoma cells, we found that cells carrying the C282Y HFE variant do not differentiate when exposed to retinoic acid. Therefore, we hypothesized HFE variants would impact response to therapeutic agents. Both the human neuroblastoma and glioma cells that express the C282Y HFE variant are resistant to Temodar, geldanamycin and γ-radiation. A gene array analysis revealed that p16INK4A (p16) expression was increased in association with C282Y expression. Decreasing p16 protein by siRNA resulted in increased vulnerability to all of the therapeutic agents suggesting that p16 is responsible for the resistance. Decreasing HFE expression by siRNA resulted in a 85% decrease in p16 expression in the neuroblastoma cells but not the astrocytoma cells. These data suggest a potential direct relationship between HFE and p16 that may be cell specific or mediated by different pathways in the different cell types. In conclusion, the C282Y HFE variant impacts the vulnerability of cancer cells to current treatment strategies apparently by increasing expression of p16. Although best known as a tumor suppressor, there are multiple reports that p16 is elevated in some forms of cancer. Given the frequency of the HFE gene variants, as high as 10% of the Caucasian population, these data provide compelling evidence that the C282Y HFE variant should be part of a pharmacogenetic strategy for evaluating treatment efficacy in cancer cells. Copyright © 2011 UICC.

Paper-based immunosensor with signal amplification by enzyme-labeled anti-p16INK4a multifunctionalized gold nanoparticles for cervical cancer screening.

PubMed

Yokchom, Ruchuon; Laiwejpithaya, Somsak; Maneeprakorn, Weerakanya; Tapaneeyakorn, Satita; Rabablert, Jundee; Dharakul, Tararaj

2018-04-01

The aim of this study was to develop a paper-based immunosensor for cervical cancer screening, with signal amplification by multifunctionalized gold nanoparticles (AuNPs). The AuNPs were functionalized with a highly specific antibody to the p16 INK4a cancer biomarker. The signal was amplified using a combination of the peroxidase activity of horseradish peroxidase (HRP) enzyme-antibody conjugate and the peroxidase-like activity of the AuNPs. The immune complex of p16 INK4a protein and multifunctionalized AuNPs was deposited on the nitrocellulose membrane, and a positive result was generated by catalytic oxidation of peroxidase enzyme substrate 3,3',5,5'-Tetramethylbenzidine (TMB). The entire reaction occurred on the membrane within 30 min. Evaluation in clinical samples revealed 85.2% accuracy with a kappa coefficient of 0.69. This proof of concept study demonstrates the successful development of a highly accurate, paper-based immunosensor that is easy to interpret using the naked eye and that is suitable for cervical cancer screening in low-resource settings. Copyright © 2018 Elsevier Inc. All rights reserved.

Novel Insights into p63 Expression and Function in Prostate

DTIC Science & Technology

2004-07-15

Gene Family, In Metastatic Prostate Cancer Cells (1999) 49. Kononen, J., Bubendorf, L., Kallioniemi, A., Barlund, M., Schraml, P., Leighton , S... Christiansen -Weber, T., and Haas, M. Independent induction of senescence by p16INK4a and p21CIP1 in spontaneously immortalized human fibroblasts. Cell

The negative cell cycle regulators, p27Kip1, p18Ink4c, and GSK-3, play critical role in maintaining quiescence of adult human pancreatic β-cells and restrict their ability to proliferate

PubMed Central

Stein, Jeffrey; Milewski, Wieslawa M; Dey, Arunangsu

2013-01-01

Adult human pancreatic β-cells are primarily quiescent (G0) yet the mechanisms controlling their quiescence are poorly understood. Here, we demonstrate, by immunofluorescence and confocal microscopy, abundant levels of the critical negative cell cycle regulators, p27(Kip1) and p18(Ink4c), 2 key members of cyclin-dependent kinase (CDK) inhibitor family, and glycogen synthase kinase-3 (GSK-3), a serine-threonine protein kinase, in islet β-cells of adult human pancreatic tissue. Our data show that p27(Kip1) localizes primarily in β-cell nuclei, whereas, p18(Ink4c) is mostly present in β-cell cytosol. Additionally, p-p27(S10), a phosphorylated form of p27(Kip1), which was shown to interact with and to sequester cyclinD-CDK4/6 in the cytoplasm, is present in substantial amounts in β-cell cytosol. Our immunofluorescence analysis displays similar distribution pattern of p27(Kip1), p-p27(S10), p18(Ink4c) and GSK-3 in islet β-cells of adult mouse pancreatic tissue. We demonstrate marked interaction of p27(Kip1) with cyclin D3, an abundant D-type cyclin in adult human islets, and vice versa as well as with its cognate kinase partners, CDK4 and CDK6. Likewise, we show marked interaction of p18(Ink4c) with CDK4. The data collectively suggest that inhibition of CDK function by p27(Kip1) and p18(Ink4c) contributes to human β-cell quiescence. Consistent with this, we have found by BrdU incorporation assay that combined treatments of small molecule GSK-3 inhibitor and mitogen/s lead to elevated proliferation of human β-cells, which is caused partly due to p27(Kip1) downregulation. The results altogether suggest that ex vivo expansion of human β-cells is achievable via increased proliferation for β-cell replacement therapy in diabetes. PMID:23896637

Wolf-Hirschhorn (4p-) syndrome: prenatal diagnosis, molecular cytogenetic characterization and association with a 1.2-Mb microduplication at 8p22-p21.3 and a 1.1-Mb microduplication at 10p15.3 in a fetus with an apparently pure 4p deletion.

PubMed

Chen, Chih-Ping; Su, Yi-Ning; Chen, Yi-Yung; Su, Jun-Wei; Chern, Schu-Rern; Chen, Yu-Ting; Chen, Wen-Lin; Chen, Li-Feng; Wang, Wayseen

2011-12-01

To present prenatal diagnosis and molecular cytogenetic characterization of Wolf-Hirschhorn syndrome (WHS) associated with microduplications at 8p and 10p in a fetus with an apparently pure 4p deletion. A 35-year-old gravida 2, para 1 woman underwent amniocentesis at 18 weeks of gestation because of advanced maternal age. Her husband was 38 years of age. There was no family history of congenital malformations. Amniocentesis revealed a karyotype of 46,XY,del(4p16.1). The parental karyotypes were normal. Array comparative genomic hybridization (aCGH) analysis revealed a 6.5-Mb deletion at 4p16.3-p16.1, a 1.2-Mb microduplication at 8p22-p21.3, and a 1.1-Mb microduplication at 10p15.3, or arr cgh 4p16.3p16.1 (0-6,531,998 bp)×1, 8p22p21.3 (18,705,388-19,940,445 bp)×3, 10p15.3 (0-1,105,065 bp)×3. Polymorphic DNA marker analysis confirmed a paternal origin of 4p deletion. Prenatal ultrasound revealed facial dysmorphism and hypospadias. The aCGH analysis of the parents revealed no genomic imbalance. Fluorescence in situ hybridization study showed an unbalanced reciprocal translocation between chromosomes 4 and 10 at bands 4p16.1 and 10p15.3. The cytogenetic result, thus, was 46,XY,der(4)t(4;10)(p16.1;p15.3),dup(8)(p21.3p22). The parents elected to terminate the pregnancy, and a 470-g malformed fetus was delivered. The present case provides evidence that an apparently pure 4p deletion can be associated with subtle chromosome imbalances in other chromosomes. Copyright © 2011. Published by Elsevier B.V.

The Expression Pattern of the Cell Cycle Inhibitor p19INK4d by Progenitor Cells of the Rat Embryonic Telencephalon and Neonatal Anterior Subventricular Zone

PubMed Central

Coskun, Volkan; Luskin, Marla B.

2014-01-01

In this study we investigated whether the pattern of expression of the cyclin-dependent kinase inhibitor p19INK4d by the unique progenitor cells of the neonatal anterior subventricular zone (SVZa) can account for their ability to divide even though they express phenotypic characteristics of differentiated neurons. p19INK4d was chosen for analysis because it usually acts to block permanently the cell cycle at the G1 phase. p19INK4d immunoreactivity and the incorporation of bromodeoxyuridine (BrdU) by SVZa cells were compared with that of the more typical progenitor cells of the prenatal telencephalic ventricular zone. In the developing telencephalon, p19INK4d is expressed by postmitotic cells and has a characteristic perinuclear distribution depending on the laminar position and state of differentiation of a cell. Moreover, the laminar-specific staining of the developing cerebral cortex revealed that the ventricular zone (VZ) is divided into p19INK4d(+) and p19INK4d(−) sublaminae, indicating that the VZ has a previously unrecognized level of functional organization. Furthermore, the rostral migratory stream, traversed by the SVZa-derived cells, exhibits an anteriorhigh–posteriorlow gradient of p19INK4d expression. On the basis of the p19INK4d immunoreactivity and BrdU incorporation, SVZa-derived cells appear to exit and reenter the cell cycle successively. Thus, in contrast to telencephalic VZ cells, SVZa cells continue to undergo multiple rounds of division and differentiation before becoming postmitotic. PMID:11312294

Protein p16 as a marker of dysplastic and neoplastic alterations in cervical epithelial cells

PubMed Central

Volgareva, Galina; Zavalishina, Larisa; Andreeva, Yulia; Frank, Georgy; Krutikova, Ella; Golovina, Darya; Bliev, Alexander; Spitkovsky, Dimitry; Ermilova, Valeriya; Kisseljov, Fjodor

2004-01-01

Background Cervical carcinomas are second most frequent type of women cancer. Success in diagnostics of this disease is due to the use of Pap-test (cytological smear analysis). However Pap-test gives significant portion of both false-positive and false-negative conclusions. Amendments of the diagnostic procedure are desirable. Aetiological role of papillomaviruses in cervical cancer is established while the role of cellular gene alterations in the course of tumor progression is less clear. Several research groups including us have recently named the protein p16INK4a as a possible diagnostic marker of cervical cancer. To evaluate whether the specificity of p16INK4a expression in dysplastic and neoplastic cervical epithelium is sufficient for such application we undertook a broader immunochistochemical registration of this protein with a highly p16INK4a-specific monoclonal antibody. Methods Paraffin-embedded samples of diagnostic biopsies and surgical materials were used. Control group included vaginal smears of healthy women and biopsy samples from patients with cervical ectopia. We examined 197 samples in total. Monoclonal antibody E6H4 (MTM Laboratories, Germany) was used. Results In control samples we did not find any p16INK4a-positive cells. Overexpression of p16INK4a was detected in samples of cervical dysplasia (CINs) and carcinomas. The portion of p16INK4a-positive samples increased in the row: CIN I – CIN II – CIN III – invasive carcinoma. For all stages the samples were found to be heterogeneous with respect to p16INK4a-expression. Every third of CINs III and one invasive squamous cell carcinoma (out of 21 analyzed) were negative. Conclusions Overexpression of the protein p16INK4a is typical for dysplastic and neoplastic epithelium of cervix uteri. However p16INK4a-negative CINs and carcinomas do exist. All stages of CINs and carcinomas analyzed are heterogeneous with respect to p16INK4a expression. So p16INK4a-negativity is not a sufficient reason to

Alterations in RDINK4/ARF-mediated en bloc regulation of the INK4-ARF locus in human squamous cell carcinoma of the head and neck

PubMed Central

Poi, Ming J.; Knobloch, Thomas J.; Sears, Marta T.; Warner, Blake M.; Uhrig, Lana K.; Weghorst, Christopher M.; Li, Junan

2014-01-01

The presence of RDINK4/ARF (RD) enhancer in the INK4-ARF locus provides a novel mechanism to simultaneously increase the transcription of p15INK4b (p15), p14ARF (p14), and p16INK4a (p16). While such up-regulation can be repressed through interactions between RD and oncoproteins CDC6 and BMI1, little is known about the involvement of RD in cancer. In this study we investigated RD deletions in 30 squamous cell carcinoma of the head and neck (SCCHN) and the patient-matched High At-Risk Mucosa specimens (HARM, “phenotypically normal” tissues neighboring SCCHN foci but beyond the surgical resection margin). RD was deleted (homozygously/heterozygously) in SCCHN and HARM at the incidence of 36.7% (11/30) and 13.3% (4/30), respectively. In comparison, no RD deletion was detected in 26 oral buccal brush biopsy specimens from healthy donors. Both p16 and p14 were lowly expressed in SCCHN and HARM, and their mRNA expression levels were positively associated with each other (p<0.01). Moreover, BMI1 was highly expressed in both SCCHN and HARM, and BMI1 over-expression was associated with p16 down-regulation in SCCHN (p<0.05). These results indicate that RD deletion and BMI1 overexpression frequently occur in the early stage of oral carcinogenesis and BMI1 overexpression may down-regulate the transcription of p16 and p14 through interfering with RD. PMID:24302590

Simultaneous knockdown of BRAF and expression of INK4A in melanoma cells leads to potent growth inhibition and apoptosis

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

Zhao Yanhua; Zhang Yan; Yang Zhen

2008-06-06

Abnormal BRAF and p16INK4A co-exist in 60% of melanomas. BRAF mutation also occurs in 80% of benign nevi where it turns-on p16INK4A resulting in proliferative senescence; loss of p16INK4A removes the inhibitory block leading to melanoma development. Since only melanomas with wild-type BRAF have amplified CDK4 and cyclin D1 genes, p16INK4A-CDK4/6-cyclin D pathway is viewed as linearly downstream of BRAF. Thus, co-occurrence of aberrant BRAF and INK4A may be remnant of changes during melanoma formation without functional significance. To explore this notion, we simultaneously knocked down BRAF (via siRNA) and expressed INK4A cDNA in melanoma cells and observed enhanced growthmore » inhibition. Notably, although each alone had no statistically significant effect on apoptosis, co-expression of BRAF siRNA and INK4A cDNA caused potent apoptosis, which was associated with up-regulation of BIM and down-regulation of BCL2. Our results suggest that aberrant BRAF and INK4A cooperate to promote proliferation and survival of melanoma cells.« less

Sexual behaviour, HPV status and p16INK4a expression in oropharyngeal and oral cavity squamous cell carcinomas: a case-case comparison study.

PubMed

Emmett, Sarah; Boros, Samuel; Whiteman, David C; Porceddu, Sandro V; Panizza, Benedict J; Antonsson, Annika

2018-06-01

A significant proportion of mucosal squamous cell carcinomas of the head and neck (HNSCC; particularly of the oropharynx) are directly attributable to the human papillomavirus (HPV). The increase in the incidence of HPV-related tumours has been postulated to be due to changing sexual practices in the community. We analysed 136 formalin-fixed paraffin-embedded squamous cell carcinomas from the oral cavity (n=40) and oropharynx (n=96) recruited from the Princess Alexandra Hospital (Brisbane, Australia). Samples were analysed for the presence of HPV DNA using a combination of mucosal HPV general primer GP+ PCR and sequencing; p 16INK4a expression was assessed by immunohistochemistry. Each patient completed a questionnaire detailing their lifestyle factors, such as tobacco smoking and alcohol consumption, marital status, and sexual behaviour and history. The HPV DNA prevalence was 5 % in the oral cavity cancers and 72 % in the oropharyngeal cancers (P<0.0001). HPV-16 was the most commonly detected HPV type (found in 91 % of all HPV-positive tumours). There was a strong correlation between HPV DNA positivity and positive p16 INK4a staining in oropharyngeal tumours (P<0.0001). Having an HPV-related tumour was associated with being married or having been married previously (P=0.046), an increasing number of passionate kissing partners (P=0.046), ever having given oral sex (P=0.0007) and an increasing number of oral sex partners (P=0.0015). This study found a higher prevalence of HPV in oropharyngeal compared to oral cavity tumours, with a strong association being identified between oral sex behaviours and HPV-positive tumours. Further research is needed to establish that vaccines will reduce the transmission and carriage of oropharyngeal HPV infections.

SUMO regulates p21Cip1 intracellular distribution and with p21Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage.

PubMed

Brun, Sonia; Abella, Neus; Berciano, Maria T; Tapia, Olga; Jaumot, Montserrat; Freire, Raimundo; Lafarga, Miguel; Agell, Neus

2017-01-01

We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus.

SUMO regulates p21Cip1 intracellular distribution and with p21Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage

PubMed Central

Brun, Sonia; Abella, Neus; Berciano, Maria T.; Tapia, Olga; Jaumot, Montserrat; Freire, Raimundo; Lafarga, Miguel

2017-01-01

We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus. PMID:28582471

Age-specific functional epigenetic changes in p21 and p16 in injury-activated satellite cells

PubMed Central

Li, Ju; Han, Suhyoun; Cousin, Wendy; Conboy, Irina M.

2014-01-01

The regenerative capacity of muscle dramatically decreases with age because old muscle stem cells fail to proliferate in response to tissue damage. Here we uncover key age-specific differences underlying this proliferative decline: namely, the genetic loci of CDK inhibitors (CDKI) p21 and p16 are more epigenetically silenced in young muscle stem cells, as compared to old, both in quiescent cells and those responding to tissue injury. Interestingly, phosphorylated ERK (pERK) induced in these cells by ectopic FGF-2 is found in association with p21 and p16 promoters, and moreover, only in the old cells. Importantly, in the old satellite cells FGF-2/pERK silences p21 epigenetically and transcriptionally, which leads to reduced p21 protein levels and enhanced cell proliferation. In agreement with the epigenetic silencing of the loci, young muscle stem cells do not depend as much as old on ectopic FGF/pERK for their myogenic proliferation. In addition, other CDKIs, such asp15INK4B and p27KIP1, become elevated in satellite cells with age, confirming and explaining the profound regenerative defect of old muscle. This work enhances our understanding of tissue aging, promoting strategies for combating age-imposed tissue degeneration. PMID:25447026

The PPARα/p16INK4a Pathway inhibits Vascular Smooth Muscle Cell Proliferation by repressing Cell Cycle-dependent Telomerase Activation

PubMed Central

Gizard, Florence; Nomiyama, Takashi; Zhao, Yue; Findeisen, Hannes M.; Heywood, Elizabeth B.; Jones, Karrie L.; Staels, Bart; Bruemmer, Dennis

2009-01-01

Peroxisome Proliferator-Activated Receptor (PPAR) α, the molecular target for fibrates used to treat dyslipidemia, exerts pleiotropic effects on vascular cells. In vascular smooth muscle cells (VSMCs), we have previously demonstrated that PPARα activation suppresses G1→S cell cycle progression by targeting the cyclin-dependent kinase inhibitor p16INK4a (p16). In the present study, we demonstrate that this inhibition of VSMC proliferation by PPARα is mediated through a p16-dependent suppression of telomerase activity, which has been implicated in key cellular functions including proliferation. PPARα activation inhibited mitogen-induced telomerase activity by repressing the catalytic subunit telomerase reverse transcriptase (TERT) through negative cross-talk with an E2F-1-dependent trans-activation of the TERT promoter. This trans-repression involved the recruitment of the retinoblastoma (RB) family proteins p107 and p130 to the TERT promoter resulting in impaired E2F-1 binding, an effect which was dependent on p16. The inhibition of cell proliferation by PPARα activation was lost in VSMC following TERT overexpression or knock-down, pointing to a key role of telomerase as a target for the antiproliferative effects of PPARα. Finally, we demonstrate that PPARα agonists suppress telomerase activation during the proliferative response following vascular injury indicating that these findings are applicable in vivo. In concert, these results demonstrate that the anti-proliferative effects of PPARα in VSMCs depend on the suppression of telomerase activity by targeting the p16/RB/E2F transcriptional cascade. PMID:18818403

Usefulness of p16ink4a, ProEX C, and Ki-67 for the diagnosis of glandular dysplasia and adenocarcinoma of the cervix uteri.

PubMed

Negri, Giovanni; Bellisano, Giulia; Carico, Elisabetta; Faa, Gavino; Kasal, Armin; Antoniazzi, Sonia; Egarter-Vigl, Eduard; Piccin, Andrea; Dalla Palma, Paolo; Vittadello, Fabio

2011-07-01

Although the diagnostic criteria of in-situ and invasive adenocarcinomas of the cervix uteri are well established, the differentiation from benign mimics may be difficult and the morphologic features of the precursors of endocervical adenocarcinoma are still debated. In this study, we evaluated the usefulness of p16ink4a (p16), ProEX C, and Ki-67 for the diagnosis of endocervical adenocarcinoma and its precursors. Immunohistochemistry with p16, ProEX C, and Ki-67 was performed in 82 glandular lesions including 15 invasive adenocarcinomas, 29 adenocarcinomas in situ (AIS), 22 non-neoplastic samples, and 16 cases of glandular dysplasia (GD), which showed significant nuclear abnormalities but did not meet the diagnostic criteria for AIS. The immunohistochemical expression pattern was scored according to the percentage of the stained cells (0, 1+, 2+, and 3+ when 0% to 5%, 6% to 25%, 26% to 50%, and more than 50% of the cells were stained, respectively) and was evaluated for each antibody. p16 was at least focally expressed (1+ or more) in 14 of 15 invasive adenocarcinomas, in all AIS and in 7 negative samples. ProEX C and Ki-67 both scored 1+ or more in all adenocarcinomas and AIS and in 8 and 6 negative samples, respectively. Of the GD 15, 14, and 15 expressed p16, ProEX C, and Ki-67, respectively. The score differences between neoplastic and non-neoplastic samples were highly significant for each marker (P<0.001); however, the score distribution by marker differed significantly only in GD (P=0.006) in which, compared with the other markers, p16 showed more often a 3+ pattern. Our study shows that p16, Ki-67, and ProEX C may be helpful for the diagnosis of glandular lesions of the cervix uteri and may also improve the diagnostic accuracy of endocervical GD. In particularly problematic cases, the combination of p16 and a proliferation marker can provide additional help for the interpretation of these lesions.

Combination of p16(INK4a) /Ki67 immunocytology and HPV polymerase chain reaction for the noninvasive analysis of HPV involvement in head and neck cancer.

PubMed

Linxweiler, Maximilian; Bochen, Florian; Wemmert, Silke; Lerner, Cornelia; Hasenfus, Andrea; Bohle, Rainer Maria; Al-Kadah, Basel; Takacs, Zoltan Ferenc; Smola, Sigrun; Schick, Bernhard

2015-04-01

High-risk human papillomavirus (HPV) infection has been identified as a relevant risk for the development of head and neck squamous cell carcinomas (HNSCCs). As HPV status has also gained a role as a prognostic and predictive biomarker for this entity, there is a growing demand for valid HPV testing in HNSCC patients Liquid-based cytological smears from 45 HNSCC and 20 control patients were collected and used for simultaneous immunocytochemical p16(INK4a) /Ki67 staining using a CINtec PLUS kit after the presence of tumor cells was verified in a Papanicolaou-stained slide. The same cytological suspension was used for the detection of HPV DNA by specific polymerase chain reaction (PCR). Tumor cells were detected in the swab material of 44 HNSCC patients corresponding to a sensitivity of 98% (44 of 45). PCR analysis revealed the presence of HPV DNA in the cytological suspension of 13 patients (13 of 65, 20%) with simultaneous p16(INK4a) /Ki67 expression by the tumor cells in 11 of these HPV DNA-positive samples (11 of 13, 85%) - a staining pattern that is strongly associated with a carcinogenic HPV infection. A simultaneous immunocytochemical detection of p16(INK4a) and Ki67 can reliably be performed on liquid-based cytological smears from HNSCC patients using a CINtec PLUS kit. In addition, the same cytological material can be used for the detection of HPV DNA by specific PCR. The combined results of both techniques enable better discrimination between latent and carcinogenic HPV infections as well as HPV-negative cases and thus can provide information on the prognosis of HNSCC patients and facilitate therapeutic decisions. © 2014 American Cancer Society.

The KIP/CIP family members p21^{Waf1/Cip1} and p57^{Kip2} as diagnostic markers for breast cancer.

PubMed

Zohny, Samir F; Baothman, Othman A; El-Shinawi, Mohamed; Al-Malki, Abdulrahman L; Zamzami, Mazin A; Choudhry, Hani

2017-01-01

We examined the expression status of p21^{Waf1/Cip1} and p57^{Kip2} in breast cancer as well as their relationship with clinicopathological factors. Moreover, the diagnostic value of gene promoter methylation of p21^Waf1/Cip1 and p57^Kip2 was assessed in breast cancer patients. This study involved 85 patients diagnosed with breast cancer and 36 patients with benign breast lesions. The expression of p21^{Waf1/Cip1} and p57^{Kip2} in cell lysates was analyzed by ELISA and Western blot, respectively. The gene promoter methylation of p21^Waf1/Cip1 and p57^Kip2 was examined in cell lysates by methylation specific PCR. p21^{Waf1/Cip1} expression was higher while p57^{Kip2} level was lower in breast cancer patients compared to patients with benign breast lesions. The combined use of p21^{Waf1/Cip1} and p57^{Kip2} provided sensitivity and specificity of 82.35% and 86.11%, respectively. None of the malignant and benign breast tumors were found to be hypermethylated at p21^Waf1/Cip1 gene promoter. However, aberrant methylation of p57^Kip2 gene promoter was detected in 49 of 85 (57.65%) of breast cancer tumors. High p21^{Waf1/Cip1} level was associated with high grade, late stages and lymph node involvement, whereas low p57^{Kip2} level was correlated with high grade and HER2 overexpressing breast cancer. Moreover, hypermethylated p57^Kip2 gene promoter was associated with high grade. Our findings show that the overexpression of p21^{Waf1/Cip1}, down-expression of p57^{Kip2} and gene promoter methylation of p57^Kip2 could be considered as promising diagnostic markers for breast cancer.

Down-regulation of p21 (CDKN1A/CIP1) is inversely associated with microsatellite instability and CpG island methylator phenotype (CIMP) in colorectal cancer.

PubMed

Ogino, S; Kawasaki, T; Kirkner, G J; Ogawa, A; Dorfman, I; Loda, M; Fuchs, C S

2006-10-01

p21 (CDKN1A/CIP1/WAF1), one of the cyclin-dependent kinase inhibitors, plays a key role in regulating the cell cycle and is transcriptionally regulated by p53. Down-regulation of p21 is caused by TP53 mutations in colorectal cancer. CpG island methylator phenotype (CIMP) appears to be a distinct subtype of colorectal cancer with concordant methylation of multiple gene promoters and is associated with a high degree of microsatellite instability (MSI-H) and BRAF mutations. However, no study to date has evaluated the relationship between p21 expression and CIMP in colorectal cancer. The purpose of this study was to examine the inter-relationships between p21, p53, CIMP, MSI and KRAS/BRAF status in colorectal cancer. We utilized 737 relatively unbiased samples of colorectal cancers from two large prospective cohort studies. Using quantitative real-time PCR (MethyLight), we measured DNA methylation in five CIMP-specific gene promoters [CACNA1G, CDKN2A (p16/INK4A), CRABP1, MLH1 and NEUROG1]. CIMP-high (>or=4/5 methylated promoters) was diagnosed in 118 (16%) of the 737 tumours. We also assessed expression of p21 and p53 by immunohistochemistry. Among the 737 tumours, 371 (50%) showed p21 loss. Both p21 loss and p53 positivity were inversely associated with CIMP-high, MSI-H and BRAF mutations. The associations of p21 with these molecular features were still present after tumours were stratified by p53 status. In contrast, the associations of p53 positivity with the molecular features were no longer present after tumours were stratified by p21 status. When CIMP-high and non-CIMP-high tumours were stratified by MSI or KRAS/BRAF status, CIMP-high and MSI-H (but not BRAF mutations) were still inversely associated with p21 loss. In conclusion, down-regulation of p21 is inversely correlated with CIMP-high and MSI-H in colorectal cancer, independent of TP53 and BRAF status.

Use of Cytology, E6/E7 mRNA, and p16INK4a-Ki-67 to Define the Management of Human Papillomavirus (HPV)-Positive Women in Cervical Cancer Screening.

PubMed

Gustinucci, Daniela; Giorgi Rossi, Paolo; Cesarini, Elena; Broccolini, Massimo; Bulletti, Simonetta; Carlani, Angela; D'angelo, Valentina; D'amico, Maria Rosaria; Di Dato, Eugenio; Galeazzi, Paola; Malaspina, Morena; Martinelli, Nadia; Spita, Nicoletta; Tintori, Beatrice; Giaimo, Maria Donata; Passamonti, Basilio

2016-01-01

We measured the accuracy of p16(INK4a)-Ki67 (CINtec PLUS, Roche, Mannheim, Germany), and E6/E7mRNA (types 16/18/31/33/45 NucliSENS easyQ, bioMérieux, Boxtel, The Netherlands) as triage test, alone and combined with cytology. Six thousand two hundred and seventy two women were recruited in a population-based screening using HPV DNA as primary test; 396 were positive and were tested for cytology and biomarkers. All tests were performed on the same sample. Cytology-positive women were referred to colposcopy; cytology-negative women were referred to one-year HPV re-testing. The endpoint was CIN2+ at baseline or follow up. Sensitivity was 77.6% (95% confidence interval (CI) 65.3-86.7) and 53.2% (95%CI: 40.3-65.4) for cytology at atypical squamous cells of undetermined significance (ASC-US) and high-grade threshold, and 87.6% (95%CI:75.7-93.6), and 80.8% (95%CI: 67.6-89.8) for p16INK4a-Ki67, and E6/E7mRNA, respectively. Colposcopy referral was 36% (95%CI: 31.2-40.9) and 11.2% (95%CI: 7.8-14.1) for cytology at ASC-US and high-grade threshold, respectively, and 36.0% (95%CI: 29.9-29.6), and 47.5% (95%CI: 32.5-42.4) for p16(INK4a)-Ki67, and E6/E7mRNA, respectively. Strategies referring high-grade cytology or biomarker positive women to colposcopy reached sensitivity close to 100%, with modest increase in colposcopy referral. The high sensitivity of combined strategies probably allows longer intervals in HPV-positive, triage-negative women. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Involvement of Bmi-1 gene in the development of gastrointestinal stromal tumor by regulating p16Ink4A/p14ARF gene expressions: An in vivo and in vitro study.

PubMed

Wang, Jiang-Li; Wu, Jiang-Hong; Hong, Cai; Wang, Ya-Nong; Zhou, Ye; Long, Zi-Wen; Zhou, Ying; Qin, Hai-Shu

2017-12-01

This study was conducted in order to explore the role that Bmi-1 plays during the development of a gastrointestinal stromal tumor (GIST) by regulation of the p16 Ink4A and p14 ARF expressions. Eighty-six patients diagnosed with GIST were selected to take part in this experiment. The Bmi-1 protein expressions in GIST and adjacent normal tissues were detected using immunohistochemistry and further analyzed by using photodensitometry. To monitor and track the progression of the GIST, a 3-year follow-up was conducted for all affected patients. After cell transfection, the GIST cells were assigned into the control group (without transfection), the negative control (NC) group (transfected with Bmi-1-Scramble plasmid), and the Bmi-1 shRNA group (transfected with the pcDNA3.1-Bmi-1 shRNA plasmid). Protein and mRNA expressions collected from Bmi-1, p16 lnk4A , P14 ARF , cyclin D1, and CDK4 were measured using both the RT-qPCR and western blotting methods Cell senescence was assessed and obtained by using the β-Galactosidase (β-Gal) activity assay. The use of a Soft agar colony formation assay and CCK-8 assay were performed in order to detect the cell growth and subsequent proliferation. Cell invasion and migration were analyzed using the Transwell assay and scratch test. Bmi-1 in the GIST tissues was found to be significantly higher and the p16 lnk4A and P14 ARF expressions were lower than those in the adjacent normal tissues. Bmi-1 was negatively correlated with p16 lnk4A and P14 ARF expressions according to the correlation analysis. Bmi-1 expression was associated with the TNM stage, postoperative recurrence, metastasis, tumor size, and the 5-year survival rate. Area under ROC curve was calculated at 0.884, and sensitivity, specificity, and accuracy of Bmi-1 predicting the GIST were 67.44%, 97.67%, and 65.12%, respectively. Patients exhibiting a high Bmi-1 expression in the GIST tissues had lower survival rates than those with low Bmi-1 expression. In comparison with

Immortalization of Human Fetal Cells: The Life Span of Umbilical Cord Blood-derived Cells Can Be Prolonged without Manipulating p16INK4a/RB Braking PathwayD⃞

PubMed Central

Terai, Masanori; Uyama, Taro; Sugiki, Tadashi; Li, Xiao-Kang; Umezawa, Akihiro; Kiyono, Tohru

2005-01-01

Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are expected to serve as an excellent alternative to bone marrow-derived human mesenchymal stem cells. However, it is difficult to study them because of their limited life span. To overcome this problem, we attempted to produce a strain of UCBMSCs with a long life span and to investigate whether the strain could maintain phenotypes in vitro. UCBMSCs were infected with retrovirus carrying the human telomerase reverse transcriptase (hTERT) to prolong their life span. The UCBMSCs underwent 30 population doublings (PDs) and stopped dividing at PD 37. The UCBMSCs newly established with hTERT (UCBTERTs) proliferated for >120 PDs. The p16INK4a/RB braking pathway leading to senescence can be inhibited by introduction of Bmi-1, a polycomb-group gene, and human papillomavirus type 16 E7, but the extension of the life span of the UCBMSCs with hTERT did not require inhibition of the p16INK4a/RB pathway. The characteristics of the UCBTERTs remained unchanged during the prolongation of life span. UCBTERTs provide a powerful model for further study of cellular senescence and for future application to cell-based therapy by using umbilical cord blood cells. PMID:15647378

Somatic INK4a-ARF locus mutations: a significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck.

PubMed

Poi, M J; Yen, T; Li, J; Song, H; Lang, J C; Schuller, D E; Pearl, D K; Casto, B C; Tsai, M D; Weghorst, C M

2001-01-01

The INK4a-ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor-suppressor genes. The p16(INK4a) (p16) tumor-suppressor gene product is a negative regulator of cyclin-dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14(ARF) tumor-suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G(1) arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0-10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14(ARF) genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single-stranded conformational polymorphism (SSCP) analysis termed "cold" SSCP. Exons 1alpha, 1beta, and 2 of INK4a/ARF were amplified using intron-based primers or a combination of intron- and exon-based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1alpha. No mutations were found in exon 1beta. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14(ARF) proteins. Specific somatic alterations included microdeletions or

Cytoplasmic localization and ubiquitination of p21{sup Cip1} by reactive oxygen species

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

Hwang, Chae Young; Kim, Ick Young; Kwon, Ki-Sun

2007-06-22

Reactive oxygen species were previously shown to trigger p21{sup Cip1} protein degradation through a proteasome-dependent pathway, however the detailed mechanism of degradation remains to be elucidated. In this report, we showed that p21{sup Cip1} was degraded at an early phase after low dose H{sub 2}O{sub 2} treatment of a variety of cell types and that preincubation of cells with the antioxidant, N-acetylcysteine, prolonged p21{sup Cip1} half-life. A mutant p21{sup Cip1} in which all six lysines were changed to arginines was protected against H{sub 2}O{sub 2} treatment. Direct interaction between p21{sup Cip1} and Skp2 was elevated in the H{sub 2}O{sub 2}-treatedmore » cells. Disruption of the two nuclear export signal (NES) sequences in p21{sup Cip1}, or treatment with leptomycin B blocked H{sub 2}O{sub 2}-induced p21{sup Cip1} degradation. Altogether, these results demonstrate that reactive oxygen species induce p21{sup Cip1} degradation through an NES-, Skp2-, and ubiquitin-dependent pathway.« less

Ubiquitylation and proteasomal degradation of the p21(Cip1), p27(Kip1) and p57(Kip2) CDK inhibitors.

PubMed

Lu, Zhimin; Hunter, Tony

2010-06-15

The expression levels of the p21(Cip1) family CDK inhibitors (CKIs), p21(Cip1), p27(Kip1) and p57(Kip2), play a pivotal role in the precise regulation of cyclin-dependent kinase (CDK) activity, which is instrumental to proper cell cycle progression. The stabilities of p21(Cip1), p27(Kip1) and p57(Kip2) are all tightly and differentially regulated by ubiquitylation and proteasome-mediated degradation during various stages of the cell cycle, either in steady state or in response to extracellular stimuli, which often elicit site-specific phosphorylation of CKIs triggering their degradation.

miR-146b-5p mediates p16-dependent repression of IL-6 and suppresses paracrine procarcinogenic effects of breast stromal fibroblasts.

PubMed

Al-Ansari, Mysoon M; Aboussekhra, Abdelilah

2015-10-06

Increasing evidence support the critical roles of active stromal fibroblasts in breast cancer development and spread. However, the mediators and the mechanisms of regulation are still not well defined. We have shown here that the tumor suppressor p16(INK4A) protein inhibits the pro-carcinogenic effects of breast stromal fibroblasts through repressing the expression/secretion of IL-6. Indeed, p16(INK4A) suppresses IL-6 at the mRNA and protein levels. This effect is mediated trough miR-146b-5p, which inhibits IL-6 expression through a specific sequence at the IL-6 3'UTR. In addition, we present clear evidence that miR-146b-5p inhibition is sufficient to transactivate breast stromal fibroblasts, which promote epithelial-to-mesenchymal-transition in breast cancer cells in a paracrine manner. By contrast, ectopic expression of miR-146b-5p in active fibroblasts abrogated their pro-carcinogenic effects. The physiological importance of miR-146b-5p inhibition was revealed by showing that the levels of pre-miR-146b-5p as well as its mature form are reduced in cancer-associated fibroblasts as compared with their normal adjacent counterparts from cancer-free tissues isolated from the same patients. Interestingly, treatment of active breast stromal fibroblasts with curcumin increased the level of the p16(INK4A) coding CDKN2A mRNA and miR-146b-5p and suppressed IL-6, which confirms the repressive effect of these two tumor suppressor molecules on IL-6, and shows the possible "normalization" of cancer-related active fibroblasts. These results show that miR-146b-5p has non-cell-autonomous tumor suppressor function through inhibition of IL-6, suggesting that targeting this microRNA in breast stromal fibroblasts could be of great therapeutic value.

Meiotic and pedigree segregation analyses in carriers of t(4;8)(p16;p23.1) differing in localization of breakpoint positions at 4p subband 4p16.3 and 4p16.1.

PubMed

Midro, Alina T; Zollino, Marcella; Wiland, Ewa; Panasiuk, Barbara; Iwanowski, Piotr S; Murdolo, Marina; Śmigiel, Robert; Sąsiadek, Maria; Pilch, Jacek; Kurpisz, Maciej

2016-02-01

The purpose of this study was to compare meiotic segregation in sperm cells from two carriers with t(4;8)(p16;p23.1) reciprocal chromosome translocations (RCTs), differing in localization of the breakpoint positions at the 4p subband-namely, 4p16.3 (carrier 1) and 4p16.1 (carrier 2)-and to compare data of the pedigree analyses performed by direct method. Three-color fluorescent in situ hybridization (FISH) on sperm cells and FISH mapping for the evaluation of the breakpoint positions, data from pedigrees, and direct segregation analysis of the pedigrees were performed. Similar proportions of normal/balanced and unbalanced sperm cells were found in both carriers. The most common was an alternate type of segregation (about 52 % and about 48 %, respectively). Unbalanced adjacent I and adjacent II karyotypes were found in similar proportions about 15 %. The direct segregation analysis (following Stengel-Rutkowski) of the pedigree of carriers of t(4;8)(p16.1;p23.1) was performed and results were compared with the data of the pedigree segregation analysis obtained earlier through the indirect method. The probability of live-born progeny with unbalanced karyotype for carriers of t(4;8)(p16.1;p23.1) was moderately high at 18.8 %-comparable to the value obtained using the indirect method for the same carriership, which was 12 %. This was, however, markedly lower than the value of 41.2 % obtained through the pedigree segregation indirect analysis estimated for carriers of t(4;8)(p16.3;p23.1), perhaps due to the unique composition of genes present within the 4p16.1-4p 16.3 region. Revealed differences in pedigree segregation analysis did not correspond to the very similar profile of meiotic segregation patterns presented by carrier 1 and carrier 2. Most probably, such discordances may be due to differences in embryo survival rates arising from different genetic backgrounds.

Mule/Huwe1/Arf-BP1 suppresses Ras-driven tumorigenesis by preventing c-Myc/Miz1-mediated down-regulation of p21 and p15.

PubMed

Inoue, Satoshi; Hao, Zhenyue; Elia, Andrew J; Cescon, David; Zhou, Lily; Silvester, Jennifer; Snow, Bryan; Harris, Isaac S; Sasaki, Masato; Li, Wanda Y; Itsumi, Momoe; Yamamoto, Kazuo; Ueda, Takeshi; Dominguez-Brauer, Carmen; Gorrini, Chiara; Chio, Iok In Christine; Haight, Jillian; You-Ten, Annick; McCracken, Susan; Wakeham, Andrew; Ghazarian, Danny; Penn, Linda J Z; Melino, Gerry; Mak, Tak W

2013-05-15

Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule(flox/flox(y)) (Mule kKO) mice and subjected them to DMBA/PMA-induced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Mule-deficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.

Mitomycin C and decarbamoyl mitomycin C induce p53-independent p21WAF1/CIP1 activation

PubMed Central

Cheng, Shu-Yuan; Seo, Jiwon; Huang, Bik Tzu; Napolitano, Tanya; Champeil, Elise

2016-01-01

Mitomycin C (MC), a commonly used anticancer drug, induces DNA damage via DNA alkylation. Decarbamoyl mitomycin C (DMC), another mitomycin lacking the carbamate at C10, generates similar lesions as MC. Interstrand cross-links (ICLs) are believed to be the lesions primarily responsible for the cytotoxicity of MC and DMC. The major ICL generated by MC (α-ICL) has a trans stereochemistry at the guanine-drug linkage whereas the major ICL from DMC (β-ICL) has the opposite, cis, stereochemistry. In addition, DMC can provoke strong p53-independent cell death. Our hypothesis is that the stereochemistry of the major unique β-ICL generated by DMC is responsible for this p53-independent cell death signaling. p53 gene is inactively mutated in more than half of human cancers. p21WAF1/CIP1 known as a major effector of p53 is involved in p53-dependent and -independent control of cell proliferation and death. This study revealed the role of p21WAF1/CIP1 on MC and DMC triggered cell damage. MCF-7 (p53-proficient) and K562 (p53-deficient) cells were used. Cell cycle distributions were shifted to the G1/S phase in MCF-7 treated with MC and DMC, but were shifted to the S phase in K562. p21WAF1/CIP1 activation was observed in both cells treated with MC and DMC, and DMC triggered more significant activation. Knocking down p53 in MCF-7 did not attenuate MC and DMC induced p21WAF1/CIP1 activation. The α-ICL itself was enough to cause p21WAF1/CIP1 activation. PMID:27666201

Low p16INK4a Expression in Early Passage Human Prostate Basal Epithelial Cells Enables Immortalization by Telomerase Expression Alone.

PubMed

Graham, Mindy Kim; Principessa, Lorenzo; Antony, Lizamma; Meeker, Alan K; Isaacs, John T

2017-03-01

There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16 INK4a /retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten. Once telomeres shorten to a critical length, the DNA damage response pathway is activated, and the tumor suppressor p53 pathway triggers replicative senescence. Exogenous expression of telomerase in normal human epithelial cells extends the replicative capacity of cells, and in some cases, immortalizes cells. However reliable immortalization of epithelial cells usually requires telomerase activity coupled with inactivation of the p16/Rb pathway. A lentiviral vector, pLOX-TERT-iresTK (Addgene #12245), containing a CMV promoter upstream of a bicistronic coding cassette that includes loxP sites flanking the catalytic subunit of human telomerase gene (TERT) and herpes simplex virus type-1 thymidine kinase gene (HSV1-tk) was used to transduce normal prostate basal epithelial cells (PrECs) initiated in cell culture from prostate cancer patients undergoing radical prostatectomies. Transduction of early (i.e., <7) passage PrECs with TERT led to successful immortalization. However, attempts to immortalize late (i.e., >7) passage PrECs were unsuccessful. Late passage PrECs, which acquired elevated p16, were unable to overcome the senescence barrier. Immortalized PrECs (TERT-PrECs) retained a normal male karyotype and low p16 expression. Additionally, TERT-PrECs were non-tumorigenic when inoculated into intact male immunodeficient NSG mice. The present studies document that early passage human PrECs have sufficiently low p16 to permit immortalization by

Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus.

PubMed

Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

2016-07-28

The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adopted for a wide range of biological applications including genome editing. In some cases, dissection of genome functions requires allele-specific genome editing, but the use of CRISPR for this purpose has not been studied in detail. In this study, using the p16INK4a gene in HCT116 as a model locus, we investigated whether chromatin states, such as CpG methylation, or a single-nucleotide gap form in a target site can be exploited for allele-specific locus binding and genome editing by CRISPR in vivo. First, we showed that allele-specific locus binding and genome editing could be achieved by targeting allele-specific CpG-methylated regions, which was successful for one, but not all guide RNAs. In this regard, molecular basis underlying the success remains elusive at this stage. Next, we demonstrated that an allele-specific single-nucleotide gap form could be employed for allele-specific locus binding and genome editing by CRISPR, although it was important to avoid CRISPR tolerance of a single nucleotide mismatch brought about by mismatched base skipping. Our results provide information that might be useful for applications of CRISPR in studies of allele-specific functions in the genomes.

Bmi1 represses Ink4a/Arf and Hox genes to regulate stem cells in the rodent incisor

PubMed Central

Biehs, Brian; Hu, Jimmy Kuang-Hsien; Strauli, Nicolas B.; Sangiorgi, Eugenio; Jung, Heekyung; Heber, Ralf-Peter; Ho, Sunita; Goodwin, Alice F.; Dasen, Jeremy S.; Capecchi, Mario R.; Klein, Ophir D.

2013-01-01

The polycomb group gene Bmi1 is required for maintenance of adult stem cells in many organs1, 2. Inactivation of Bmi1 leads to impaired stem cell self-renewal due to deregulated gene expression. One critical target of BMI1 is Ink4a/Arf, which encodes the cell cycle inhibitors p16ink4a and p19Arf3. However, deletion of Ink4a/Arf only partially rescues Bmi1 null phenotypes4, indicating that other important targets of BMI1 exist. Here, using the continuously-growing mouse incisor as a model system, we report that Bmi1 is expressed by incisor stem cells and that deletion of Bmi1 resulted in fewer stem cells, perturbed gene expression, and defective enamel production. Transcriptional profiling revealed that Hox expression is normally repressed by BMI1 in the adult, and functional assays demonstrated that BMI1-mediated repression of Hox genes preserves the undifferentiated state of stem cells. As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated1, 2, 5–7, our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation. PMID:23728424

Anti-proliferative and pro-apoptotic activity of whole extract and isolated indicaxanthin from Opuntia ficus-indica associated with re-activation of the onco-suppressor p16{sup INK4a} gene in human colorectal carcinoma (Caco-2) cells

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

Naselli, Flores; Tesoriere, Luisa; Caradonna, Fabio

Highlights: • Cactus pear fruit extract and indicaxanthin cause apoptosis of colon cancer cells. • Indicaxanthin does not cause ROS formation, but affects epigenoma in Caco-2 cells. • Indicaxanthin reverses methylation of oncosuppressor p16{sup INK4a} gene in Caco-2 cells. • Indicaxanthin reactivates retinoblastoma in Caco-2 cells. • Bioavailable indicaxanthin may have chemopreventive activity in colon cancer. - Abstract: Phytochemicals may exert chemo-preventive effects on cells of the gastro-intestinal tract by modulating epigenome-regulated gene expression. The effect of the aqueous extract from the edible fruit of Opuntia ficus-indica (OFI extract), and of its betalain pigment indicaxanthin (Ind), on proliferation of humanmore » colon cancer Caco-2 cells has been investigated. Whole extract and Ind caused a dose-dependent apoptosis of proliferating cells at nutritionally relevant amounts, with IC{sub 50} 400 ± 25 mg fresh pulp equivalents/mL, and 115 ± 15 μM (n = 9), respectively, without toxicity for post-confluent differentiated cells. Ind accounted for ∼80% of the effect of the whole extract. Ind did not cause oxidative stress in proliferating Caco-2 cells. Epigenomic activity of Ind was evident as de-methylation of the tumor suppressor p16{sup INK4a} gene promoter, reactivation of the silenced mRNA expression and accumulation of p16{sup INK4a}, a major controller of cell cycle. As a consequence, decrease of hyper-phosphorylated, in favor of the hypo-phosphorylated retinoblastoma was observed, with unaltered level of the cycline-dependent kinase CDK4. Cell cycle showed arrest in the G2/M-phase. Dietary cactus pear fruit and Ind may have chemo-preventive potential in intestinal cells.« less

IGFBP2 is a candidate biomarker for Ink4a-Arf status and a therapeutic target for high-grade gliomas.

PubMed

Moore, Lynette M; Holmes, Kristen M; Smith, Sarah M; Wu, Ying; Tchougounova, Elena; Uhrbom, Lene; Sawaya, Raymond; Bruner, Janet M; Fuller, Gregory N; Zhang, Wei

2009-09-29

The levels of insulin-like growth factor-binding protein 2 (IGFBP2) are elevated during progression of many human cancers. By using a glial-specific transgenic mouse system (RCAS/Ntv-a), we reported previously that IGFBP2 is an oncogenic factor for glioma progression in combination with platelet-derived growth factor-beta (PDGFB). Because the INK4a-ARF locus is often deleted in high-grade gliomas (anaplastic oligodendroglioma and glioblastoma), we investigated the effect of the Ink4a-Arf-null background on IGFBP2-mediated progression of PDGFB-initiated oligodendroglioma. We demonstrate here that homozygous deletion of Ink4a-Arf bypasses the requirement of exogenously introduced IGFBP2 for glioma progression. Instead, absence of Ink4a-Arf resulted in elevated endogenous tumor cell IGFBP2. An inverse relationship between p16(INK4a) and IGFBP2 expression was also observed in human glioma tissue samples and in 90 different cancer cell lines by using Western blotting and reverse-phase protein lysate arrays. When endogenous IGFBP2 expression was attenuated by an RCAS vector expressing antisense IGFBP2 in our mouse model, a decreased incidence of anaplastic oligodendroglioma as well as prolonged survival was observed. Thus, p16(INK4a) is a negative regulator of the IGFBP2 oncogene. Loss of Ink4a-Arf results in increased IGFBP2, which contributes to glioma progression, thereby implicating IGFBP2 as a marker and potential therapeutic target for Ink4a-Arf-deleted gliomas.

IGFBP2 is a candidate biomarker for Ink4a-Arf status and a therapeutic target for high-grade gliomas

PubMed Central

Moore, Lynette M.; Holmes, Kristen M.; Smith, Sarah M.; Wu, Ying; Tchougounova, Elena; Uhrbom, Lene; Sawaya, Raymond; Bruner, Janet M.; Fuller, Gregory N.; Zhang, Wei

2009-01-01

The levels of insulin-like growth factor-binding protein 2 (IGFBP2) are elevated during progression of many human cancers. By using a glial-specific transgenic mouse system (RCAS/Ntv-a), we reported previously that IGFBP2 is an oncogenic factor for glioma progression in combination with platelet-derived growth factor-β (PDGFB). Because the INK4a-ARF locus is often deleted in high-grade gliomas (anaplastic oligodendroglioma and glioblastoma), we investigated the effect of the Ink4a-Arf-null background on IGFBP2-mediated progression of PDGFB-initiated oligodendroglioma. We demonstrate here that homozygous deletion of Ink4a-Arf bypasses the requirement of exogenously introduced IGFBP2 for glioma progression. Instead, absence of Ink4a-Arf resulted in elevated endogenous tumor cell IGFBP2. An inverse relationship between p16INK4a and IGFBP2 expression was also observed in human glioma tissue samples and in 90 different cancer cell lines by using Western blotting and reverse-phase protein lysate arrays. When endogenous IGFBP2 expression was attenuated by an RCAS vector expressing antisense IGFBP2 in our mouse model, a decreased incidence of anaplastic oligodendroglioma as well as prolonged survival was observed. Thus, p16INK4a is a negative regulator of the IGFBP2 oncogene. Loss of Ink4a-Arf results in increased IGFBP2, which contributes to glioma progression, thereby implicating IGFBP2 as a marker and potential therapeutic target for Ink4a-Arf-deleted gliomas. PMID:19805356

Stratification of HPV-Induced Cervical Pathology using the Virally-Encoded Molecular Marker E4 in Combination with p16 or MCM

PubMed Central

Griffin, Heather; Soneji, Yasmina; Van Baars, Romy; Arora, Rupali; Jenkins, David; van de Sandt, Miekel; Wu, Zhonglin; Quint, Wim; Jach, Robert; Okon, Krzysztof; Huras, Hubert; Singer, Albert; Doorbar, John

2015-01-01

High-risk HPV types cause cervical lesions of varying severity, ranging from transient productive infections to high-grade neoplasia. Disease stratification requires the examination of lesional pathology, and possibly also the detection of biomarkers. P16INK4a and MCM are established surrogates of high-risk HPV E6/E7 activity, and can be extensively expressed in high-grade lesions. Here we have combined these two cellular biomarkers with detection of the abundant HPV-encoded E4 protein in order to identify both productive and transforming lesions. This approach has allowed us to distinguish true papillomavirus infections from similar pathologies, and has allowed us to divide the heterogeneous CIN2 category into those that are CIN1-like and express E4, and those that more closely resemble non-productive CIN3. To achieve this, 530 lesional areas were evaluated according to standard pathology criteria and by using a multiple staining approach that allows us to superimpose biomarker patterns either singly or in combination onto an annotated haematoxylin & eosin image. Conventional grading of neoplasia was established by review panel, and compared directly to the composite molecular pathology visualised on the same tissue section. The detection of E4 coincided with the onset of vacuolation, becoming abundant in koilocytes as the MCM marker declined and cells lost their defined nuclear margins as visualised by standard H&E staining. Of the dual marker approaches, p16INK4a and E4 appeared most promising, with E4 generally identifying areas of low-grade disease even when p16INK4a was present. Extensive p16INK4a expression usually coincided with an absence of E4 expression or its focal retention in sporadic cells within the lesion. Our results suggest that a straightforward molecular evaluation of HPV life-cycle deregulation in cervical neoplasia may help improve disease stratification, and that this can be achieved using complementary molecular biomarker pairs such as MCM

Cytoplasmic p21(Cip1/WAF1) regulates neurite remodeling by inhibiting Rho-kinase activity.

PubMed

Tanaka, Hiroyuki; Yamashita, Toshihide; Asada, Minoru; Mizutani, Shuki; Yoshikawa, Hideki; Tohyama, Masaya

2002-07-22

p21(Cip1/WAF1) has cell cycle inhibitory activity by binding to and inhibiting both cyclin/Cdk kinases and proliferating cell nuclear antigen. Here we show that p21(Cip1/WAF1) is induced in the cytoplasm during the course of differentiation of chick retinal precursor cells and N1E-115 cells. Ectopic expression of p21(Cip1/WAF1) lacking the nuclear localization signal in N1E-115 cells and NIH3T3 cells affects the formation of actin structures, characteristic of inactivation of Rho. p21(Cip1/WAF1) forms a complex with Rho-kinase and inhibits its activity in vitro and in vivo. Neurite outgrowth and branching from the hippocampal neurons are promoted if p21(Cip1/WAF1) is expressed abundantly in the cytoplasm. These results suggest that cytoplasmic p21(Cip1/WAF1) may contribute to the developmental process of the newborn neurons that extend axons and dendrites into target regions.

Cytoplasmic p21Cip1/WAF1 regulates neurite remodeling by inhibiting Rho-kinase activity

PubMed Central

Tanaka, Hiroyuki; Yamashita, Toshihide; Asada, Minoru; Mizutani, Shuki; Yoshikawa, Hideki; Tohyama, Masaya

2002-01-01

p21Cip1/WAF1 has cell cycle inhibitory activity by binding to and inhibiting both cyclin/Cdk kinases and proliferating cell nuclear antigen. Here we show that p21Cip1/WAF1 is induced in the cytoplasm during the course of differentiation of chick retinal precursor cells and N1E-115 cells. Ectopic expression of p21Cip1/WAF1 lacking the nuclear localization signal in N1E-115 cells and NIH3T3 cells affects the formation of actin structures, characteristic of inactivation of Rho. p21Cip1/WAF1 forms a complex with Rho-kinase and inhibits its activity in vitro and in vivo. Neurite outgrowth and branching from the hippocampal neurons are promoted if p21Cip1/WAF1 is expressed abundantly in the cytoplasm. These results suggest that cytoplasmic p21Cip1/WAF1 may contribute to the developmental process of the newborn neurons that extend axons and dendrites into target regions. PMID:12119358

Inhibitory Effect of Ginseng on Breast Cancer Cell Line Growth Via Up-Regulation of Cyclin Dependent Kinase Inhibitor, p21 and p53

PubMed

AL Shabanah, Othman A; Alotaibi, Moureq rashed; Al Rejaie, Salim S; Alhoshani, Ali R; Almutairi, Mashal M; Alshammari, Musaad A; Hafez, Mohamed M

2016-11-01

Objective: Breast cancer is global female health problem worldwide. Most of the currently used agents for breast cancer treatment have toxic side-effects. Ginseng root, an oriental medicine, has many health benefits and may exhibit direct anti-cancer properties. This study was performed to assess the effects of ginseng on breast cancer cell lines. Materials and Methods: Cytotoxicity of ginseng extract was measured by MTT assay after exposure of MDA-MB-231, MCF-10A and MCF-7 breast cancer cells to concentrations of 0.25, 0.5, 1, 1.5, 2 and 2.5 mg/well. Expression levels of p21WAF, p16INK4A, Bcl-2, Bax and P53 genes were analyzed by quantitative real time PCR. Results: The treatment resulted in inhibition of cell proliferation in a dose-and time-dependent manner. p53, p21WAF1and p16INK4A expression levels were up-regulated in ginseng treated MDA-MB-231 and MCF-7 cancer cells compared to untreated controls and in MCF-10A cells. The expression levels of Bcl2 in the MDA-MB-231 and MCF-7 cells were down-regulated. In contrast, that of Bax was significantly up-regulated. Conclusion: The results of this study revealed that ginseng may inhibit breast cancer cell growth by activation of the apoptotic pathway. Creative Commons Attribution License

CDK inhibitors, p21{sup Cip1} and p27{sup Kip1}, participate in cell cycle exit of mammalian cardiomyocytes

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

Tane, Shoji; Ikenishi, Aiko; Okayama, Hitomi

2014-01-17

Highlights: •Expression of p21 and p27 in the hearts showed a peak during postnatal stages. •p21 and p27 bound to cyclin E, cyclin A and CDK2 in the hearts at postnatal stages. •Cardiomyocytes in both KO mice showed failure in the cell cycle exit at G1-phase. •These data show the first apparent phenotypes in the hearts of Cip/Kip KO mice. -- Abstract: Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remainsmore » largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21{sup Cip1} and p27{sup Kip1} but not p57{sup Kip2} showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21{sup Cip1} and p27{sup Kip1} bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21{sup Cip1} and p27{sup Kip1} knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21{sup Cip1} and p27{sup Kip} play important roles in the cell cycle exit of postnatal cardiomyocytes.« less

The ethanol extract from Artemisia princeps Pampanini induces p53-mediated G1 phase arrest in A172 human neuroblastoma cells.

PubMed

Park, Eun Young; Lee, Kyung-Won; Lee, Heon-Woo; Cho, Young-Wuk; Baek, Nam-In; Chung, Hae-Gon; Jeong, Tae-Sook; Choi, Myung-Sook; Lee, Kyung-Tae

2008-06-01

In the present study, the antiproliferative effects of the ethanol extract of Artemisia princeps Pampanini (EAPP) and the mechanism involved were investigated. Of the various cancer cells examined, human neuroblastoma A172 cells were most sensitive to EAPP, and their proliferation was dose- and time-dependently inhibited by EAPP. DNA flow cytometry analysis indicated that EAPP notably induced the G(1) phase arrest in A172 cells. Of the G(1) phase cycle-related proteins examined, the expressions of cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 and of cyclin D(1), D(2), and D(3) were found to be markedly reduced by EAPP, whereas cyclin E was unaffected. Moreover, the protein and mRNA levels of the CDK inhibitors p16(INK4a), p21(CIP1/WAF1), and p27(KIP1) were increased, and the activities of CDK2, CDK4, and CDK6 were reduced. Furthermore, the expressions of E2F-1 and of phosphorylated pRb were also decreased, and the protein levels of p53 and pp53 (Ser15) were increased. Up-regulation of p21(CIP1/WAF1) was found to be mediated by a p53-dependent pathway in EAPP-induced G(1)-arrested A172 cells. When these data are taken together, the EAPP was found to potently inhibit the proliferation of human neuroblastoma A172 cells via G(1) phase cell cycle arrest.

Modifications in cell cycle kinetics and in expression of G1 phase-regulating proteins in human amniotic cells after exposure to electromagnetic fields and ionizing radiation.

PubMed

Lange, S; Viergutz, T; Simkó, M

2004-10-01

Low-frequency electromagnetic fields are suspected of being involved in carcinogenesis, particularly in processes that could be related to cancer promotion. Because development of cancer is associated with deregulated cell growth and we previously observed a magnetic field-induced decrease in DNA synthesis [Lange et al. (2002) Alterations in the cell cycle and in the protein level of cyclin D1p, 21CIP1, and p16INK4a after exposure to 50 HZ. MF in human cells. Radiat. Environ. Biophys.41, 131], this study aims to document the influence of 50 Hz, 1 mT magnetic fields (MF), with or without initial gamma-ionizing radiation (IR), on the following cell proliferation-relevant parameters in human amniotic fluid cells (AFC): cell cycle distribution, expression of the G1 phase-regulating proteins Cdk4, cyclin D1, p21CIP1 and p16INK4a, and Cdk4 activity. While IR induced a G1 delay and a dose-dependent G2 arrest, no discernible changes in cell cycle kinetics were observed due to MF exposure. However, a significant decrease in the protein expression of cyclin D1 and an increase in p21CIP1- and p16INK4a-expression could be detected after exposure to MF alone. IR-exposure caused an augmentation of p21CIP1- and p16INK4a- levels as well, but did not alter cyclin D1 expression. A slight diminution of Cdk4 activity was noticed after MF exposure only, indicating that Cdk4 appears not to act as a mediator of MF- or IR-induced changes in the cell cycle of AFC cells. Co-exposure to MF/IR affected neither cell cycle distribution nor protein expression or kinase activity additionally or synergistically, and therefore MF seems not to modify the mutagenic potency of IR.

A new molecular targeted therapeutic approach for renal cell carcinoma with a p16 functional peptide using a novel transporter system.

PubMed

Zennami, Kenji; Yoshikawa, Kazuhiro; Kondo, Eisaku; Nakamura, Kogenta; Upsilonamada, Yoshiaki; De Velasco, Marco A; Tanaka, Motoyoshi; Uemura, Hirotsugu; Shimazui, Toru; Akaza, Hideyuki; Saga, Shinsuke; Ueda, Ryuzo; Honda, Nobuaki

2011-08-01

Molecular targeting agents have become formidable anticancer weapons showing much promise against refractory tumors and functional peptides and are among the more desirable of these nanobio-tools. Intracellular delivery of multiple functional peptides forms the basis for a potent, non-invasive mode of delivery, providing distinctive therapeutic advantages. We examine the growth suppression efficiency of human renal cell carcinoma (RCC) by single-peptide targeting. We simultaneously introduced p16INK4a tumor suppressor peptides by Wr-T-mediated peptide delivery. Wr-T-mediated transport of p16INK4a functional peptide into 10 RCC lines, lacking expression of the p16INK4a molecule, reversed the specific loss of p16 function, thereby drastically inhibiting tumor growth in all but 3 lines by >95% within the first 96 h. In vivo analysis using SK-RC-7 RCC xenografts in nude mice demonstrated tumor growth inhibition by the p16INK4a peptide alone, however, inoculation of Wr-T and the p16INK4a functional peptide mixture, via the heart resulted in complete tumor regression. Thus, restoration of tumor suppressor function with Wr-T peptide delivery represents a powerful approach, with mechanistic implications for the development of efficacious molecular targeting therapeutics against intractable RCC.

Anti-proliferative and pro-apoptotic activity of whole extract and isolated indicaxanthin from Opuntia ficus-indica associated with re-activation of the onco-suppressor p16(INK4a) gene in human colorectal carcinoma (Caco-2) cells.

PubMed

Naselli, Flores; Tesoriere, Luisa; Caradonna, Fabio; Bellavia, Daniele; Attanzio, Alessandro; Gentile, Carla; Livrea, Maria A

2014-07-18

Phytochemicals may exert chemo-preventive effects on cells of the gastro-intestinal tract by modulating epigenome-regulated gene expression. The effect of the aqueous extract from the edible fruit of Opuntia ficus-indica (OFI extract), and of its betalain pigment indicaxanthin (Ind), on proliferation of human colon cancer Caco-2 cells has been investigated. Whole extract and Ind caused a dose-dependent apoptosis of proliferating cells at nutritionally relevant amounts, with IC50 400±25 mg fresh pulp equivalents/mL, and 115±15 μM (n=9), respectively, without toxicity for post-confluent differentiated cells. Ind accounted for ∼80% of the effect of the whole extract. Ind did not cause oxidative stress in proliferating Caco-2 cells. Epigenomic activity of Ind was evident as de-methylation of the tumor suppressor p16(INK4a) gene promoter, reactivation of the silenced mRNA expression and accumulation of p16(INK4a), a major controller of cell cycle. As a consequence, decrease of hyper-phosphorylated, in favor of the hypo-phosphorylated retinoblastoma was observed, with unaltered level of the cycline-dependent kinase CDK4. Cell cycle showed arrest in the G2/M-phase. Dietary cactus pear fruit and Ind may have chemo-preventive potential in intestinal cells. Copyright © 2014 Elsevier Inc. All rights reserved.

p21(WAF1/CIP1) and cancer: a shifting paradigm?

PubMed

Gartel, Andrei L

2009-01-01

The cyclin-dependent kinase inhibitor p21(WAF1/CIP1) is a key mediator of p53-dependent cell cycle arrest and may play the role of a tumor suppressor in cancer. However, it has been shown that p21 may also act as an oncogene, because it inhibits apoptosis and may promote cell proliferation in some tumors. These data point out to "antagonistic duality" of p21, because it possesses anticancer and procancer properties at the same time. New data suggest that more and more proteins also may play contradictory roles in cancer thus challenging current paradigm of established oncogenes and tumor suppressors. (c) 2009 International Union of Biochemistry and Molecular Biology, Inc.

Chromosomal instability, telomere shortening, and inactivation of p21(WAF1/CIP1) in dysplastic nodules of hepatitis B virus-associated multistep hepatocarcinogenesis.

PubMed

Lee, Yoon Hee; Oh, Bong-Kyeong; Yoo, Jeong Eun; Yoon, So-Mi; Choi, Jinsub; Kim, Kyung Sik; Park, Young Nyun

2009-08-01

Systemic analysis for chromosomal instability and inactivation of cell cycle checkpoints are scarce during hepatocarcinogenesis. We studied 24 patients with chronic B viral cirrhosis including 30 cirrhotic regenerative nodules, 35 low-grade dysplastic nodules, 15 high-grade dysplastic nodules, 7 dysplastic nodules with hepatocellular carcinoma foci, and 18 hepatocellular carcinomas. Eight normal livers were studied as the control group. Telomere length and micronuclei were detected by Southern blot and Feulgen-fast green dyeing technique, respectively, and p21(WAF1/CIP1) expression was studied by immunohistochemistry. Micronuclei >1 per 3000 hepatocytes were found in 17% of low-grade dysplastic nodules, 87% of high-grade dysplastic nodules, and 100% of high-grade dysplastic nodules with hepatocellular carcinoma foci and hepatocellular carcinomas in contrast to those of all normal livers, and 90% of cirrhosis showed no micronuclei. The micronuclei index showed a gradual increase during hepatocarcinogenesis and there was a significant increase between cirrhosis and low-grade dysplastic nodules, low-grade dysplastic nodules and high-grade dysplastic nodules, and high-grade dysplastic nodules and hepatocellular carcinomas. Telomere length showed a gradual shortening during hepatocarcinogenesis and a significant reduction was found in high-grade dysplastic nodules (P=0.024) and hepatocellular carcinomas (P=0.031) compared with normal and cirrhotic livers. The micronuclei index was correlated with telomere shortening (P=0.016). The p21(WAF1/CIP1) labeling index was significantly higher in cirrhosis than in normal livers (P=0.024) and markedly decreased in low-grade dysplastic nodules, high-grade dysplastic nodules, and hepatocellular carcinomas compared with cirrhosis (P<0.05). The p21(WAF1/CIP1) labeling index was associated with telomere length (P<0.001) but not micronuclei index. This study shows that telomere shortening, chromosomal instability, and inactivation of p

The role of p21Waf1/CIP1 as a Cip/Kip type cell-cycle regulator in oral squamous cell carcinoma (Review).

PubMed

Pérez-Sayáns, Mario; Suárez-Peñaranda, José-Manuel; Gayoso-Diz, Pilar; Barros-Angueira, Francisco; Gándara-Rey, José-Manuel; García-García, Abel

2013-03-01

Oral Squamous Cell Carcinoma (OSCC) is biologically characterized by the accumulation of multiple genetic and molecular alterations that end up clinically characterized as a malignant neoplasm through a phenomenon known as multistep. The members of the Cip/Kip family, specifically p21Waf1/CIP1, are responsible for cell cycle control, blocking the transition from phase G1 to phase S. We made a search of articles of peer-reviewed Journals in PubMed/ Medline, crossing the keywords. The goal of this paper is to determine the relationship between p21Waf1/CIP1 expression and several clinical and pathological aspects of OSCC, their relationship with p53 and HPV, as well as genetic alterations in their expression pattern, their use as a prognosis market in the evolution of precancerous lesions and their roles in anticancer treatments. The results of p21WAF1/CIP1 expression in OSCC showed mixed results in terms of positivity/negativity throughout different studies. It seems that, although p21Waf1/CIP1 expression is controlled in a p53-dependent manner, coexpression of both in OSCC is not intrinsically related. Although the presence of HPV viral oncoproteins increases p21Waf1/CIP1 levels, the small number of studies, have forced us to disregard the hypothesis that HPV infected lesions that present better prognosis are due to a p21Waf1/CIP1-dependent control. The role of p21WAF1/CIP1 as cell-cycle regulator has been well described; however, its relationship to OSCC, the clinical and pathological variables of tumors, HPV and different treatments are not entirely clear. Thus, it would be very interesting to pursue further study of this protein, which may have a significant value for the diagnosis, prognosis and therapy of this type of tumors.

4p16.1-p15.31 duplication and 4p terminal deletion in a 3-years old Chinese girl: Array-CGH, genotype-phenotype and neurological characterization.

PubMed

Piccione, Maria; Salzano, Emanuela; Vecchio, Davide; Ferrara, Dante; Malacarne, Michela; Pierluigi, Mauro; Ferrara, Ines; Corsello, Giovanni

2015-07-01

Microscopically chromosome rearrangements of the short arm of chromosome 4 include the two known clinical entities: partial trisomy 4p and deletions of the Wolf-Hirschhorn critical regions 1 and 2 (WHSCR-1 and WHSCR-2, respectively), which cause cranio-facial anomalies, congenital malformations and developmental delay/intellectual disability. We report on clinical findings detected in a Chinese patient with a de novo 4p16.1-p15.32 duplication in association with a subtle 4p terminal deletion of 6 Mb in size. This unusual chromosome imbalance resulted in WHS classical phenotype, while clinical manifestations of 4p trisomy were practically absent. This observation suggests the hypothesis that haploinsufficiency of sensitive dosage genes with regulatory function placed in WHS critical region, is more pathogenic than concomitant 4p duplicated segment. Additionally clinical findings in our patient confirm a variable penetrance of major malformations and neurological features in Chinese children despite of WHS critical region's deletion. Copyright © 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells.

PubMed

Cereseto, A; Diella, F; Mulloy, J C; Cara, A; Michieli, P; Grassmann, R; Franchini, G; Klotman, M E

1996-09-01

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.

Differential effects of cell cycle regulatory protein p21(WAF1/Cip1) on apoptosis and sensitivity to cancer chemotherapy.

PubMed

Liu, Suxing; Bishop, W Robert; Liu, Ming

2003-08-01

p21(WAF1/Cip1) was initially identified as a cell cycle regulatory protein that can cause cell cycle arrest. It is induced by both p53-dependent and p53-independent mechanisms. This mini-review briefly discusses its currently known functions in apoptosis and drug sensitivity. As an inhibitor of cell proliferation, p21(WAF1/Cip1) plays an important role in drug-induced tumor suppression. Nevertheless, a number of recent studies have shown that p21(WAF1/Cip1) can assume both pro- or anti-apoptotic functions in response to anti-tumor agents depending on cell type and cellular context. This dual role of p21(WAF1/Cip1) in cancer cells complicates using p21(WAF1/Cip1) status to predict response to anti-tumor agents. However, it is possible to develop p21(WAF1/Cip1)-targeted reagents or p21(WAF1/Cip1) gene transfer techniques to have a beneficial effect within a well-defined therapeutic context. Better understanding of the roles of p21(WAF1/Cip1) in tumors should enable a more rational approach to anti-tumor drug design and therapy.

Cell Attachment to the Extracellular Matrix Induces Proteasomal Degradation of p21CIP1 via Cdc42/Rac1 Signaling

PubMed Central

Bao, Wenjie; Thullberg, Minna; Zhang, Hongquan; Onischenko, Anatoli; Strömblad, Staffan

2002-01-01

The cyclin-dependent kinase 2 (Cdk2) inhibitors p21CIP1 and p27KIP1 are negatively regulated by anchorage during cell proliferation, but it is unclear how integrin signaling may affect these Cdk2 inhibitors. Here, we demonstrate that integrin ligation led to rapid reduction of p21CIP1 and p27KIP1 protein levels in three distinct cell types upon attachment to various extracellular matrix (ECM) proteins, including fibronectin (FN), or to immobilized agonistic anti-integrin monoclonal antibodies. Cell attachment to FN did not rapidly influence p21CIP1 mRNA levels, while the protein stability of p21CIP1 was decreased. Importantly, the down-regulation of p21CIP1 and p27KIP1 was completely blocked by three distinct proteasome inhibitors, demonstrating that integrin ligation induced proteasomal degradation of these Cdk2 inhibitors. Interestingly, ECM-induced proteasomal proteolysis of a ubiquitination-deficient p21CIP1 mutant (p21K6R) also occurred, showing that the proteasomal degradation of p21CIP1 was ubiquitin independent. Concomitant with our finding that the small GTPases Cdc42 and Rac1 were activated by attachment to FN, constitutively active (ca) Cdc42 and ca Rac1 promoted down-regulation of p21CIP1. However, dominant negative (dn) Cdc42 and dn Rac1 mutants blocked the anchorage-induced degradation of p21CIP1, suggesting that an integrin-induced Cdc42/Rac1 signaling pathway activates proteasomal degradation of p21CIP1. Our results indicate that integrin-regulated proteasomal proteolysis might contribute to anchorage-dependent cell cycle control. PMID:12052868