Expression Of p16INK4A Gene Research Articles

The ETS Transcription Factor ETV7 Exhausts Hematopoietic Stem Cells By Enhancing The Cell Cycle Entry and Cell Proliferation

Although ETS-transcription factors play a role in normal and malignant hematopoiesis, their function in hematopoietic stem cells (HSCs) and leukemia initiating cells (LICs) is largely unknown. We originally identified the novel ETS transcription factor ETV7, which is highly homologous to ETV6/TEL, a frequent target of chromosomal translocation in human leukemia. Previously we have shown that ETV7 is a hematopoietic oncoprotein that requires cooperating mutations to induce leukemic transformation. Microarray analysis revealed that ETV7 expression is upregulated in 70% of pediatric ALL and AML samples. This indicates a possible oncogenic function of ETV7 in a variety of leukemias, although the molecular mechanism of ETV7-mediated leukemogenesis remains to be elucidated. ETV7 is widely but not abundantly expressed in various human tissues. Recently we found that overexpression of ETV7 in human cord blood-derived CD34+ cells depletes the number of CD34+CD38- HSCs. In addition, ETV7-transduced cells slightly accerelated cell proliferation. These results suggest that overexpression of ETV7 activates cell proliferation in primary human CD34+cells and depletes the number of HSCs. Here, by using a mouse model, we show that ectopic expression of ETV7 in quiescent HSCs accelerates their cell cycle entry and proliferation, leading to the exhaust of HSCs in mice.The ETV7 gene locus is deleted in part of the rodents including the mouse despite its high level of conservation among vertebrates. To circumvent this limitation, we have generated an ETV7 BAC transgenic mouse that carries a single copy of a human BAC DNA containing the ETV7 gene locus. In flow cytometry (FCM) analysis of wild type (WT) and ETV7 bone marrow (BM)-derived Lin-Sca1+cKit+(LSK) cells, the size and frequency of LT(long term)-HSCs (CD48-CD150+LSK) in ETV7 LSK was 2-fold lower than that in WT LSK, while the frequency of LSK and hematopoietic common progenitor cells in WT and ETV7 BM are similar. As compared with WT-LSK, ETV7-LSK showed a significantly decreased number of myeloid progenitor colonies in both the initial plating (MC1) and replating of MC1 colonies (MC2) in methylcellulose colony formation assay in vitro. To assess the ETV7 HSC function contributing to blood cell generation in vivo, we performed competitive repopulation assays. In agreement with the in vitro results, the repopulation ability of HSC is significantly compromised in ETV7 mice as measured 7 weeks post transplantation. This defect was even more pronounced 16 weeks post transplantation. Since enhanced cell cycle entry is known to cause loss of hematopoietic stem/progenitor cells (HSPCs) through the activation of a tumor suppressor response, we quantified p19ARF, p16INK4a, and p21CDKN1A gene expression in LSK cells by qRT-PCR. At day 6 and day 9 of in vitro culture, ETV7 LSK cells activated the p19ARF, p16INK4a, and p21CDKN1A genes about 2-fold greater than WT LSK cells. To measure the de novo DNA replication of HSPCs in vivo, BrdU-pulse labeled BM cells were harvested and BrdU incorporation was quantified by FCM analysis. ETV7 LSK cells showed elevated BrdU incorporation compared with that of WT. In addition, Hoechst33342/Pyronin Y staining revealed that ETV7 LSK enhanced transition from G0 to the G1 phase of the cell cycle, suggesting that ETV7 forced cell cycle entry of quiescent HSCs. Finally to clarify the involvement of the CDKN2A tumor suppressor in ETV7-associated HSC exhaustion, we examined the frequency of HSPCs in CDKN2A-/- and ETV7+/-CDKN2A-/- LSK cells in vivo by FCM analysis. Loss of CDKN2A but not ARF restored the depletion of ETV7 LT-HSCs. Moreover, loss of CDKN2A rescued the defect of repopulation ability in vivo and self-renewal activity in vitro of ETV7 HSPCs. These results indicate that exhaustion of HSC in ETV7 BM occurred through ETV7-induced activation of cell proliferation and the CDKN2A tumor suppressor pathway in mice. Disclosures:No relevant conflicts of interest to declare.

Promoter and histone methylation and p16INK4A gene expression in colon cancer

The inactivation of the cyclin-dependent kinase inhibitor p16INK4A gene by hypermethylation is observed in numerous types of cancer. New findings indicate that DNA and histone methylation act in concert in gene silencing. In this study, we investigated the methylation status of the p16INK4A gene promoter and the histone 3 lysine 9 residue in the tumors and matched normal tissue samples from patients with colorectal cancer and analyzed their association with gene expression. The methylation and expression of the p16INK4A gene were analyzed by real-time PCR, and histone methylation was analyzed by chromatin immunoprecipitation followed by real-time PCR. p16INK4A expression was significantly higher in the tumors compared to normal tissue. Mono-, di- and trimethylation levels of the H3K9 residue were similar in the tumor and normal tissue samples. We did not observe any significant correlation between p16INK4A methylation or expression and clinical parameters. Our results suggest that epigenetic modifications of the p16INK4A gene and histone lysine methylation do not play a major role in colon carcinogenesis.

Histone deacetylase 3 (HDAC3) participates in the transcriptional repression of the p16INK4a gene in mammary gland of the female rat offspring exposed to an early-life high-fat diet

Maternal exposure to environmental agents throughout pregnancy and lactation may affect offspring’s mammary gland growth and alter the epigenome. This may predispose the offspring’s mammary glands to be more susceptible to carcinogenesis. The purpose of this study was to examine the effect of a maternal high-fat diet on the regulation of p16INK4a gene expression in the mammary gland of rat offspring. Timed-pregnant Sprague-Dawley rats were fed one of the two diets, a control (C, 16% of fat) or a high fat (HF, 45% of fat) diet, throughout gestation and lactation and sacrificed at 12 weeks of age. Compared with C, HF offspring showed a decrease of p16INK4a gene expression in the mammary gland at both mRNA and protein levels. Chromatin immunoprecipitation (ChIP) assay demonstrated that the downregulation of p16INK4a transcription in HF offspring was associated with reduced acetylation of histone H4 and increased recruitment of histone deacetylase 3 (HDAC3) within the p16INK4a promoter region, but was not associated with acetylation of histone H3 or HDAC1. Methylated DNA immunoprecipitation (MeDIP) did not detect differences in methylation at different regions of the p16INK4a gene between C and HF offspring. We conclude that maternal high fat exposure represses p16INK4a gene expression in the mammary gland of offspring through changes of histone modifications and HDAC3 binding activity within the regulatory regions of the p16INK4a gene.

Visualizing the dynamics of senescence stress response in living animals

Although the role of p16INK4a tumor suppressor gene expression is well documented in various cell culture studies, its in vivo roles are poorly understood. To gain further insight into the roles and mechanisms regulating p16INK4a gene expression in vivo, we attempted to visualize the dynamics of p16INK4a gene expression using bioluminescence in vivo imaging technique in living mice. By monitoring and quantifying the p16INK4a gene expression repeatedly in the same mouse throughout its entire lifespan, we were able to unveil the dynamics of p16INK4a gene expression in the aging process. This system can also be applied to chemically- or genetically- induced carcinogenesis. Here, we introduce you a novel approach to study senescence stress response in vivo and its potential towards understanding molecular link between aging and cancer.

Regulation of Multilineage Gene Expression and Apoptosis during in vitro Expansion of Human Bone Marrow Stromal Cells with Different Cell Culture Media

The aim of the present study was to analyze the effect of 3 different expansion media on the expression of marker genes of mesenchymal differentiation (bone, cartilage, fat) as well as apoptosis and senescence during repeated passaging in human bone marrow stromal cells (hBMSCs) in order to identify potential expansion strategies for the use of these cells into tissue-engineered growth of bone. Medium 1 (EGF, PDGF, low Glc, 2% FCS) was associated with the highest proliferation rate compared to medium 2 (β-mercaptoethanol, high Glc DMEM, 15% FCS) and medium 3 (low Glc DMEM, 10% FCS). Real time RT-PCR indicated the lowest levels of expression of osteonectin, core binding factor-α 1, lipoprotein lipase and cartilage oligo matrix protein in medium-1 cultures as compared to media 2 and 3. Early passages expressed higher levels of peroxisome proliferator-activator receptor-γ2 in medium 1 than in media 2 and 3, whereas no difference of Sox-9 expression was noticed among the 3 media. Expression of apoptosis- and senescence-related genes (Bax, BCL-2 and P16INK4a) exhibited the lowest level of Bax/BCL-2 ratio and P16INK4a gene expression of hBMSC in medium 1. In conclusion, the replacement of FCS by recombinant EGF and PDGF promoted rapid proliferation of hBMSCs without inducing differentiation of hBMSCs. It also inhibited expression of apoptosis-related genes and limited replicative senescence during repeated passaging. Media with the lowest possible FCS content and replacement by EGF and PDGF thus should be used for 2D culturing during expansion of hBMSCs, whereas β-mercaptoethanol and high concentrations of FCS can help to commence osteogenic differentiation.

Abstract 2622: C282Y HFE polymorphism imparts temozolomide and 17-DMAG resistance via p16INK4A

Abstract Cancer cells have a robust requirement for iron. The HFE protein interacts with transferrin receptor and limits the cellular iron uptake. HFE polymorphisms - the most common genetic variants in Caucasians - have been associated with a variety of cancers. Therefore we hypothesized that HFE polymorphisms would impact the phenotype of cancer cells. Using stably transfected human neuroblastoma cell lines and a number of human glioma cell lines we determined the effect of HFE polymorphisms on the therapy resistance. In the HFE variants stably transfected neuroblastoma cells, cells carrying the C282Y allelic variant were significantly more resistant to the chemotherapeutic agent temozolomide (currently the standard of care for brain tumors) than the same cell line carrying wildtype or H63D variant of HFE. The resistance to temozolomide was also found in commercially available human astrocytoma cell lines with the C282Y allele. The mechanism of greater resistance in the cells with the C282Y allele did not appear to involve O6-methylguanine-DNA methyltransferase traditionally associated with temozolomide resistance. In addition to temozolomide, the C282Y allele expressing glioma cells were also resistant to geldanamycin and its derivatives, 17-allylaminogeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). These ansamycin compounds inhibit heat shock protein 90 (Hsp90) and thus have a different mechanism of action on the cancer cells than temozolomide. To discover the mechanism by which C282Y HFE variant cells were resistant to different therapeutic agents, we performed functional gene arrays in HFE stably transfected neuroblastoma cells. The expression of p16INK4A (cyclin dependent kinase inhibitor 2A) gene was found significantly increased in the C282Y cells compared to the wildtype cells. Consistent with the gene expression, p16INK4A protein expression was elevated in association with the C282Y allele relative to wildtype HFE. C282Y expressing astrocytoma cells also expressed high levels of p16INK4A protein. Therefore, we further determined the role of p16INK4A in drug resistance using p16INK4A siRNA, and found that p16INK4A knockdown sensitized C282Y expressing cells to temozolomide and 17-DMAG. Furthermore, siRNA of HFE decreased p16INK4A expression in C282Y cells. Taking together, these data suggest that C282Y HFE polymorphism impacts the cancer phenotype such as drug resistance, and the mechanism appears to be via increased expression of p16INK4A. Given the frequency of the HFE variants in the general population, these data provide compelling evidence that C282Y HFE polymorphism should be a pharmacogenetic consideration for evaluating treatment efficacy in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2622.

Real-time in vivo imaging of p16Ink4a gene expression: a new approach to study senescence stress signaling in living animals

Oncogenic proliferative signals are coupled to a variety of growth inhibitory processes. In cultured primary human fibroblasts, for example, ectopic expression of oncogenic Ras or its downstream mediator initiates cellular senescence, the state of irreversible cell cycle arrest, through up-regulation of cyclin-dependent kinase (CDK) inhibitors, such as p16INK4a. To date, much of our current knowledge of how human p16INK4a gene expression is induced by oncogenic stimuli derives from studies undertaken in cultured primary cells. However, since human p16INK4a gene expression is also induced by tissue culture-imposed stress, it remains unclear whether the induction of human p16INK4a gene expression in tissue-cultured cells truly reflects an anti-cancer process or is an artifact of tissue culture-imposed stress. To eliminate any potential problems arising from tissue culture imposed stress, we have recently developed a bioluminescence imaging (BLI) system for non-invasive and real-time analysis of human p16INK4a gene expression in the context of a living animal. Here, we discuss the molecular mechanisms that direct p16INK4a gene expression in vivo and its potential for tumor suppression.

Adaptor Protein Crk Induces Src-Dependent Activation of p38 MAPK in Regulation of Synovial Sarcoma Cell Proliferation

The adaptor protein Crk mediates intracellular signaling related to cell motility and proliferation and is implicated in human tumorigenesis. The role of Crk in the growth of human sarcoma has remained unclear, however. The present study shows that Crk-induced activation of Src and subsequent signaling by p38 mitogen-activated protein kinase (MAPK) contribute to the enhanced proliferation of human synovial sarcoma cells. Depletion of Crk by RNA interference markedly inhibited proliferation of the synovial sarcoma cell lines HS-SYII, SYO-1, and Fuji as well as prevented anchorage-independent growth. Conversely, reconstitution with CrkII by authentic small interfering RNA-resistant Crk gene restored proliferation in Crk-silenced SYO-1 cells. Crk-depleted synovial sarcoma cells manifested enhanced transcriptional activity and expression of the p16(INK4A) gene, resulting in their accumulation in G(1) phase of the cell cycle. In response to hepatocyte growth factor stimulation, Crk prominently induced the tyrosine phosphorylation of Grb2-associated binder 1 through activation of Src and focal adhesion kinase, and the Src family kinase inhibitor PP2 almost completely inhibited the proliferation of SYO-1 cells. Crk also induced the phosphorylation of p38 MAPK, and SB203580, a p38 MAPK-specific inhibitor, increased expression of p16(INK4A) gene in SYO-1 cells. Furthermore, SB203580 or depletion of p38 MAPK by small interfering RNA suppressed both the phosphorylation of Akt triggered by hepatocyte growth factor and the proliferation of SYO-1 cells. These results suggest that Crk promotes proliferation of human synovial sarcoma cells through activation of Src and its downstream signaling by a novel p38 MAPK-Akt pathway, with these signaling molecules providing potent new targets for molecular therapeutics.

Caveolin-1 expression and stress-induced premature senescence in human intervertebral disc degeneration

IntroductionChronic and debilitating low back pain is a common condition and a huge economic burden. Many cases are attributed to age-related degeneration of the intervertebral disc (IVD); however, age-related degeneration appears to occur at an accelerated rate in some individuals. We have previously demonstrated biomarkers of cellular senescence within the human IVD and suggested a role for senescence in IVD degeneration. Senescence occurs with ageing but can also occur prematurely in response to stress. We hypothesised that stress-induced premature senescence (SIPS) occurs within the IVD and here we have investigated the expression and production of caveolin-1, a protein that has been shown previously to be upregulated in SIPS.MethodsCaveolin-1 gene expression in human nucleus pulposus (NP) cells was assessed by conventional and quantitative real-time polymerase chain reaction (PCR), and caveolin-1 protein expression was examined within human IVDs using immunohistochemistry. The correlation between caveolin-1 and p16INK4a (biomarker of cellular senescence) gene expression was investigated using quantitative real-time PCR.ResultsCaveolin-1 gene expression and protein expression were demonstrated within the human IVD for the first time. NP cells from degenerate discs exhibited elevated levels of caveolin-1 which did not relate to increasing chronological age. A negative correlation was observed between gene expression for caveolin-1 and donor age, and no correlation was found between caveolin-1 protein expression and age. A positive correlation was identified between gene expression of caveolin-1 and p16INK4a.ConclusionOur findings are consistent with a role for caveolin-1 in degenerative rather than age-induced changes in the NP. Its expression in IVD tissue and its association with the senescent phenotype suggest that caveolin-1 and SIPS may play a prominent role in the pathogenesis of IVD degeneration.

Expression of p16INK4A gene in human pituitary tumours

Pituitary adenomas comprise 10-15% of primary intracranial tumours but the mechanisms leading to tumour development are yet to be clearly established. The retinoblastoma pathway, which regulates the progression through the cell cycle, is often deregulated in different types of tumours. We studied the cyclin-dependent kinase inhibitor p16(INK4A) gene expression at mRNA level in human pituitary adenomas. Forty-six tumour specimens of different subtypes, 21 clinically non-functioning, 12 growth hormone-secreting, 6 prolactin-secreting, 6 adrenocorticotropin-secreting, and 1 thyrotropin-secreting tumours were studied. All clinically non-functioning and most of the hormone-secreting tumours were macroadenomas (38/46). The RT-PCR assay and electrophoresis of the PCR-products showed that p16(INK4A) mRNA was undetectable in: 62% of non-functioning, 8% of growth hormone-secreting, 17% of prolactin-secreting and 17% of adrenocorticotropin-secreting adenomas. Forty percent of all macroadenomas and 25% of microadenomas had negative p16(INK4A) mRNA, the latter results suggest that the absence of p16(INK4A) product might be an early event in tumours with no expression of this suppressor gene. Within the non-functioning adenomas 63% were "null cell" and 37% were positive for some hormone, both subgroups showed similar percentage of cases with absence of p16(INK4A) mRNA. Our results show that clinically non-functioning macroadenomas have impaired p16(INK4A) expression in a clearly higher proportion than any other pituitary tumour subtype investigated. Other regulatory pathways may be implicated in the development of tumours with positive p16(INK4A) expression.

Expression of the p16INK4A Gene Is Associated Closely with Senescence of Human Mesenchymal Stem Cells and Is Potentially Silenced by DNA Methylation During In Vitro Expansion

The precise biological characteristics of human mesenchymal stem cells (hMSCs), including growth regulatory mechanisms, have not yet been defined. Using 29 strains of hMSCs isolated from bone marrow, we have performed extensive analyses of the growth profiles of hMSCs in vitro. All 29 strains stopped proliferating with a mean population doubling (PD) of 28, although there was a considerable difference among strains. The mean telomere restriction fragment length of the cells passaged twice correlated well with the final number of PDs in each strain, suggesting the value of this measurement to be predictive of the growth potential of hMSCs. The expression level of the p16INK4A gene was associated closely with the PD number of each strain (p = .00000001). Most of the p16INK4A-positive cells were Ki67-negative and senescence associated beta-galactosidase-positive, and the suppression of p16INK4A gene expression by small interfering RNA in senescent hMSCs reduced the number of senescent cells and endowed them with the ability to proliferate. Twenty-five of the 29 strains showed a steady gradual increase in the expression of p16INK4A. The remaining four strains (13.8%) showed different profiles, in which DNA methylation in the promoter region occurred in vitro. One of the four strains continued to proliferate for much longer than the others and showed chromosomal aberrations in the later stages. These results indicated p16INK4A to be a key factor in the regulation of hMSC growth, and, most importantly, careful monitoring of DNA methylation should be considered during the culture of hMSCs, particularly when a prolonged and extended propagation is required.

Accelerated cellular senescence in degenerate intervertebral discs: a possible role in the pathogenesis of intervertebral disc degeneration

Current evidence implicates intervertebral disc degeneration as a major cause of low back pain, although its pathogenesis is poorly understood. Numerous characteristic features of disc degeneration mimic those seen during ageing but appear to occur at an accelerated rate. We hypothesised that this is due to accelerated cellular senescence, which causes fundamental changes in the ability of disc cells to maintain the intervertebral disc (IVD) matrix, thus leading to IVD degeneration. Cells isolated from non-degenerate and degenerate human tissue were assessed for mean telomere length, senescence-associated β-galactosidase (SA-β-gal), and replicative potential. Expression of P16INK4A (increased in cellular senescence) was also investigated in IVD tissue by means of immunohistochemistry. RNA from tissue and cultured cells was used for real-time polymerase chain reaction analysis for matrix metalloproteinase-13, ADAMTS 5 (a disintegrin and metalloprotease with thrombospondin motifs 5), and P16INK4A. Mean telomere length decreased with age in cells from non-degenerate tissue and also decreased with progressive stages of degeneration. In non-degenerate discs, there was an age-related increase in cellular expression of P16INK4A. Cells from degenerate discs (even from young patients) exhibited increased expression of P16INK4A, increased SA-β-gal staining, and a decrease in replicative potential. Importantly, there was a positive correlation between P16INK4A and matrix-degrading enzyme gene expression. Our findings indicate that disc cell senescence occurs in vivo and is accelerated in IVD degeneration. Furthermore, the senescent phenotype is associated with increased catabolism, implicating cellular senescence in the pathogenesis of IVD degeneration.

P‐22 P16INK4A AS A MARKER FOR CERVICAL DYSKARYOSIS IN THINPREP® LIQUID BASED CYTOLOGY SAMPLES

Cervical cancer is the second most common cancer amongst women worldwide. It is now accepted that almost all cervical cancers arise through the disruption of the pathways of p53 and the product of the retinoblastoma gene (pRb) by high‐risk Human Papilloma Virus (HPV) oncoproteins E6 and E7. In cervical cancer p16INK4a is strongly over expressed when pRb is inactivated by HPV E7. This over expression highlights the potential of p16INK4a as a marker for cervical intraepithelial neoplasia and cervical cancer. Immunocytochemical (ICC) analysis of P16INK4a expression was performed on 30 negative, 30 Borderline, 30 CIN1 and 30 High Grade ThinPrep® cervical samples selected from 1094 consented samples received for routine cervical screening assessment. The results were correlated with HR‐HPV status as determined by a PCR assay and the cytology findings. In addition a real time RT‐PCR technique was employed to evaluate P16INK4a gene expression in 50 of the 120 study samples. Eighty‐five of the 120 cervical samples were found positive by the ICC assay, 13/30 Negative, 23/30 Borderline, 20/30 CIN 1 and 29/30 High Grade. The ICC staining results were supported by the results of the P16INK4a real time RT‐PCR analysis and the HR‐HPV PCR assay. Interestingly not all HR‐HPV positive samples were P16INK4a ICC positive, suggesting the marker may be capable of discriminating between productive viral infections and active cell transformation. In conclusion P16INK4a is a useful marker of HR‐HPV mediated cervical dyskaryosis in ThinPrep® samples and may be utilized as an adjunct to the Pap smear to aid in the identification of patients with lesions which are more likely to progress to cervical cancer. Additional studies correlating p16INK4a ICC with viral load, viral integration and E6/E7 expression are required to establish how discriminatory a marker p16INK4a truly is.

The relationship between methylation and expression defect of tumor suppressor gene p16INK4A in epithelial ovarian cancer

Objective: To evaluate the expression of p16INK4A gene in ovarian cancer and analyze the relation between this alteration and the promoter methylation of p16INK4A DNA. Methods: Seven ovarian cancer cell lines and eighteen ovarian cancer specimens were selected for the study. Genomic DNA and RNA were extracted from fresh tissues and cell lines, DNA was treated with sodium bisulfite and then analyzed with methylation-specific PCR (MSP) to detect p16INK4A methylation. The expression of p16INK4A mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). In addition, the proliferation of methylated cell lines before and after treatment of demethylating agent 5-Aza-2'-deoxycytidine (5-ADC) was examined with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay in vivo. Results: Compared with the control, the expression of p16INK4A mRNA decreased significantly or absolutely defaulted in 10 of 18 (55.56%) ovarian cancer specimens and 71.4% (5/7) ovarian cancer cell lines (P0.05), and the expression of p16INK4A protein also decreased (P0.05). The decrease of p16INK4A was due, in part, to p16INK4A methylation, which was found in the first exon of three cell lines and six ovarian cancer specimens and the rate was 42.86% and 33.33% in ovarian cancer cell lines and specimens respectively. All the methylated cells and tissues showed expression defect of p16INK4A, but the treatment of 5-ADC reactivated the expression of pl6INK4A in methylated cells and decreased the proliferation of tumor cells in vitro and in vivo. Conclusion: The expression defect of p16INK4A gene possibly has an important role in the development of ovarian cancer, and this alteration is due, in part, to the methylation of the first exon in p16INK4A.

The expression of p16 INK4a tumor suppressor is upregulated by human cytomegalovirus infection and required for optimal viral replication

The human cytomegalovirus (HCMV) induces a replicative senescence program after arresting host cell cycle progression so as to create a favorable environment for its replication. Here, we report that HCMV infection stimulates the expression of p16 INK4a, a direct effector of the senescence phenotype. The increase in p16 INK4a gene expression was due to an increase in gene transcription, since the expression of a reporter gene driven by the p16 INK4a-encoding CDKN2A gene promoter was strongly induced by HCMV infection. The results of deletion and mutational analysis of the CDKN2A promoter further suggest the involvement of Ets transcription factors in HCMV-mediated stimulation of p16 INK4a gene expression. The significance of p16 INK4a upregulation during the HCMV replicative cycle is underscored by the finding that virus replication was severely impaired in fibroblasts homozygous for an intragenic deletion in CDKN2A locus and devoid of functional p16 INK4a. Moreover, a retrovirus-mediated p16 INK4a small interfering RNA (p16-siRNA) effectively reduced viral replication, thus providing direct evidence that p16 INK4a upregulation plays a positive role for HCMV replication.

The hypermethylation and protein expression of p16 INK4A and DNA repair gene O 6-methylguanine-DNA methyltransferase in various uterine cervical lesions

This study is aimed at investigating the significance of gene promoter methylation status and protein expression of p16 INK4A and O6-methylguanine-DNA methyltransferase (MGMT) in the various uterine cervical lesions. Methylation status by using methylation-specific polymerase chain reaction (MS-PCR) and protein expression by using immunohistochemistry for p16 INK4A and MGMT genes were performed in cervical squamous intraepithelial neoplasms (CIN), invasive squamous cell carcinomas (SCC), adenocarcinomas and non-neoplastic cervices. None of 20 non-neoplastic cervices showed p16 INK4A and MGMT gene hypermethylation, whereas at least one of these genes was hypermethylated with 50.0% (5/10) of CIN I, 65.0% (13/20) of CIN II-III, 70.2% (33/47) of SCC and 85.0% (17/20) of adenocarcinoma. p16 INK4A protein was totally negative in non-neoplastic cervices, but positive with 90.0% of CIN I, 100% of CIN II-III and adenocarcinoma, and 78.7% of SCC. MGMT protein was expressed in 10% of non-neoplastic cervices, but significantly increased in SCC (42.5%) and adenocarcinoma (70.0%). The protein expression of p16 INK4A and MGMT was not related to their gene promoter methylation status. The hypermethylation of p16 INK4A and MGMT genes in the uterine cervix may indicate the presence of malignant cells, and p16 INK4A immunostaining is useful in grading CIN and diagnosing invasive SCC and adenocarcinoma.

Frequent promoter hypermethylation of RASSF1A and p16INK4a and infrequent allelic loss other than 9p21 in betel‐associated oral carcinoma in a Vietnamese non‐smoking/non‐drinking female population

Betel-chewing, a risk factor for oral carcinoma, is a common habit of elderly Vietnamese females, but concomitant habits of tobacco and alcohol are uncommon. In the present study, 36 paraffin-embedded betel-associated oral carcinoma samples including 27 squamous cell carcinoma (SCC) and nine verrucous carcinomas (VC) were analyzed for the hypermethylation of tumor suppressor genes (TSGs) and loss of heterozygosity (LOH) of important TSG loci. Methylation-specific polymerase chain reaction (MSP) was used to identify promoter hypermethylation of p16INK4a and RASSF1A. For LOH analysis, 39 microsatellite markers at 12 chromosomal arms were examined by p olymerase chain reaction (PCR)-based microsatellite assay. Hypermethylation of p16IKN4a was detected in 63% of SCC and 67% of VC. In addition, LOH at 9p21 (locus for p16INK4a) was 58% for SCC and 22% for VC, and hypermethylation of RASSF1A was 93% for SCC and 100% for VC. LOH at 3p21.3-3p22.1 (where RASSF1A is located) was detected in only 12% of SCC and 0% of VC. LOH of other chromosomal arms were infrequent. As LOH for chromosomes other than 9p was uncommon, epigenetic silencing of RASSF1A and p16INK4a gene expression by promoter hypermethylation may play a critical role in betel-associated oral carcinogenesis.

Expression of the p16INK4a gene and methylation pattern of CpG sites in the promoter region in rat tumor cell lines.

Loss of p16(INK4a) protein expression has frequently been related to DNA methylation in association with gene silencing. Although the methylation status of exon1alpha for p16(INK4a) involvement in various cancers has been extensively analyzed, it has been pointed out that some inconsistencies existed in its relationship to gene silencing of p16(INK4a). In this study, we focused on the expression and methylation status in the regions of nt -478 to -201, containing a putative TATA box (nt -401 to -396), and nt -233 to 26, both in a recently cloned 5' upstream region of rat p16(INK4a). We showed that rat lung adenocarcinoma RLCNR did not express the p16(INK4a) gene, whereas rat osteosarcoma COS1NR and malignant fibrous histiocytoma MFH1NR both expressed it at levels similar to normal fibroblasts, even though the region of nt -233 to 26 was hypermethylated in COS1NR rather than RLCNR. In contrast, the CpG islands near the putative TATA box region were consistently methylated in RLCNR, but not in COS1NR and MFH1NR, as well as in normal fibroblasts. Treatment with 5-aza 2'-deoxycytidine induced expression of p16(INK4a) gene in RLCNR after 48 h, but no changes were observed in COS1NR and MFH1NR. The results indicated that methylation of CpG islands near a TATA box region played a critical role for gene silencing of the rat p16(INK4a) gene, rather than that of other regions.

Epstein-Barr virus LMP1 blocks p16INK4a-RB pathway by promoting nuclear export of E2F4/5.

The p16INK4a–RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a–RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a–RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a–RB pathway.

Involvement of p-15INK4b and p-16INK4a Gene Expression in Saikosaponin a and TPA-Induced Growth Inhibition of HepG2 Cells

Saikosaponin a, a purified ingredients of Chinese herb with known antitumor activity can inhibit cell growth and DNA synthesis of hepatoma cell line HepG2. Both mRNA and protein of the CDK inhibitor p-16INK4a and p-15INK4b in HepG2 were greatly induced by saikosaponin a while that of p-21CIP, p-27KIP and other cell cycle related genes were not. In addition, reduced phosphorylation of RB protein is observed in saikosaponin a-treated HepG2. Staurosporin, one of the PKC inhibitors, significantly prevented the saikosaponin a induced growth inhibition suggesting PKC pathway be involved. On the other hand, the phorbol ester tumor promoter TPA (12-O-Tetredecanolyphorbol 13-acetate) also inhibited HepG2 growth and specifically induced p-16INK4a and p-15INK4b mRNA expression. The results suggest that both saikosaponin a and TPA-induced HepG2 growth inhibition are associated with p-15INK4a and p-16INK4b gene expression and might be mediated by PKC signaling pathway.