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Sample records for p15ink4b p16ink4a p21cip1

  1. Roles of p15Ink4b and p16Ink4a in myeloid differentiation and RUNX1-ETO-associated acute myeloid leukemia

    PubMed Central

    Ko, Rose M.; Kim, Hyung-Gyoon; Wolff, Linda; Klug, Christopher A.

    2008-01-01

    Inactivation of p15Ink4b expression by promoter hypermethylation occurs in up to 80% of acute myeloid leukemia (AML) cases and is particularly common in the FAB-M2 subtype of AML, which is characterized by the presence of the RUNX1-ETO translocation in 40% of cases. To establish whether the loss of p15Ink4b contributes to AML progression in association with RUNX1-ETO, we have expressed the RUNX1-ETO fusion protein from a retroviral vector in hematopoietic progenitor cells isolated from wild-type, p15Ink4b or p16Ink4a knockout bone marrow. Analysis of lethally irradiated recipient mice reconstituted with RUNX1-ETO-expressing cells showed that neither p15Ink4b or p16Ink4a loss significantly accelerated disease progression over the time period of one year post-transplantation. Loss of p15Ink4b alone resulted in increased myeloid progenitor cell frequencies in bone marrow by 10 months post-transplant and a 19-fold increase in the frequency of Lin-c-Kit+Sca-1+ (LKS) cells that was not associated with expansion of long-term reconstituting HSC. These results strongly suggest that p15Ink4b loss must be accompanied by additional oncogenic changes for RUNX1-ETO-associated AML to develop. PMID:18037485

  2. The p15(INK4b)/p16(INK4a)/RB1 pathway is frequently deregulated in human pituitary adenomas.

    PubMed

    Ogino, Akiyoshi; Yoshino, Atsuo; Katayama, Yoichi; Watanabe, Takao; Ota, Takashi; Komine, Chiaki; Yokoyama, Takakazu; Fukushima, Takao

    2005-05-01

    Pituitary adenomas are common benign intracranial neoplasms. However, their tumorigenesis is not yet clearly defined. Inactivation of genes involved in the negative cell-cycle regulatory p15(INK4b) - p16(INK4a) -cyclin D/CDK4-RB1-mediated pathway (RB1 pathway) is one of the most common and important mechanisms in the growth advantage of tumor cells. Recently, much attention has been focused on the importance of alternative mechanisms of gene inactivation, particularly promoter hypermethylation in the transcriptional silencing of such tumor-suppressor genes. Based on the rare occurrence of inactivation by gene mutations and deletions of the RB1 pathway in pituitary adenomas, we investigated the deregulation of the RB1 pathway in 42 sporadic human pituitary adenomas, especially focusing on the methylation status of this pathway as determined by a methylation-specific polymerase chain reaction assay. Homozygous deletion of the p15(INK4b) or p16(INK4a) gene was detected in one adenoma each. Amplification of the CDK4 gene was not apparent in any of the pituitary adenomas presently examined. Promoter hypermethylation of the p15(INK4b), p16(INK4a), and RB1 genes was detected in 15 (35.7%), 30 (71.4%), and 12 (28.6%) of the adenomas, respectively. Promoter hypermethylation of the p15(INK4b) gene coincided with p16(INK4a) alteration and/or RB1 methylation, whereas p16(INK4a) and RB1 methylations tended to be mutually exclusive (p = 0.019). Thus, the vast majority of the adenomas (38 of 42, 90.5%) displayed alterations of the RB1 pathway. None of the clinicopathologic features, including the proliferation cell index, was significantly correlated with any particular methylation status. Our results suggest that inactivation of the RB1 pathway may play a causal role in pituitary tumorigenesis, with hypermethylation of the p16(INK4a) gene being the most common deregulation, and further provide evidence that RB1 and p16(INK4a) methylations tend to be mutually exclusive but

  3. Methylation of CpG island of p14(ARK), p15(INK4b) and p16(INK4a) genes in coke oven workers.

    PubMed

    Zhang, H; Li, X; Ge, L; Yang, J; Sun, J; Niu, Q

    2015-02-01

    To detect the blood genomic DNA methylation in coke oven workers and find a possible early screening index for occupational lung cancer, 74 coke oven workers as the exposed group and 47 water pump workers as the controls were surveyed, and urine samples and peripheral blood mononuclear cells (PBMCs) were collected. Airborne benzo[a]pyrene (B[a]P) levels in workplace and urinary 1-hydroxypyrene (1-OH-Py) levels were determined by high-performance liquid chromatography. DNA damage of PBMCs and the p14(ARK), p15(INK4b) and p16(INK4a) gene CpG island methylation in the promoter region were detected by comet assay and methylation-specific polymerase chain reaction techniques, respectively. Results show that compared with the controls, concentration of airborne B[a]Ps was elevated in the coke plant, and urinary 1-OH-Py's level and DNA olive tail moment in comet assay were significantly increased in the coke oven workers, and p14(ARK), p15(INK4b) and p16(INK4a) gene methylation rates were also significantly increased. With the working years and urinary 1-OH-Py's level, the rates of p14(ARK) and p16(INK4a) gene methylation were significantly increased while that of p15(INK4b) gene methylation displayed no statistical change. We conclude that PBMCs' p14(ARK) and p16(INK4a) gene methylation may be used for screening and warning lung cancer in coke oven workers.

  4. Expression of p14(ARF), p15(INK4b), p16(INK4a) and skp2 increases during esophageal squamous cell cancer progression.

    PubMed

    Bai, Peng; Xiao, Xue; Zou, Juan; Cui, Lin; Bui Nguyen, Tri M; Liu, Jinsong; Xiao, Jianguo; Chang, Bin; Wu, Jin; Wang, He

    2012-06-01

    Esophageal carcinoma is the sixth most common cause of cancer-related mortality in the world. Senescence and apoptosis are assumed to be two main mechanisms that inhibit age-related carcinogenesis. p14(ARF), p15(INK4b) and p16(INK4a), which are known to induce senescence by regulating G(1) cell cycle arrest, have been identified as senescence markers. However, the mechanism by which senescence and apoptosis causes neoplasia in esophageal squamous cell carcinoma (ESCC) has not been identified. In this study, 20 cases of normal esophageal tissues, 11 cases of esophageal intraepithelial dysplasia (EID) and 60 cases of ESCC were obtained and pathologically diagnosed. Immunohistochemical staining was performed to assess the expression of p14(ARF), p15(INK4b), p16(INK4a), skp2, bcl-2 and ki-67. The senescence markers p14(ARF) and p16(INK4a) were found to be expressed in 15 and 10% of the normal tissues, 82 and 73% of the EID cases and 100 and 88% of the ESCC cases, respectively. The expression of p15(INK4b) was low in normal tissues, while 92% of the ESCC specimens were diffusely and markedly stained, involving the basal, middle and upper portion of the epithelium. The nuclear expression markers ki-67 and skp2 were highly expressed in ESCC tissues (100 and 72%, respectively). bcl-2 was expressed weakly in normal tissues (10%) and demonstrated various staining patterns in carcinoma specimens (strong in 60%, negative in 40%). MI was 0.09% in normal tissues and 0.95% in the ESCC specimens. Apart from the increased proliferation in esophageal carcinogenesis, as indicated in the ki-67 and skp2 indices, there was an increased expression of senescence-associated molecular markers in the ESCC specimens, which indicates that the senescence pathway may be activated and become a part of cancer development. Of greatest interest to us was that, when compared with clinical information, the expression of the senescence markers was markedly high in the poorly differentiated specimens

  5. Mutational analysis of genes p14ARF, p15INK4b, p16INK4a, and PTEN in human nervous system tumors.

    PubMed

    Almeida, L O; Custódio, A C; Araújo, J J; Rey, J A; Almeida, J R W; Santos, M J; Clara, C A; Casartelli, C

    2008-05-27

    Cancer is one of the most common and severe problems in clinical medicine, and nervous system tumors represent about 2% of the types of cancer. The central role of the nervous system in the maintenance of vital activities and the functional consequences of the loss of neurons can explain how severe brain cancers are. The cell cycle is a highly complex process, with a wide number of regulatory proteins involved, and such proteins can suffer alterations that transform normal cells into malignant ones. The INK4 family members (CDK inhibitors) are the cell cycle regulators that block the progression of the cycle through the R point, causing an arrest in G1 stage. The p14ARF (alternative reading frame) gene is a tumor suppressor that inhibits p53 degradation during the progression of the cell cycle. The PTEN gene is related to the induction of growth suppression through cell cycle arrest, to apoptosis and to the inhibition of cell adhesion and migration. The purpose of the present study was to assess the mutational state of the genes p14ARF, p15INK4b, p16INK4a, and PTEN in 64 human nervous system tumor samples. Homozygous deletions were found in exon 2 of the p15INK4b gene and exon 3 of the p16INK4a gene in two schwannomas. Three samples showed a guanine deletion (63 codon) which led to a loss of heterozygosity in the p15 gene, and no alterations could be seen in the PTEN gene. Although the group of patients was heterogeneous, our results are in accordance with other different studies that indicate that homozygous deletion and loss of heterozygosity in the INK4 family members are frequently observed in nervous system tumors.

  6. Analysis of the methylation patterns of the p16 INK4A, p15 INK4B, and APC genes in gastric adenocarcinoma patients from a Brazilian population.

    PubMed

    do Nascimento Borges, Bárbara; Burbano, Rommel Mario Rodriguez; Harada, Maria Lúcia

    2013-08-01

    Gastric cancer is a major public health problem in Pará state, where studies suggest complex genetic and epigenetic profiles of the population, indicating the need for the identification of molecular markers for this tumor type. In the present study, the methylation patterns of three genes [p16 (INK4A), p15 (INK4B), and adenomatous polyposis coli (APC)] were assessed in patients with gastric adenocarcinoma from Pará state in order to identify possible molecular markers of gastric carcinogenesis. DNA samples from tumoral and non-tumoral gastric tissues were modified with sodium bisulfite. A fragment of the promoter region of each gene was amplified and sequenced, and samples with more than 20 % of methylated CpG sites were considered hypermethylated. The correlation between the methylation pattern of the selected genes and the MTHFR C677T polymorphism, as well as the relationship between APC and CDH1 methylation, were evaluated. The results suggest that APC hypermethylation is an age-specific marker of gastric carcinogenesis, and the concordance of this event with CDH1 hypermethylation suggests that the Wnt pathway has an important role in gastric carcinogenesis. While the hypermethylation pattern of p15 (INK4B) seems to be an earlier event in this type of tumor, the hypomethylated status of this gene seems to be correlated to the C677T MTHFR TT genotype. On the other hand, the observed pattern of p16 (INK4A) hypermethylation suggests that this event is a good marker for the gastric cancer pathway in the Pará state population.

  7. Conspirators in a capital crime: co-deletion of p18INK4c and p16INK4a/p14ARF/p15INK4b in glioblastoma multiforme.

    PubMed

    Solomon, David A; Kim, Jung-Sik; Jean, Walter; Waldman, Todd

    2008-11-01

    Glioblastoma multiforme (GBM) is one of the most dreaded cancer diagnoses due to its poor prognosis and the limited treatment options. Homozygous deletion of the p16(INK4a)/p14(ARF)/p15(INK4b) locus is among the most common genetic alterations in GBM. Two recent studies have shown that deletion and mutation of another INK4 family member, p18(INK4c), also drives the pathogenesis of GBM. This minireview will discuss the known roles for p18(INK4c) in the initiation and progression of cancer and suggest opportunities for future studies.

  8. Deregulated E2F-1 blocks terminal differentiation and loss of leukemogenicity of M1 myeloblastic leukemia cells without abrogating induction of p15(INK4B) and p16(INK4A).

    PubMed

    Amanullah, A; Hoffman, B; Liebermann, D A

    2000-07-15

    The transcription factor E2F-1 has been postulated to play a crucial role in the control of cell cycle progression because of its ability to be bound and regulated by the retinoblastoma gene product (pRb). Exogenous expression of E2F-1, under growth restrictive conditions, was shown to result in p53-dependent programmed cell death. The consequences of deregulated expression of E2F-1 on terminal differentiation of hematopoietic cells in the absence of E2F-1-mediated apoptosis, as well as mechanistic insights into how deregulated E2F-1 may affect terminal differentiation, have not been established. The autonomously proliferating M1 myeloblastic leukemia cell line, which is null for p53 expression and can be induced by interleukin-6 (IL-6) to undergo terminal macrophage differentiation with concomitant loss of leukemogenicity, provides a particularly attractive model system to address these issues. Deregulated and continued expression of E2F-1 blocked the IL-6-induced terminal differentiation program at an early blast stage, giving rise to immature cells, which continued to proliferate without undergoing apoptosis and retained their leukemogenic phenotype. Although E2F-1 blocked IL-6-mediated terminal differentiation and its associated growth arrest, it did not prevent the rapid induction of both p15(INK4B) and p16(INK4A), inhibition of cdk4 kinase activity, and subsequent hypophosphorylation of pRb. The results obtained imply that genetic alterations that both impair p53 function and deregulate E2F-1 expression may render hematopoietic cells refractory to the induction of differentiation and are, thereby, likely to play a major role in the progression of leukemias. (Blood. 2000;96:475-482)

  9. Inactivation of p16INK4a, with retention of pRB and p53/p21cip1 function, in human MRC5 fibroblasts that overcome a telomere-independent crisis during immortalization.

    PubMed

    Taylor, Lisa M; James, Alexander; Schuller, Christine E; Brce, Jesena; Lock, Richard B; Mackenzie, Karen L

    2004-10-15

    Recent investigations, including our own, have shown that specific strains of fibroblasts expressing telomerase reverse transcriptase (hTERT) have an extended lifespan, but are not immortal. We previously demonstrated that hTERT-transduced MRC5 fetal lung fibroblasts (MRC5hTERTs) bypassed senescence but eventually succumbed to a second mortality barrier (crisis). In the present study, 67 MRC5hTERT clones were established by limiting dilution of a mass culture. Whereas 39/67 clones had an extended lifespan, all 39 extended lifespan clones underwent crisis. 11 of 39 clones escaped crisis and were immortalized. There was no apparent relationship between the fate of clones at crisis and the level of telomerase activity. Telomeres were hyperextended in the majority of the clones analyzed. There was no difference in telomere length of pre-crisis compared with post-crisis and immortal clones, indicating that hyperextended telomeres were conducive for immortalization and confirming that crisis was independent of telomere length. Immortalization of MRC5hTERT cells was associated with repression of the cyclin-dependent kinase inhibitor p16INK4a and up-regulation of pRB. However, the regulation of pRB phosphorylation and the response of the p53/p21cip1/waf1 pathway were normal in immortal cells subject to genotoxic stress. Overexpression of oncogenic ras failed to de-repress p16INK4a in immortal cells. Furthermore, expression of ras enforced senescent-like growth arrest in p16INK4a-positive, but not p16INK4a-negative MRC5hTERT cells. Immortal cells expressing ras formed small, infrequent colonies in soft agarose, but were non-tumorigenic. Overall, these results implicate the inactivation of p16INK4a as a critical event for overcoming telomere-independent crisis, immortalizing MRC5 fibroblasts and overcoming ras-induced premature senescence.

  10. Tumor Suppressor and Aging Biomarker p16INK4a Induces Cellular Senescence without the Associated Inflammatory Secretory Phenotype*

    PubMed Central

    Coppé, Jean-Philippe; Rodier, Francis; Patil, Christopher K.; Freund, Adam; Desprez, Pierre-Yves; Campisi, Judith

    2011-01-01

    Cellular senescence suppresses cancer by preventing the proliferation of cells that experience potentially oncogenic stimuli. Senescent cells often express p16INK4a, a cyclin-dependent kinase inhibitor, tumor suppressor, and biomarker of aging, which renders the senescence growth arrest irreversible. Senescent cells also acquire a complex phenotype that includes the secretion of many cytokines, growth factors, and proteases, termed a senescence-associated secretory phenotype (SASP). The SASP is proposed to underlie age-related pathologies, including, ironically, late life cancer. Here, we show that ectopic expression of p16INK4a and another cyclin-dependent kinase inhibitor, p21CIP1/WAF1, induces senescence without a SASP, even though they induced other features of senescence, including a stable growth arrest. Additionally, human fibroblasts induced to senesce by ionizing radiation or oncogenic RAS developed a SASP regardless of whether they expressed p16INK4a. Cells induced to senesce by ectopic p16INK4a expression lacked paracrine activity on epithelial cells, consistent with the absence of a functional SASP. Nonetheless, expression of p16INK4a by cells undergoing replicative senescence limited the accumulation of DNA damage and premature cytokine secretion, suggesting an indirect role for p16INK4a in suppressing the SASP. These findings suggest that p16INK4a-positive cells may not always harbor a SASP in vivo and, furthermore, that the SASP is not a consequence of p16INK4a activation or senescence per se, but rather is a damage response that is separable from the growth arrest. PMID:21880712

  11. Loss of p15/Ink4b accompanies tumorigenesis triggered by complex DNA double-strand breaks

    PubMed Central

    Camacho, Cristel V.; Mukherjee, Bipasha; McEllin, Brian; Ding, Liang-Hao; Hu, Burong; Habib, Amyn A.; Xie, Xian-Jin; Nirodi, Chaitanya S.; Saha, Debabrata; Story, Michael D.; Balajee, Adayabalam S.; Bachoo, Robert M.; Boothman, David A.; Burma, Sandeep

    2010-01-01

    DNA double-strand breaks (DSBs) are the most deleterious lesion inflicted by ionizing radiation. Although DSBs are potentially carcinogenic, it is not clear whether complex DSBs that are refractory to repair are more potently tumorigenic compared with simple breaks that can be rapidly repaired, correctly or incorrectly, by mammalian cells. We previously demonstrated that complex DSBs induced by high-linear energy transfer (LET) Fe ions are repaired slowly and incompletely, whereas those induced by low-LET gamma rays are repaired efficiently by mammalian cells. To determine whether Fe-induced DSBs are more potently tumorigenic than gamma ray-induced breaks, we irradiated ‘sensitized’ murine astrocytes that were deficient in Ink4a and Arf tumor suppressors and injected the surviving cells subcutaneously into nude mice. Using this model system, we find that Fe ions are potently tumorigenic, generating tumors with significantly higher frequency and shorter latency compared with tumors generated by gamma rays. Tumor formation by Fe-irradiated cells is accompanied by rampant genomic instability and multiple genomic changes, the most interesting of which is loss of the p15/Ink4b tumor suppressor due to deletion of a chromosomal region harboring the CDKN2A and CDKN2B loci. The additional loss of p15/Ink4b in tumors derived from cells that are already deficient in p16/Ink4a bolsters the hypothesis that p15 plays an important role in tumor suppression, especially in the absence of p16. Indeed, we find that reexpression of p15 in tumor-derived cells significantly attenuates the tumorigenic potential of these cells, indicating that p15 loss may be a critical event in tumorigenesis triggered by complex DSBs. PMID:20663777

  12. 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.

  13. Inactivation of p15(INK4b) in chronic arsenic poisoning cases.

    PubMed

    Zhang, Aihua; Gao, Chen; Han, Xue; Wang, Lifang; Yu, Chun; Zeng, Xiaowen; Chen, Liping; Li, Daochuan; Chen, Wen

    2014-01-01

    Arsenic exposure from burning high arsenic-containing coal has been associated with human skin lesion and cancer. However, the mechanisms of arsenic-related carcinogenesis are not fully understood. Inactivation of critical tumor suppression genes by epigenetic regulation or genetic modification might contribute to arsenic-induced carcinogenicity. This study aims to clarify the correlation between arsenic pollution and functional defect of p15(INK4b) gene in arsenic exposure residents from a region of Guizhou Province, China. To this end, 103 arsenic exposure residents and 105 control subjects were recruited in this study. The results showed that the exposure group exhibited higher levels of urinary and hair arsenic compared with the control group (55.28 vs 28.87 μg/L, 5.16 vs 1.36 μg/g). Subjects with higher arsenic concentrations are more likely to have p15(INK4b) methylation and gene deletion (χ(2) = 4.28, P = 0.04 and χ(2) = 4.31, P = 0.04). We also found that the degree of p15(INK4b) hypermethylation and gene deletion occurred at higher incidence in the poisoning cases with skin cancer (3.7% and 14.81% in non-skin cancer group, 41.18% and 47.06 in skin cancer group), and were significantly associated with the stage of skin lesions (χ(2) = 12.82, P < 0.01 and χ(2) = 7.835, P = 0.005). These observations indicate that inactivation of p15(INK4b) through genetic alteration or epigenetic modification is a common event that is associated with arsenic exposure and the development of arsenicosis.

  14. Nuclear p16INK4a expression predicts enhanced radiation response in head and neck cancers

    PubMed Central

    Dok, Rüveyda; Asbagh, Layka Abbasi; Van Limbergen, Evert Jan; Sablina, Anna; Nuyts, Sandra

    2016-01-01

    Immunohistochemistry analysis of p16INK4a in head and neck squamous cell carcinomas (HNSCC) tumor samples revealed that 28% of tumors showed nuclear/cytoplasmic p16INK4a localization, while 37% of tumors had cytoplasmic p16INK4a. Our previous study showed that p16INK4a inhibits the DNA repair response independently of its function in the cell cycle, suggesting that p16INK4a subcellular localization should be considered during stratification of HNSCC patients. Using p16INK4a mutants with different localization signals, we found that expression of nuclear p16INK4a, but not cytoplasmic p16INK4a impaired RAD51 foci formation, indicating that nuclear localization of p16INK4a is crucial for its function in DNA repair. We next investigated the role of p16INK4a subcellular localization in radiation response in a retrospective cohort of 261 HNSCC patients treated with chemoradiation. We found that only HNSCC patients expressing nuclear p16INK4a expression showed better outcome, locoregional control and disease free survival, after chemoradiation. In concordance with the patient data, only expression of nuclear p16INK4a increased radiosensitivity of HNSCC cells. These results implicate nuclear p16INK4a expression as a potent marker to predict radiation response of HNSCC patients and should be taken into account in intensification or de-escalation studies. PMID:27246975

  15. Ablation of the p16(INK4a) tumour suppressor reverses ageing phenotypes of klotho mice.

    PubMed

    Sato, Seidai; Kawamata, Yuka; Takahashi, Akiko; Imai, Yoshinori; Hanyu, Aki; Okuma, Atsushi; Takasugi, Masaki; Yamakoshi, Kimi; Sorimachi, Hiroyuki; Kanda, Hiroaki; Ishikawa, Yuichi; Sone, Saburo; Nishioka, Yasuhiko; Ohtani, Naoko; Hara, Eiji

    2015-04-29

    The p16(INK4a) tumour suppressor has an established role in the implementation of cellular senescence in stem/progenitor cells, which is thought to contribute to organismal ageing. However, since p16(INK4a) knockout mice die prematurely from cancer, whether p16(INK4a) reduces longevity remains unclear. Here we show that, in mutant mice homozygous for a hypomorphic allele of the α-klotho ageing-suppressor gene (kl(kl/kl)), accelerated ageing phenotypes are rescued by p16(INK4a) ablation. Surprisingly, this is due to the restoration of α-klotho expression in kl(kl/kl) mice and does not occur when p16(INK4a) is ablated in α-klotho knockout mice (kl(-/-)), suggesting that p16(INK4a) is an upstream regulator of α-klotho expression. Indeed, p16(INK4a) represses α-klotho promoter activity by blocking the functions of E2Fs. These results, together with the observation that the expression levels of p16(INK4a) are inversely correlated with those of α-klotho throughout ageing, indicate that p16(INK4a) plays a previously unrecognized role in downregulating α-klotho expression during ageing.

  16. Ageing as developmental decay: insights from p16(INK4a.).

    PubMed

    Martin, Nadine; Beach, David; Gil, Jesús

    2014-12-01

    The p16(INK4a) cell cycle regulator is one of the best ageing biomarkers because it is suppressed in early embryogenesis and progressively induced during ageing. p16(INK4a) plays a crucial role in key cell fate decisions which contribute to ageing, such as cellular senescence and stem cell dynamics. Detailed examination of the pathways regulating p16(INK4a) expression has revealed an overlap with those regulating early development. We present the hypothesis that ageing might be primarily driven by gradual functional decay of developmental pathways. To support this, we summarise the role of p16(INK4a) in ageing and our current knowledge on p16(INK4a) regulation. The developmental decay hypothesis implies that the much-evidenced damage associated with all aspects of ageing might be secondary to such decay. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. p16(Ink4a)-induced senescence of pancreatic beta cells enhances insulin secretion.

    PubMed

    Helman, Aharon; Klochendler, Agnes; Azazmeh, Narmen; Gabai, Yael; Horwitz, Elad; Anzi, Shira; Swisa, Avital; Condiotti, Reba; Granit, Roy Z; Nevo, Yuval; Fixler, Yaakov; Shreibman, Dorin; Zamir, Amit; Tornovsky-Babeay, Sharona; Dai, Chunhua; Glaser, Benjamin; Powers, Alvin C; Shapiro, A M James; Magnuson, Mark A; Dor, Yuval; Ben-Porath, Ittai

    2016-04-01

    Cellular senescence is thought to contribute to age-associated deterioration of tissue physiology. The senescence effector p16(Ink4a) is expressed in pancreatic beta cells during aging and limits their proliferative potential; however, its effects on beta cell function are poorly characterized. We found that beta cell-specific activation of p16(Ink4a) in transgenic mice enhances glucose-stimulated insulin secretion (GSIS). In mice with diabetes, this leads to improved glucose homeostasis, providing an unexpected functional benefit. Expression of p16(Ink4a) in beta cells induces hallmarks of senescence--including cell enlargement, and greater glucose uptake and mitochondrial activity--which promote increased insulin secretion. GSIS increases during the normal aging of mice and is driven by elevated p16(Ink4a) activity. We found that islets from human adults contain p16(Ink4a)-expressing senescent beta cells and that senescence induced by p16(Ink4a) in a human beta cell line increases insulin secretion in a manner dependent, in part, on the activity of the mechanistic target of rapamycin (mTOR) and the peroxisome proliferator-activated receptor (PPAR)-γ proteins. Our findings reveal a novel role for p16(Ink4a) and cellular senescence in promoting insulin secretion by beta cells and in regulating normal functional tissue maturation with age.

  18. The molecular balancing act of p16(INK4a) in cancer and aging.

    PubMed

    LaPak, Kyle M; Burd, Christin E

    2014-02-01

    p16(INK4a), located on chromosome 9p21.3, is lost among a cluster of neighboring tumor suppressor genes. Although it is classically known for its capacity to inhibit cyclin-dependent kinase (CDK) activity, p16(INK4a) is not just a one-trick pony. Long-term p16(INK4a) expression pushes cells to enter senescence, an irreversible cell-cycle arrest that precludes the growth of would-be cancer cells but also contributes to cellular aging. Importantly, loss of p16(INK4a) is one of the most frequent events in human tumors and allows precancerous lesions to bypass senescence. Therefore, precise regulation of p16(INK4a) is essential to tissue homeostasis, maintaining a coordinated balance between tumor suppression and aging. This review outlines the molecular pathways critical for proper p16(INK4a) regulation and emphasizes the indispensable functions of p16(INK4a) in cancer, aging, and human physiology that make this gene special.

  19. Expression of p16(INK4a) prevents cancer and promotes aging in lymphocytes.

    PubMed

    Liu, Yan; Johnson, Soren M; Fedoriw, Yuri; Rogers, Arlin B; Yuan, Hong; Krishnamurthy, Janakiraman; Sharpless, Norman E

    2011-03-24

    Previous authors have suggested that tumor suppressor expression promotes aging while preventing cancer, but direct experimental support for this cancer-aging hypothesis has been elusive. Here, by using somatic, tissue-specific inactivation of the p16(INK4a) tumor suppressor in murine T- or B-lymphoid progenitors, we report that ablation of p16(INK4a) can either rescue aging or promote cancer in a lineage-specific manner. Deletion of p16(INK4a) in the T lineage ameliorated several aging phenotypes, including thymic involution, decreased production of naive T cells, reduction in homeostatic T-cell proliferation, and attenuation of antigen-specific immune responses. Increased T-cell neoplasia was not observed with somatic p16(INK4a) inactivation in T cells. In contrast, B lineage-specific ablation of p16(INK4a) was associated with a markedly increased incidence of systemic, high-grade B-cell neoplasms, which limited studies of the effects of somatic p16(INK4a) ablation on B-cell aging. Together, these data show that expression of p16(INK4a) can promote aging and prevent cancer in related lymphoid progeny of a common stem cell.

  20. Expression of p16INK4A and p14ARF in hematological malignancies.

    PubMed

    Taniguchi, T; Chikatsu, N; Takahashi, S; Fujita, A; Uchimaru, K; Asano, S; Fujita, T; Motokura, T

    1999-11-01

    The INK4A/ARF locus yields two tumor suppressors, p16INK4A and p14ARF, and is frequently deleted in human tumors. We studied their mRNA expressions in 41 hematopoietic cell lines and in 137 patients with hematological malignancies; we used a quantitative reverse transcription-PCR assay. Normal peripheral bloods, bone marrow and lymph nodes expressed little or undetectable p16INK4A and p14ARF mRNAs, which were readily detected in 12 and 17 of 41 cell lines, respectively. Patients with hematological malignancies frequently lacked p16INK4A expression (60/137) and lost p14ARF expression less frequently (19/137, 13.9%). Almost all patients without p14ARF expression lacked p16INK4A expression, which may correspond to deletions of the INK4A/ARF locus. Undetectable p16INK4A expression with p14ARF expression in 41 patients may correspond to p16INK4A promoter methylation or to normal expression status of the p16INK4A gene. All patients with follicular lymphoma (FL), myeloma or acute myeloid leukemia (AML) expressed p14ARF while nine of 23 patients with diffuse large B cell lymphoma (DLBCL) lost p14ARF expression. Patients with ALL, AML or blast crisis of chronic myelogenous leukemia expressed abundant p16INK4A mRNAs more frequently than patients with other diseases (12/33 vs 6/104, P < 0.01). Patients with FL and high p14ARF expression had a significantly shorter survival time while survival for patients with DLBCL and increased p14ARF expression tended to be longer. These observations indicate that p16INK4A and p14ARF expression is differentially affected among hemato- logical malignancies and that not only inactivation but also increased expression may have clinical significance.

  1. p16INK4a expression in basal-like breast carcinoma

    PubMed Central

    Bohn, Olga L; Fuertes-Camilo, Mariana; Navarro, Leticia; Saldivar, Jesus; Sanchez-Sosa, Sergio

    2010-01-01

    BLBC represents a distinctive group of invasive breast carcinomas with specific genotype and immunopro-file. BLBC is usually defined by gene expression profiling and is currently associated with poor outcome. BLBCs are estrogen receptor (ER) negative, progesterone receptor (PgR) negative, HER2 negative, and usually show a variable expression of basal cytokeratins (CKs), EGFR and CD117. p16 INK4a is a tumor suppressor protein, encoded by the CDKN2A gene, which regulates cell cycle. The reported association of abnormalities in the p16/Rb pathway with increased risk of malignancy prompted us to determine the expression of p16INK4a in a group of BLBC; the results were compared with a group of high-grade invasive carcinoma (HG-IC) of breast. Tissue microarrays (TMA) were constructed in triplicate including 18 BLBC and 18 HG-IC. All BLBC cases were ER-/PgR-/HER2-. Seventeen (94%) BLBC were CK 5/6+/CK 14+; 14 (78%) BLCB showed EGFR expression and 13 (72%) were CD117 positive. BLBCs showed a strong positive reaction with p16 INK4a antibody in 16 of 18 (89%) cases. Although the significance of p16 INK4a expression in breast cancer is not fully understood, we have shown that p16INK4a is strongly expressed in breast cancers with basal-like phenotype. Since it is known that p16INK4a is associated with aggressive behavior in human carcinomas, these data suggest that p16INK4a play a role in the poor prognosis of BLBC. PMID:20661408

  2. Human Papillomaviruses, p16INK4a and Akt expression in basal cell carcinoma

    PubMed Central

    2011-01-01

    Background The pathogenic role of beta-HPVs in non melanoma skin cancer (NMSC), is not still completely understood, and literature data indicate that they might be at least cofactors in the development of certain cutaneous squamous cell carcinomas. However, only few reports contain data on basal cell carcinoma (BCC). The HPVs interact with many cellular proteins altering their function or the expression levels, like the p16INK4a and Akt. Our study aimed to determine the presence of different beta -HPV types and the expression of p16INK4a and Akt in BCC, the commonest NMSC, in the normal appearing perilesional skin and in forehead swab of 37 immunocompetent patients. Methods The expression of p16INK4a and Akt, by immunohistochemistry, and the HPV DNA, by nested PCR, were investigated in each sample. Results No correspondence of HPV types between BCC and swab samples was found, whereas a correspondence between perilesional skin and BCC was ascertained in the 16,7% of the patients. In BCC, 16 different types of beta HPV were found and the most frequent types were HPV107 (15,4%), HPV100 (11,5%) and HPV15 (11,5%) all belonging to the beta HPV species 2. Immunohistochemistry detected significant p16INK4a expression in almost all tumor samples (94,3%) with the highest percentages (> 30%) of positive cells detected in 8 cases. A statistically significant (p = 0,012) increase of beta HPV presence was detected in p16INK4a strongly positive samples, in particular of species 2. pAkt expression was detected in all tumor samples with only 2 cases showing rare positive cells, whereas Akt2 expression was found in 14 out of 35 BCC (40%); in particular in HPV positive samples over-expressing p16INK4a. Conclusions Our data show that p16INK4a and pAkt are over-expressed in BCC and that the high expression of p16INK4a and of Akt2 isoform is often associated with the presence of beta-HPV species 2 (i.e. HPV 15). The association of these viruses with the up-regulation of p16INK4a and Akt

  3. Hematopoietic stem cell ageing is uncoupled from p16 INK4A-mediated senescence.

    PubMed

    Attema, J L; Pronk, C J H; Norddahl, G L; Nygren, J M; Bryder, D

    2009-06-04

    Somatic stem cells are ultimately responsible for mediating appropriate organ homeostasis and have therefore been proposed to represent a cellular origin of the ageing process-a state often characterized by inappropriate homeostasis. Specifically, it has been suggested that ageing stem cells might succumb to replicative senescence by a mechanism involving the cyclin-dependent kinase inhibitor p16(INK4A). Here, we tested multiple functional and molecular parameters indicative of p16(INK4A) activity in primary aged murine hematopoietic stem cells (HSCs). We found no evidence that replicative senescence accompanies stem cell ageing in vivo, and in line with p16(INK4A) being a critical determinant of such processes, most aged HSCs (>99%) failed to express p16(INK4A) at the mRNA level. Moreover, whereas loss of epigenetically guided repression of the INK4A/ARF locus accompanied replicative senescent murine embryonic fibroblasts, such repression was maintained in aged stem cells. Taken together, these studies indicate that increased senescence as mediated by the p16(INK4A) tumor suppressor has only a minor function as an intrinsic regulator of steady-state HSC ageing in vivo.

  4. p16(Ink4a) overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors.

    PubMed

    Romagosa, C; Simonetti, S; López-Vicente, L; Mazo, A; Lleonart, M E; Castellvi, J; Ramon y Cajal, S

    2011-05-05

    p16(Ink4a) is a protein involved in regulation of the cell cycle. Currently, p16(Ink4a) is considered a tumor suppressor protein because of its physiological role and downregulated expression in a large number of tumors. Intriguingly, overexpression of p16(Ink4a) has also been described in several tumors. This review attempts to elucidate when and why p16(Ink4a) overexpression occurs, and to suggest possible implications of p16(Ink4a) in the diagnosis, prognosis and treatment of cancer.

  5. Prognostic significance of p16 INK4a alteration for Ewing sarcoma: a meta-analysis.

    PubMed

    Honoki, Kanya; Stojanovski, Elizabeth; McEvoy, Mark; Fujii, Hiromasa; Tsujiuchi, Toshifumi; Kido, Akira; Takakura, Yoshinori; Attia, John

    2007-09-15

    Despite findings from individual studies regarding prognostic factors for Ewing sarcoma, no conclusive results have been produced, partly because of small sample sizes. The objective of the current study was to evaluate whether the presence of p16(INK4a) alteration is associated with a poorer prognosis in patients with Ewing sarcomas. A review was conducted of publications that assessed associations between p16(INK4a) status and 2-year survival among patients with Ewing sarcoma. The association between metastatic disease at initial diagnosis and 2-year survival was evaluated by synthesizing data in the form of risk ratios. Of 11 studies that were identified in the initial search strategy, 6 studies, representing 188 patients, met the inclusion criteria and, consequently, were pooled for quantitative analyses. The estimated pooled risk ratio of p16(INK4a) aberration was 2.17 (95% confidence interval [95% CI], 1.55-3.03; P < .001), whereas the estimated pooled risk ratio of metastasis at diagnosis among the 164 eligible patients was 2.60 (95% CI, 1.71-3.97; P < .001). There was no statistically significant difference in the pooled estimated risk ratios of p16(INK4a) aberration for a poor prognosis between patients with and without metastasis at diagnosis (1.86 and 2.21, respectively; P > .59). The presence of p16(INK4a) alteration was a statistically significant predictor of prognosis for patients with Ewing sarcoma. Along with other prognostic factors, such as metastasis, the p16(INK4a) alteration may be a potential candidate for improving the risk-stratifying strategy for patients with these tumors. (c) 2007 American Cancer Society.

  6. p16INK4a expression in actinic keratosis and Bowen's disease.

    PubMed

    Salama, M E; Mahmood, M N; Qureshi, H S; Ma, C; Zarbo, R J; Ormsby, A H

    2003-11-01

    Progression of cutaneous squamous neoplasms from actinic keratosis (AK) to Bowen's disease (BD; squamous cell carcinoma in situ) has important implications for clinical management and treatment, thus requiring accurate diagnosis. p16INK4a is a cell cycle regulatory tumour suppressor protein that negatively regulates D-type cyclins in the G1 cell cycle phase via intimate interplay with the retinoblastoma gene. Expression of a paraffin-reactive p16INK4a marker has recently been shown to increase in cervical squamous neoplasms as lesions progress from low-grade dysplasia to squamous cell carcinoma in situ. p16INK4a expression in the progression of squamous cutaneous neoplasia, however, has not been evaluated. To evaluate p16INK4a expression in the progression of squamous cutaneous neoplasia. Biopsies of 203 squamous cutaneous neoplasms with unequivocal features of AK (n = 87) and BD (n = 116) as well as a benign squamous control group (verruca vulgaris: n = 10; seborrhoeic keratosis: n = 11; scar tissue: n = 8) obtained between January and December 2001 at Henry Ford Hospital (Detroit, MI, U.S.A.) were immunostained for p16INK4a (Dako; clone E6H4; dilution 1 : 50) using large core (1.5 mm) tissue microarray analysis. Nuclear/cytoplasmic immunoreactivity in > 10% of neoplastic cells was considered positive. Of 203 cases, 166 (81.8%) were interpretable (AK 59; BD 107). Mean patient age was 71.0 years (range 33-93); 57% were male. Sites of involvement were: head and extremities 75.9%, trunk/buttocks 21.7%, genital region 2.4%. p16INK4a immunostaining was positive in 90 of 107 (84.1%) BD cases, four of 59 (6.8%) AK cases and none of 29 benign squamous controls. The sensitivity and specificity of p16INK4a for a diagnosis of BD (vs. benign squamous controls/AK) was 84.1% and 95.5%, respectively (P < 0.0001, Fisher's exact test, two-sided). p16INK4a is a sensitive and specific marker for distinguishing BD from AK/benign squamous cutaneous lesions and may be helpful as an

  7. p16(INK4a) /Ki-67 co-expression specifically identifies transformed cells in the head and neck region.

    PubMed

    Prigge, Elena-Sophie; Toth, Csaba; Dyckhoff, Gerhard; Wagner, Steffen; Müller, Franziska; Wittekindt, Claus; Freier, Kolja; Plinkert, Peter; Hoffmann, Jürgen; Vinokurova, Svetlana; Klussmann, Jens Peter; von Knebel Doeberitz, Magnus; Reuschenbach, Miriam

    2015-04-01

    p16(INK4a) immunohistochemical overexpression is an overall reliable surrogate marker of human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCC). However, cases of ambiguous p16(INK4a) overexpression are regularly detected in the head and neck: p16(INK4a) expression can be observed in non-malignant tissue, such as tonsillar crypt epithelium and a proportion of branchial cleft cysts. Additionally, diverse patterns of p16(INK4) expression can complicate interpretation of "p16(INK4a) -positivity". These aspects impede the unrestricted application of p16(INK4a) as a diagnostic marker in the head and neck. We hypothesized that combined detection of p16(INK4a) and the proliferation marker Ki-67 could support clarification of ambiguous p16(INK4a) expression in the head and neck by specifically indicating p16(INK4a) -expressing cells with proliferative activity. p16(INK4a) /Ki-67 co-expression in a combined staining procedure was correlated to distinct p16(INK4a) expression patterns and HPV status (HPV DNA followed by E6*I oncogene mRNA detection) in 147 HNSCC and 50 non-malignant head and neck samples. p16(INK4a) /Ki-67 co-expression only occurred in transformed cells of the head and neck. Co-expression was never detected in non-transformed cells. Combined p16(INK4a) /Ki-67 expression was stringently associated with a diffuse p16(INK4a) expression pattern. All HPV oncogene-expressing HNSCC showed p16(INK4a) /Ki-67 co-expression. We demonstrate that p16(INK4a) /Ki-67 co-expression occurs exclusively in transformed cells of the head and neck. Our findings indicate a substantial impact of combined p16(INK4a) /Ki-67 expression in the assessment of ambiguous p16(INK4a) expression in the head and neck by specifically identifying p16(INK4a) -expressing cells with proliferative activity. This property will be of considerable significance for head and neck histo- and cytopathology. © 2014 UICC.

  8. Naturally occurring p16Ink4a-positive cells shorten healthy lifespan

    PubMed Central

    Baker, Darren J.; Childs, Bennett G.; Durik, Matej; Wijers, Melinde E.; Sieben, Cynthia J.; Zhong, Jian; Saltness, Rachel; Jeganathan, Karthik B.; Versoza, Grace C.; Pezeshki, Abdul-Mohammad; Khazaie, Khashayarsha; Miller, Jordan D.; van Deursen, Jan M.

    2016-01-01

    Cellular senescence, a stress-induced irreversible growth arrest often characterized by p16Ink4a expression and a distinctive secretory phenotype, prevents the proliferation of preneoplastic cells and has beneficial roles in tissue remodelling during embryogenesis and wound healing. Senescent cells accumulate in various tissues and organs over time and have been speculated to play a role in aging. To explore the physiological relevance and consequences of naturally occurring senescent cells, we used a previously established transgene, INK-ATTAC, to induce apoptosis in p16Ink4a-expressing cells of wild-type mice by injection of AP20187 twice a week starting at one year of age. Here we show that compared to vehicle alone, AP20187 treatment extended median lifespan in both male and female mice of two distinct genetic backgrounds. Clearance of p16Ink4a-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart and fat, where clearance preserved the functionality of glomeruli, cardio-protective KATP channels, and adipocytes, respectively. Thus, p16Ink4a-positive cells that accumulate during adulthood negatively influence lifespan and promote age-dependent changes in multiple organs, and their therapeutic removal may be an attractive approach to extend healthy lifespan. PMID:26840489

  9. CREG1 enhances p16(INK4a) -induced cellular senescence.

    PubMed

    Moolmuang, Benchamart; Tainsky, Michael A

    2011-02-01

    Cellular senescence is an irreversible growth arrest that is activated in normal cells upon shortening of telomere and other cellular stresses. Bypassing cellular senescence is a necessary step for cells to become immortal during oncogenic transformation. During the spontaneous immortalization of Li-Fraumeni Syndrome (LFS) fibroblasts, we found that CREG1 (Cellular Repressor of E1A-stimulated Genes 1) expression was decreased during immortalization and increased in senescence. Moreover, we found that repression of CREG1 expression occurs via an epigenetic mechanism, promoter DNA methylation. Ectopic expression of CREG1 in the immortal LFS cell lines decreases cell proliferation but does not directly induce senescence. We confirmed this in osteosarcoma and fibrosarcoma cancer cell lines, cancers commonly seen in Li-Fraumeni Syndrome. In addition, we found that p16 (INK4a) is also downregulated in immortal cells and that coexpression of CREG1 and p16 (INK4a) , an inhibitor of CDK4/6 and Rb phosphorylation, has a greater effect than either CREG1 and p16 (INK4a) alone to reduce cell growth, induce cell cycle arrest and cellular senescence in immortal LFS fibroblasts, osteosarcoma and fibrosarcoma cell lines. Moreover, cooperation of CREG1 and p16 (INK4a) inhibits the expression of cyclin A and cyclin B by inhibiting promoter activity thereby decreasing mRNA and protein levels; these proteins are required for S-phase entry and G2/M transition. In conclusion, this is the first evidence to demonstrate that CREG1 enhances p16 (INK4a) -induced senescence by transcriptional repression of cell cycle-regulated genes.

  10. Prognostic Value of Overexpressed p16INK4a in Vulvar Cancer: A Meta-Analysis

    PubMed Central

    Cao, Hanyu; Wang, Si; Zhang, Zhenyu; Lou, Jiangyan

    2016-01-01

    Objective This study aimed to examine the prognostic value of overexpressed p16INK4a in vulvar cancer. Although the tumor suppressor p16INK4a has been shown to be of prognostic value in a wide variety of cancers and precancerous lesions, its role in the vulvar cancer is still unclear. Methods All publications in English language on the association between p16INK4a and clinicopathological features of vulvar cancer were searched from Pubmed, Embase, and Web of Science, and those in Chinese language were identified manually and online from the China National Knowledge Infrastructure. Strict inclusion and exclusion criteria were followed. Odds ratios(ORs) or risk ratios(RRs) with 95% confidence intervals(CIs) were pooled to assess the strength of association. Publication bias was estimated using funnel plots and the Egger’s regression test. Results A total of 17 studies with 2309 patients were included. The p16INK4a overexpression was found to correlate significantly with the lower International Federation of Gynecology and Obstetrics stage(I+II vs III+IV; OR = 0.60,95%CI:0.41–0.86,P = 0.006),negative lymph node metastasis(negative vs positive; OR = 0.61,95%CI:0.39–0.95,P = 0.029),patient’s age<55(OR = 0.54,95%CI:0.31–0.96,P = 0.034),human papillomavirus–positive status(OR = 0.01,95%CI:0.00–0.11,P<0.001),and higher overall survival(RR = 0.53,95%CI = 0.35–0.80,P = 0.003). Conclusion The p16INK4a might be associated with a higher survival and indicates better prognosis of vulvar cancer. PMID:27031618

  11. p16Ink4a Interferes with Abelson Virus Transformation by Enhancing Apoptosis

    PubMed Central

    Sachs, Zohar; Sharpless, Norman E.; DePinho, Ronald A.; Rosenberg, Naomi

    2004-01-01

    Pre-B-cell transformation by Abelson virus (Ab-MLV) is a multistep process in which primary transformants are stimulated to proliferate but subsequently undergo crisis, a period of erratic growth marked by high levels of apoptosis. Inactivation of the p53 tumor suppressor pathway is an important step in this process and can be accomplished by mutation of p53 or down-modulation of p19Arf, a p53 regulatory protein. Consistent with these data, pre-B cells from either p53 or Ink4a/Arf null mice bypass crisis. However, the Ink4a/Arf locus encodes both p19Arf and a second tumor suppressor, p16Ink4a, that blocks cell cycle progression by inhibiting Cdk4/6. To determine if p16Ink4a plays a role in Ab-MLV transformation, primary transformants derived from Arf−/− and p16Ink4a−/− mice were compared. A fraction of those derived from Arf−/− animals underwent crisis, and even though all p16Ink4a−/− primary transformants experienced crisis, these cells became established more readily than cells derived from +/+ mice. Analyses of Ink4a/Arf−/− cells infected with a virus that expresses both v-Abl and p16Ink4a revealed that p16Ink4a expression does not alter cell cycle profiles but does increase the level of apoptosis in primary transformants. These results indicate that both products of the Ink4a/Arf locus influence Ab-MLV transformation and reveal that in addition to its well-recognized effects on the cell cycle, p16Ink4a can suppress transformation by inducing apoptosis. PMID:15016851

  12. 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.

  13. Sustained p16(INK4a) expression is required to prevent IR-induced tumorigenesis in mice.

    PubMed

    Palacio, L; Krishnan, V; Le, N L O; Sharpless, N E; Beauséjour, C M

    2017-03-02

    Exposure of murine and human tissues to ionizing radiation (IR) induces the expression of p16(INK4a), a tumor suppressor gene and senescence/aging biomarker. Increased p16(INK4a) expression is often delayed several weeks post exposure to IR. In this context, it remains unclear if it occurs to suppress aberrant cellular growth of potentially transformed cells or is simply a result of IR-induced loss of tissue homeostasis. To address this question, we used a conditional p16(INK4a) null mouse model and determined the impact of p16(INK4a) inactivation long-term post exposure to IR. We found that, in vitro, bone marrow stromal cells exposed to IR enter DNA replication following p16(INK4a) inactivation. However, these cells did not resume growth; instead, they mostly underwent cell cycle arrest in G2. Similarly, delayed inactivation of p16(INK4a) in mice several weeks post exposure to IR resulted in increased BrdU incorporation and cancer incidence. In fact, we found that the onset of tumorigenesis was similar whether p16(INK4a) was inactivated before or after exposure to IR. Overall, our results suggest that IR-induced p16(INK4a) dependent growth arrest is reversible in mice and that sustained p16(INK4a) expression is necessary to protect against tumorigenesis.

  14. 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

  15. Regulatory mechanisms of tumor suppressor P16(INK4A) and their relevance to cancer.

    PubMed

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

    2011-06-28

    P16(INK4A) (also known as P16 and MTS1), a protein consisting exclusively of four ankyrin repeats, is recognized as a tumor suppressor mainly because of the prevalence of genetic inactivation of the p16(INK4A) (or CDKN2A) gene in virtually all types of human cancers. However, it has also been shown that an elevated level of expression (upregulation) 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 for the structure-function relationship of P16 and for human cancers.

  16. Management of HPV-related cervical disease: role of p16INK4a immunochemistry. Review of the literature.

    PubMed

    Savone, Delia; Carrone, Angela; Riganelli, Lucia; Merlino, Lucia; Mancino, Pasquale; Benedetti Panici, Pierluigi

    2016-10-13

    This systematic review of 43 studies aims to evaluate the absolute and relative sensitivity and specificity of p16INK4a with regard to uterine cervix lesions, describing innovations and techniques for the detection of high-grade cervical dysplasia and allowing correct treatment. Studies were identified in the PubMed database up to March 2015. The keywords hrHPV, p16INK4a gene, and uterine cervical disease (MeSH terms) were used. Only English-language articles were included. We considered retrospective and prospective studies that assessed p16INK4a or p16INK4a/Ki67 staining, with or without HPV-DNA testing (HC2/PCR) as a comparator test, in cytological/histological specimens for which the diagnosis of ASCUS, LSIL or HSIL was verified with a reference standard. The primary outcome for cervical lesions was evaluation of the absolute p16INK4a immunoreactivity; the secondary outcome was evaluation of the relative p16INK4a immunoreactivity versus HPV testing in those studies where comparator tests were available. p16INK4a was more specific than HPV-DNA test (median values of 56.1% vs. 52.25% in CIN grade ≥2 lesions; 82.5% vs. 53% in negative and CIN grade ≥1 lesions). The main limitation of this study is linked to both qualitative and quantitative p16INK4a levels of expression, while the second limitation is the lack of standardized scales. p16INK4a and HPV-DNA used together increased the sensitivity and negative predictive value for CIN detection. p16INK4a can be considered a biomarker of CIN2 or CIN3, indicating a high risk of relapse or evolution to invasive carcinoma. Also p16INK4a-negative CIN should be considered and further research should be performed.

  17. Development of Novel p16INK4a Mimetics as Anticancer Therapy

    DTIC Science & Technology

    2015-10-01

    derived from p16 INK4a that inhibit the activity of CDK4/6 and identify stabilized peptides that inhibit CDK4/6. The pharmacophore(s) of mutated peptides...DAAREGFLATLVVLHRAGAR-NH2). In addition, the native sequences were each linked to a protein transduction domain (TAT) of the HIV-1 virus ...Immunoblotting. Cells were removed from the plates by trypsinization, washed in phosphate- buffered saline (PBS) and lysed in cold lysis buffer

  18. p16INK4A expression in squamous cell carcinomas of the vagina and the vulva in Tunisian women.

    PubMed

    Missaoui, Nabiha; Abdelkarim, Soumaya Ben; Mokni, Moncef; Hmissa, Sihem

    2014-01-01

    The role of p16INK4A expression in uterine cervix cancer is well established. In the remaining female lower genital tract cancers, the importance of p16INK4A up-regulation is less clear. In our study, we analyzed the role of p16INK4A expression and HPV infection in carcinomas of the vulva and the vagina in Tunisian women. We conducted a retrospective study of 30 carcinomas including 15 vulvar squamous cell carcinomas (SCCs) and 15 vaginal SCCs. Immunohistochemistry was used to determine p16INK4A expression. HPV detection and typing was by in situ hybridization. p16INK4A expression was detected in 86.7% of vaginal SCCs with a strong and diffuse immunostaining in 60% of cases, and also in 73.3% of vulvar SCCs with focal immunoreactivity in 53.3% The association between p16INK4A expression and HPV infection was significant in vaginal SCCs (p=0.001) but not vulvar SCCs (p>0.05). p16INK4A expression could be used as a useful marker for HPV positivity in vaginal SCCs similar to that described in uterine cervix cancers. However, our data support the presence of 2 different mechanisms for p16INK4A expression in HPV-related and HPV-unrelated vulvar carcinomas.

  19. The meaning of p16(ink4a) expression in tumors: functional significance, clinical associations and future developments.

    PubMed

    Witkiewicz, Agnieszka K; Knudsen, Karen E; Dicker, Adam P; Knudsen, Erik S

    2011-08-01

    The CDKN2A gene is a tumor suppressor that encodes the CDK4/6 inhibitor p16(ink4a). Loss of this tumor suppressor contributes to the bypass of critical senescent signals and is associated with progression to malignant disease. However, the high-level expression of p16(ink4a) in tumors is associated with aggressive subtypes of disease, and in certain clinical settings elevated p16(ink4a) expression is an important determinant for disease prognosis and therapeutic response. These seemingly contradictory facets of p16(ink4a) expression have lead to confusion related to the meaning of this tumor suppression in tumor pathobiology. As reviewed here, the alternative expression of p16(ink4a) represents an ideal marker for considering RB-pathway function, tumor heterogeneity, and novel means for directing therapy.

  20. Association of CDKN2A/p16INK4A with human head and neck keratinocyte replicative senescence: relationship of dysfunction to immortality and neoplasia.

    PubMed

    Loughran, O; Malliri, A; Owens, D; Gallimore, P H; Stanley, M A; Ozanne, B; Frame, M C; Parkinson, E K

    1996-08-01

    We have previously suggested that a gene mapping to chromosome 9p21 could contribute to replicative senescence and suppress cullular immortality in squamous neoplasia. Two candidate genes, the cyclin D1/cyclindependent kinase inhibitors CDKN2A/p16INK4A (p16) and CDKN2B/p15INK4B (p15) have now been identified in this region and we show here that p16 is upregulated when normal human keratinocytes undergo replicative senescence but not when they undergo differentiation. Furthermore, all of 19 immortal neoplastic keratinocyte head and neck lines, including nine showing loss of heterozygosity (LOH) at 9p21, showed undetectable p16 expression, whereas five of six senscent neoplastic cultures showed normal levels of expression. The retinoblastoma protein (pRb) appeared functional in all the cell lines and cultures examined. The mechanism of p16 inactivation appeared to be transcriptional silencing in 10 of 18 lines and homozygous deletions in the rest. Treatment of two of the immortal cell lines which had transcriptionally silent wild type p16 genes with 5aza-2deoxycytidine resulted in the re-expression of p16, thus implicating DNA methylation as one mechanism of transcriptional silencing in the immortal SCC-HN lines. We observed no cases of p16 point mutation. In contrast, the p15 gene was rarely transcriptionally silent and was not deleted in any of the cell lines which showed p16 deletions. Our results show that p16 dysfunction correlates strongly with keratinocyte immortalisation but less strongly with the stage of tumour progression. P16 dysfunction was not related to the neoplastic state or the length of time spent in vitro. The results also suggest that p16 but not p15 is involved in the keratinocyte replicative senescence programme. However, two neoplastic cell cultures which lacked p16 expression were still mortal, suggesting that the loss of p16 is a necessary but insufficient condition for human keratinocyte immortality.

  1. Role of p14ARF and p15INK4B promoter methylation in patients with lung cancer: a systematic meta-analysis

    PubMed Central

    Yang, Xinmei; Yang, Lei; Dai, Wanrong; Ye, Bo

    2016-01-01

    Background The cyclin-dependent kinase inhibitors p14ARF and p15INK4B are tumor suppressor genes that have been reported to be silenced through promoter methylation in many human cancers. However, the strength of association between p14ARF or p15INK4B promoter methylation and lung cancer remains unclear. Thus, we first determined whether p14ARF and p15INK4B promoter methylation played a key role in the carcinogenesis of lung cancer. Methods Eligible studies were selected from the online electronic databases. The pooled odds ratios or hazard ratios and 95% confidence intervals were calculated and summarized. Results Finally, 12 studies with 625 lung cancer samples and 488 nontumor samples were included under the fixed-effects model. The pooled odds ratio showed that p14ARF promoter methylation was observed to be significantly higher in non-small-cell lung cancer (NSCLC) than in nontumor samples (P<0.001). No significant correlation was found between p15INK4B promoter methylation and lung cancer (P=0.27). Subgroup analysis of ethnicity revealed that p14ARF promoter methylation was significantly related to the risk of NSCLC in Asian and Caucasian populations. Subgroup analysis of sample type demonstrated that p14ARF promoter methylation was correlated with the risk of NSCLC in tissue samples (P<0.001), but not in bronchoalveolar lavage fluid and blood samples. P14ARF promoter methylation from one study was not significantly correlated with overall survival of patients with NSCLC. Promoter methylation of p14ARF and p15INK4B was not correlated with clinicopathological characteristics, such as gender status, smoking status, tumor differentiation, and tumor stage (P>0.05). Conclusion Our findings suggested that p14ARF promoter methylation may play an important role in the carcinogenesis of lung cancer, but not p15INK4B promoter methylation. Promoter methylation of p14ARF and p15INK4B was not associated with clinicopathological parameters. However, more extensive large

  2. 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

  3. Hydrogen peroxide induces p16(INK4a) through an AUF1-dependent manner.

    PubMed

    Guo, Gai E; Ma, Li Wei; Jiang, Bin; Yi, Jie; Tong, Tan Jun; Wang, Wen Gong

    2010-04-01

    Elevation of p16(INK4a) has been described as an important mechanism for hydrogen peroxide (H2O2)-induced replicative senescence. However, the mechanisms underlying remain unknown. In this study, we demonstrate an important role of RNA-binding protein AUF1-mediated mRNA turnover in H2O2-induced p16(INK4a) expression. The induction of p16 by H2O2 was accompanied with declined cytoplasmic AUF1 level. Accordingly, exposure of cells to H2O2 remarkably reduced the binding of AUF1 to p16 3'UTR and increased the half-life of an EGFP-p16-3'UTR chimeric transcript. In AUF1-silenced cells, the effect of H2O2 on p16 induction was abolished. Furthermore, in cells co-transfected with vectors expressing AUF1s, treatment with H2O2 failed to significantly reduce the expression of AUF1 and subsequently elevate the levels of p16. Moreover, HeLa cells overexpressing AUF1s were resistant to H2O2-induced senescence. Our results indicate that AUF1 is critical for H2O2-induced p16 expression and cellular senescence. Copyright 2010 Wiley-Liss, Inc.

  4. Methylation and Immunoexpression of p16(INK4a) Tumor Suppressor Gene in Primary Breast Cancer Tissue and Their Quantitative p16(INK4a) Hypermethylation in Plasma by Real-Time PCR.

    PubMed

    Lee, Jae Jun; Ko, Eunkyung; Cho, Junhun; Park, Ha Young; Lee, Jeong Eon; Nam, Seok Jin; Kim, Duk-Hwan; Cho, Eun Yoon

    2012-12-01

    The p16(INK4a) gene methylation has been reported to be a major tumorigenic mechanism. We evaluated the methylation status of the p16(INK4a) genes in 231 invasive breast cancer and 90 intraductal carcinoma specimens using a methylation-specific polymerase chain reaction and p16 protein expression using immunohistochemistry. The quantity of cell-free methylated p16(INK4a) DNA in the plasma samples of 200 patients with invasive breast cancer was also examined using a fluorescence-based real-time polymerase chain reaction assay. The frequencies of p16(INK4a) methylation in invasive and intraductal tumors were 52.8% (122/231) and 57.8% (52/90), respectively. The p16 protein was overexpressed in 145 of the 231 invasive carcinomas (62.8%) and 63 of the 90 intraductal carcinomas (70%). High p16 expression in invasive carcinomas correlated significantly with a high histologic grade, a negative estrogen receptor and progesterone receptor status, p53 immunoreactivity and high Ki-67 expression with immunohistochemistry. In addition, the methylation index of p16(INK4a) was significantly higher in the cancer patients than the normal controls (p<0.001). High p16 immunoreactivity correlated with a loss of differentiation in breast carcinomas and high frequency of p16(INK4a) promoter methylation in both invasive and intraductal carcinomas, suggesting it may be involved in the pathogenesis of breast cancer.

  5. p16(INK4A) enhances the transcriptional and the apoptotic functions of p53 through DNA-dependent interaction.

    PubMed

    Al-Khalaf, Huda H; Nallar, Shreeram C; Kalvakolanu, Dhananjaya V; Aboussekhra, Abdelilah

    2017-02-20

    p16(INK4A) and p53 are two important tumor suppressor proteins that play essential roles during cell proliferation and aging through regulating the expression of several genes. Here, we report that p16(INK4A) and p53 co-regulate a plethora of transcripts. Furthermore, both proteins colocalize in the nucleus of human primary skin fibroblasts and breast luminal cells, and form a heteromer whose level increases in response to genotoxic stress as well as aging of human fibroblasts and various mouse organs. CDK4 is also present in this heteromeric complex, which is formed only in the presence of DNA both in vitro using pure recombinant proteins and in vivo. We have also shown that p16(INK4A) enhances the binding efficiency of p53 to its cognate sequence presents in the CDKN1A promoter in vitro, and both proteins are present at the promoters of CDKN1A and BAX in vivo. Importantly, the fourth ankyrin repeat of p16(INK4A) and the C-terminal domain of p53 were necessary for the physical association between these two proteins. The physiologic importance of this association was revealed by the inability of cancer-associated p16(INK4A) mutants to interact with p53 and to transactivate the expression of its major targets CDKN1A and BAX in the p16-defective U2OS cells expressing either wild-type or mutated p16(INK4A) . Furthermore, the association between p16(INK4A) and p53 was capital for their nuclear colocalization, the X-ray-dependent induction of p21 and Bax proteins as well as the induction of apoptosis in various types of cells. Together, these results show DNA-dependent physical interaction between p16(INK4A) and p53.

  6. Dietary folate is associated with p16(INK4A) methylation in head and neck squamous cell carcinoma.

    PubMed

    Kraunz, Kim S; Hsiung, Debra; McClean, Michael D; Liu, Mei; Osanyingbemi, Joyce; Nelson, Heather H; Kelsey, Karl T

    2006-10-01

    Inactivation of the p16(INK4A) (CDKN2A) gene in the Rb pathway is among the most common somatic alterations observed in tobacco-related solid tumors, including head and neck squamous cell carcinoma (HNSCC). In addition, a low folate diet is an important risk factor for HNSCC. Decreased dietary folate in an animal model of hepatocellular carcinoma has been associated with the induction of epigenetic silencing of the p16(INK4A) gene. In an ongoing population-based study of HNSCC, we sought to extend this observation to human disease testing the hypothesis that p16(INK4A) methylation is associated with decreased dietary folate. We also investigated the association of methylation silencing with functional polymorphisms in the folate metabolism enzyme methylene tetrahydrofolate reductase (MTHFR). In 169 HNSCCs, the odds ratio for p16(INK4A) methylation among those with low dietary folate intake was 2.3 (95% CI = 1.1-4.8) when compared with those with high folate intake. Furthermore, this increased risk for epigenetic silencing at p16(INK4A) was modified by the MTHFR alleles previously associated with diminished serum folate levels. Hence, in HNSCC low dietary intake of folate is associated with p16(INK4A) methylation, and this relationship is modified by the MTHFR genotype. Our data provides important evidence for a mechanism of action of folate deficiency in cancer. Copyright 2006 Wiley-Liss, Inc.

  7. P16(INK 4a) and Ki-67 expression in human papilloma virus-related head and neck mucosal lesions.

    PubMed

    Gültekin, Sibel Elif; Sengüven, Burcu; Klussmann, Jens Peter; Dienes, Hans Peter

    2015-03-01

    Human papilloma virus (HPV) is postulated as a risk factor in the etiology of some specific mucosal pathologies in the head and neck regions. Despite the frequent use of p16(INK4a) as a surrogate marker for HPV-infection, there is still controversy with respect to its reliability. This study has been undertaken to assess the potential role of p16(INK 4a) and Ki-67 expression in HPV-related lesions. The study was conducted on 71 specimens of oral, tonsillar and laryngeal lesions which comprised 25 dysplasia and 46 papilloma specimens. Specimens were immunohistochemically stained for p16(INK4A) and Ki-67 proteins. HPV DNA was determined by one step multiplex polymerase chain reaction. HPV DNA was detected in 33.8% of all lesions. Tonsil and larynx lesions showed significant differences with oral lesions for HPV positivity (p < 0.001). p16(INK 4a) over-expression was seen in 56.5% of papilloma and 60% of dysplasia specimens. HPV status showed a positive correlation with p16(INK 4a) expression in tonsillar dysplasias (p < 0.001). p16(INK 4a) expression may have a value as a marker in high risk HPV induced dysplasias, but not in low risk infected lesions. The proliferation index is not related to HPV-induced lesions and may be evaluated as an independent marker in head and neck premalignant lesions.

  8. Pioglitazone promotes preadipocyte proliferation by downregulating p16(Ink4a).

    PubMed

    Hasan, Arif U; Ohmori, Koji; Hashimoto, Takeshi; Kamitori, Kazuyo; Hirata, Yuko; Ishihara, Yasuhiro; Okamoto, Naoko; Noma, Takahisa; Kosaka, Hiroaki; Tokuda, Masaaki; Kohno, Masakazu

    2011-07-29

    Pioglitazone, a synthetic ligand of peroxisome proliferator-activated receptor (PPAR)γ, causes preadipocyte proliferation through a mechanism which still remains elusive. Here, to address the mechanism, we investigated the effects of PPARγ and pioglitazone on the kinetics of cyclin-dependent kinase inhibitors, especially with p16(Ink4a) (p16) centered, by employing 3T3-L1 preadipocytes. Pioglitazone promoted preadipocyte proliferation by increasing S and G(2)/M cell-cycle entry, which was accompanied by decreased p16 mRNA expression. PPARγ overexpression along with the luciferase reporter assay confirmed that PPARγ was crucial for the downregulation of p16 mRNA transcription, and that the action was augmented by pioglitazone. Thus, pioglitazone exerted cell-cycle dependent promoting effect on preadipocyte proliferation, of which mechanisms include p16-downregulation through PPARγ. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. The protective effect of p16(INK4a) in oral cavity carcinomas: p16(Ink4A) dampens tumor invasion-integrated analysis of expression and kinomics pathways.

    PubMed

    Isayeva, Tatyana; Xu, Jie; Ragin, Camille; Dai, Qian; Cooper, Tiffiny; Carroll, William; Dayan, Dan; Vered, Marilena; Wenig, Bruce; Rosenthal, Eben; Grizzle, William; Anderson, Joshua; Willey, Christopher D; Yang, Eddy S; Brandwein-Gensler, Margaret

    2015-05-01

    A large body of evidence shows that p16(INK4a) overexpression predicts improved survival and increased radiosensitivity in HPV-mediated oropharyngeal squamous cell carcinomas.(OPSCC). Here we demonstrate that the presence of transcriptionally active HPV16 in oral cavity squamous cell carcinomas does not correlate with p16(INK4a) overexpression, enhanced local tumor immunity, or improved outcome. It is interesting that HPV-mediated oropharyngeal squamous cell carcinomas can be categorized as having a 'nonaggressive' invasion phenotype, whereas aggressive invasion phenotypes are more common in HPV-negative squamous cell carcinomas. We have developed primary cancer cell lines from resections with known pattern of invasion as determined by our validated risk model. Given that cell lines derived from HPV-mediated oropharyngeal squamous cell carcinomas are less invasive than their HPV-negative counterparts, we tested the hypothesis that viral oncoproteins E6, E7, and p16(INK4a) can affect tumor invasion. Here we demonstrate that p16(INK4a) overexpression in two cancer cell lines (UAB-3 and UAB-4), derived from oral cavity squamous cell carcinomas with the most aggressive invasive phenotype (worst pattern of invasion type 5 (WPOI-5)), dramatically decreases tumor invasiveness by altering expression of extracellular matrix remodeling genes. Pathway analysis integrating changes in RNA expression and kinase activities reveals different potential p16(INK4a)-sensitive pathways. Overexpressing p16(INK4a) in UAB-3 increases EGFR activity and increases MMP1 and MMP3 expression, possibly through STAT3 activation. Overexpressing p16(INK4a) in UAB-4 decreases PDGFR gene expression and reduces MMP1 and MMP3, possibly through STAT3 inactivation. Alternatively, ZAP70/Syk might increase MUC1 phosphorylation, leading to the observed decreased MMP1 expression.

  10. 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.

  11. 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

  12. CDKN2A/p16INK4a expression is associated with vascular progeria in chronic kidney disease

    PubMed Central

    Stenvinkel, Peter; Luttropp, Karin; McGuinness, Dagmara; Witasp, Anna; Rashid Qureshi, Abdul; Wernerson, Annika; Nordfors, Louise; Schalling, Martin; Ripsweden, Jonaz; Wennberg, Lars; Söderberg, Magnus; Bárány, Peter; Olauson, Hannes; Shiels, Paul G

    2017-01-01

    Patients with chronic kidney disease (CKD) display a progeric vascular phenotype linked to apoptosis, cellular senescence and osteogenic transformation. This has proven intractable to modelling appropriately in model organisms. We have therefore investigated this directly in man, using for the first time validated cellular biomarkers of ageing (CDKN2A/p16INK4a, SA-β-Gal) in arterial biopsies from 61 CKD patients undergoing living donor renal transplantation. We demonstrate that in the uremic milieu, increased arterial expression of CDKN2A/p16INK4a associated with vascular progeria in CKD, independently of chronological age. The arterial expression of CDKN2A/p16INK4a was significantly higher in patients with coronary calcification (p=0.01) and associated cardiovascular disease (CVD) (p=0.004). The correlation between CDKN2A/p16INK4a and media calcification was statistically significant (p=0.0003) after correction for chronological age. We further employed correlate expression of matrix Gla protein (MGP) and runt-related transcription factor 2 (RUNX2) as additional pathognomonic markers. Higher expression of CDKN2A/p16INK4a, RUNX2 and MGP were observed in arteries with severe media calcification. The number of p16INK4a and SA-β-Gal positive cells was higher in biopsies with severe media calcification. A strong inverse correlation was observed between CDKN2A/p16INK4a expression and carboxylated osteocalcin levels. Thus, impaired vitamin K mediated carboxylation may contribute to premature vascular senescence. PMID:28192277

  13. p16(INK4a) immunostaining as an alternative to histology review for reliable grading of cervical intraepithelial lesions.

    PubMed

    Dijkstra, Maaike G; Heideman, Daniëlle A M; de Roy, Sabine C; Rozendaal, Lawrence; Berkhof, Johannes; van Krimpen, Kees; van Groningen, Krijn; Snijders, Peter J F; Meijer, Chris J L M; van Kemenade, Folkert J

    2010-11-01

    Histomorphological grading of cervical intraepithelial neoplasia (CIN) is crucial for clinical management. CIN grading is however subjective and affected by substantial rates of discordance among pathologists, which may lead to overtreatment. To minimise this problem, a histology review of CIN lesions by a consensus panel of pathologists is often used. Diffuse strong p16(INK4a) immunostaining has been proposed to aid the identification of true high-grade cervical lesions (ie, CIN2/3). To assess the value of additional interpretation of p16(INK4a) immunostains for making a more reproducible diagnosis of CIN2/3 lesions. The authors used a series of 406 biopsies of cervical lesions, with known HPV status, stained for both H&E- and p16(INK4a). First, in a randomly selected set of 49 biopsies, we examined the effect of additional interpretation of p16(INK4a) immunostained slides, on the agreement of CIN diagnosis among three pathologists. Second, the full series of samples was used to assess the accuracy of p16(INK4a)-supported lesion grading by a single pathologist, by evaluating the degree of diagnostic agreement with the consensus diagnosis of expert pathologists based on H&E-stained sections only. The study shows that the interobserver agreement between three pathologists for the routine H&E-based diagnosis ranged from fair (weighted kappa 0.44 (95% CI 0.19 to 0.64)) to moderate (weighted kappa 0.66 (95% CI 0.47 to 0.79)). The concordance increased substantially for p16(INK4a)-supported grading (mean weighted kappa 0.80 (95% CI 0.66 to 0.89)). Furthermore, an almost perfect agreement was found between the p16(INK4a)-supported diagnosis of a single pathologist and the consensus diagnosis of an expert pathology panel (kappa 0.88 (95% CI 0.85 to 0.89)). These data demonstrate that additive use of p16(INK4a) immunohistochemistry significantly improves the accuracy of grading CIN lesions by a single pathologist, equalling an expert consensus diagnosis. Hence, the authors

  14. p16INK4a and p14ARF tumor suppressor genes are commonly inactivated in cutaneous squamous cell carcinoma.

    PubMed

    Brown, Victoria L; Harwood, Catherine A; Crook, Tim; Cronin, James G; Kelsell, David P; Proby, Charlotte M

    2004-05-01

    The p16(INK4a) and p14(ARF) tumor suppressor genes (TSGs) are encoded within the CDKN2A locus on chromosome 9p21 and function as cell cycle regulatory proteins in the p53 and RB pathways. Inactivation of these genes by genetic and epigenetic changes has been described in some human cancers, but their importance in cutaneous squamous cell carcinoma (SCC) has not been established. Our detailed examination of 40 cutaneous SCC revealed loss of heterozygosity of 9p21 markers in 32.5% of cases. Mutational analysis confirmed five point mutations in four of 40 SCCs. These mutations changed the amino acid sequence of p16(INK4a) in four tumors and p14(ARF) in three tumors. Promoter methylation of p16(INK4a) and p14(ARF) was detected in 13 of 36 (36%) and 16 of 38 (42%) cases, respectively. Absent protein expression was confirmed by immunohistochemistry in 13 of 16 (82%) of the tumors with biallelic inactivating events. Overall, the frequency of 9p21 alterations was 76% and for both p16(INK4a) and p14(ARF), promoter methylation is the commonest mechanism of gene inactivation. Alterations at this locus were significantly more common in tumors from immunocompetent compared with immunosuppressed individuals. These data confirm the importance of inactivation of p16(INK4a) and p14(ARF) TSGs in the pathogenesis of cutaneous SCCs.

  15. Examining how p16INK4a expression levels are linked to handgrip strength in the elderly

    PubMed Central

    Kao, Tung-Wei; Chen, Wei-Liang; Han, Der- Sheng; Huang, Ying-Hsin; Chen, Chi-Ling; Yang, Wei-Shiung

    2016-01-01

    Although many studies have shown that p16INK4a is more highly expressed in the human body during senescence, studies on its relevance to handgrip strength among old adults, are relatively sparse. We enrolled 205 community-dwelling old adults aged 65 years and older without specific medical conditions. Handgrip strength of the dominant hand was measured. Low handgrip strength was defined as the lowest quartile of handgrip strength among the participants. RNA was extracted from peripheral white blood cells. Use quantitative polymerase chain reaction to estimate the p16INK4a mRNA expression level. The average handgrip strength was 25.22 ± 8.98 kg, and gender difference was observed. In the linear regression model, the p16INK4a mRNA expression level was significantly negatively associated with handgrip strength in men but not in women. The β coefficient, representing the change of handgrip strength for each increment in the p16INK4a mRNA expression level, was −0.208 (p = 0.024) among old men. The negative association remained after additional covariates adjustment. In the multiple logistic regression model among old men, the odds ratio (OR) of low handgrip strength was 1.246 (p = 0.032). In this study, we observed the p16INK4a mRNA expression level was negative associated with handgrip strength among community-dwelling old men. PMID:27549351

  16. The Role of p16(INK4a) Pathway in Human Epidermal Stem Cell Self-Renewal, Aging and Cancer.

    PubMed

    D'Arcangelo, Daniela; Tinaburri, Lavinia; Dellambra, Elena

    2017-07-22

    The epidermis is a self-renewing tissue. The balance between proliferation and differentiation processes is tightly regulated to ensure the maintenance of the stem cell (SC) population in the epidermis during life. Aging and cancer may be considered related endpoints of accumulating damages within epidermal self-renewing compartment. p16(INK4a) is a potent inhibitor of the G1/S-phase transition of the cell cycle. p16(INK4a) governs the processes of SC self-renewal in several tissues and its deregulation may result in aging or tumor development. Keratinocytes are equipped with several epigenetic enzymes and transcription factors that shape the gene expression signatures of different epidermal layers and allow dynamic and coordinated expression changes to finely balance keratinocyte self-renewal and differentiation. These factors converge their activity in the basal layer to repress p16(INK4a) expression, protecting cells from senescence, and preserving epidermal homeostasis and regeneration. Several stress stimuli may activate p16(INK4a) expression that orchestrates cell cycle exit and senescence response. In the present review, we discuss the role of p16(INK4a) regulators in human epidermal SC self-renewal, aging and cancer.

  17. Status of p16(INK4a) and E-cadherin gene promoter methylation in Moroccan patients with cervical carcinoma.

    PubMed

    Attaleb, Mohammed; El hamadani, Wail; Khyatti, Meriem; Benbacer, Laila; Benchekroun, Nadia; Benider, Abdellatif; Amrani, Mariam; El Mzibri, Mohammed

    2009-01-01

    Aberrant methylation of tumor suppressor gene promoters has been extensively investigated in cervical cancer. Transcriptional silencing, as a main consequence of hypermethylation of CpG islands, is the predominant mechanism of p16(INK4a) and E-cadherin gene inactivation in malignant epithelial tumors. This study was conducted to evaluate the promoter methylation status of p16(INK4a) and E-cadherin genes in 22 specimens of cervical carcinomas, four cervical cancer cell lines (HeLa, SiHa, Caski, C33A), and 20 human papillomavirus negative specimens, obtained from normal cervical swabs, using the methylation-specific PCR approach. Hypermethylation of the 5' CpG island of the p16(INK4a) and E-cadherin genes were found in 13 (59.1%) and 10 (45.5%) of 22 cervical cancer samples, respectively. Furthermore, our findings did not show any correlation between promoter methylation of p16(INK4a) and E-cadherin genes and clinicopathological parameters, including HPV infection, phenotypic distribution, and stage of the disease. However, hypermethylation of E-cadherin gene promoter appears to be age related in cervical cancer, whereas the frequency of aberrant methylation of p16(INK4a) gene promoter is unchanged according to the age of patients. Thus, caution must be made to use these markers in the diagnosis of cervical cancer. However, dietary or pharmaceutical agents that can inhibit these epigenetic events may prevent or delay the development of cervical cancer.

  18. Promoter methylation and loss of p16(INK4a) gene expression in head and neck cancer.

    PubMed

    Demokan, Semra; Chuang, Alice; Suoğlu, Yusufhan; Ulusan, Murat; Yalnız, Zubeyde; Califano, Joseph A; Dalay, Nejat

    2012-10-01

    Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. In this study we aimed to evaluate aberrant p16(INK4a) gene promoter methylation in patients with head and neck cancer. Methylation of the gene was investigated by bisulfite modification/methylation-specific polymerase chain reaction and gene expression levels were analyzed by quantitative reverse transcription-polymerase chain reaction in tumors and matched normal tissue samples from Turkish patients with head and neck cancer. The promoter region of the p16(INK4a) gene was methylated in 67.5% and 28.6% of the primary tumors and the corresponding normal tissue, respectively. This difference was highly significant. In concordance, p16(INK4a) gene expression was downregulated in 67.5% of the tumor samples. Methylation and the absence of expression in the tumors were observed in 48% of the patients. Our data indicate that methylation of the p16(INK4a) gene is a frequent event in primary head and neck cancer and that it plays a major role in the silencing of p16(INK4a) gene expression during tumor development. Copyright © 2011 Wiley Periodicals, Inc.

  19. 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.

  20. Expression of p16(Ink4a) in mixed squamous cell and glandular papilloma of the lung.

    PubMed

    Miyoshi, Ryo; Menju, Toshi; Yoshizawa, Akihiko; Date, Hiroshi

    2017-06-01

    Mixed squamous cell and glandular papilloma (mixed papilloma) of the lung is an extremely rare neoplasm, with only 21 cases reported in the English literature. Although the expression of p16(Ink4a) has been analyzed in only two cases of mixed papilloma, they were negative for p16(Ink4a) . Therefore, the significance of p16(Ink4a) overexpression in mixed papilloma remains unclear. This is the first case of mixed papilloma with positive p16(Ink4a) expression in a 72-year-old male smoker. The 20 mm sized tumor was histologically diagnosed as mixed papilloma following right upper lobectomy. Immunohistochemically, cytokeratin 5 and p40 positivity was predominant in basal cells of the glandular component and squamous cells, while thyroid transcription factor-1, p53, and Ki-67 were focally positive. Both glandular and squamous components were diffusely positive for p16(Ink4a) . This finding could be important to clarify the pathogenesis and biology of mixed papilloma. © 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

  1. Radionuclides in cigarettes may lead to carcinogenesis via p16(INK4a) inactivation.

    PubMed

    Prueitt, Robyn L; Goodman, Julie E; Valberg, Peter A

    2009-02-01

    It is widely accepted that tobacco smoke is responsible for the vast majority of lung cancers worldwide. There are many known and suspected carcinogens present in cigarette smoke, including alpha-emitting radioisotopes. Epidemiologic studies have shown that increased lung cancer risk is associated with exposure to ionizing radiation, and it is estimated that the majority of smoking-induced lung cancers may be at least partly attributable to the inhaled and deposited radiation dose from radioisotopes in the cigarette smoke itself. Recent research shows that silencing of the tumor suppressor gene p16(INK4a) (p16) by promoter methylation plays a role in smoking-related lung cancer. Inactivation of p16 has also been associated with lung cancer incidence in radiation-exposed workers, suggesting that radionuclides in cigarette smoke may be acting with other compounds to cause smoking-induced lung cancer. We evaluated the mechanism of ionizing radiation as an accepted cause of lung cancer in terms of its dose from tobacco smoke and silencing of p16. Because both radiation and cigarette smoking are associated with inactivation of p16, and p16 inactivation has been shown to play a major role in carcinogenesis, ionizing radiation from cigarette smoke likely plays a role in lung cancer risk. How large a role it plays, relative to chemical carcinogens and other modes of action, remains to be elucidated.

  2. 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

  3. 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.

  4. Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation.

    PubMed Central

    Asada, M; Yamada, T; Ichijo, H; Delia, D; Miyazono, K; Fukumuro, K; Mizutani, S

    1999-01-01

    p21(Cip1/WAF1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. The cell-cycle inhibitory activity of p21(Cip1/WAF1) is correlated with its nuclear localization. Here, we report a novel cytoplasmic localization of p21(Cip1/WAF1) in peripheral blood monocytes (PBMs) and in U937 cells undergoing monocytic differentiation by in vitro treatment with vitamin D3 or ectopic expression of p21(Cip1/WAF1), and analyze the biological consequences of this cytoplasmic expression. U937 cells which exhibit nuclear p21(Cip1/WAF1) demonstrated G1 cell-cycle arrest and subsequently differentiated into monocytes. The latter event was associated with a cytoplasmic expression of nuclear p21(Cip1/WAF1), concomitantly with a resistance to various apoptogenic stimuli. Biochemical analysis showed that cytoplasmic p21(Cip1/WAF1) forms a complex with the apoptosis signal-regulating kinase 1 (ASK1) and inhibits stress-activated MAP kinase cascade. Expression of a deletion mutant of p21(Cip1/WAF1) lacking the nuclear localization signal (DeltaNLS-p21) did not induce cell cycle arrest nor monocytic differentiation, but led to an apoptosis-resistant phenotype, mediated by binding to and inhibition of the stress-activated ASK1 activity. Thus, cytoplasmic p21(Cip1/WAF1) itself acted as an inhibitor of apoptosis. Our findings highlight the different functional roles of p21(Cip1/WAF1), which are determined by its intracellular distribution and are dependent on the stage of differentiation. PMID:10064589

  5. Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney.

    PubMed

    Melk, Anette; Schmidt, Bernhard M W; Takeuchi, Oki; Sawitzki, Birgit; Rayner, David C; Halloran, Philip F

    2004-02-01

    Somatic cells in vitro have a finite life expectancy before entering a state of senescence. If this state has an in vivo counterpart, it could contribute to organ aging. We have previously shown that human kidney cortex displays telomere shortening with age. In the present study, we evaluated the relationship between renal age in humans and a number of phenomena associated with cellular senescence in vitro. Human kidney specimens were obtained at 8 weeks to 88 years of age and were assessed for changes related to aging. We found that human kidneys expressed relatively constant levels of mRNAs for genes potentially related to senescence. Among the candidate genes surveyed, the cell cycle regulator p16INK4a emerged with the strongest association with renal aging for both mRNA and protein expression. Proliferation as measured by Ki-67 expression was inversely correlated with p16INK4a expression, compatible with a role for p16INK4a as an irreversible cell cycle inhibitor. Cyclooxygenase 1 and 2 (COX-1 and COX-2) mRNA expression was elevated in older kidneys, associated with increased protein expression. Comparison of gene expression with age-related histologic changes revealed that glomerulosclerosis correlated with p16INK4a and p53, whereas interstitial fibrosis and tubular atrophy were associated with p16INK4a, p53, COX-1, transforming growth factor-beta 1 (TGF-beta 1), and heat shock protein A5 (HSPA5). We conclude that some changes observed in cellular senescence in vitro do occur in human kidney with age, particularly in the renal cortex, in some cases correlating with histologic features. P16INK4a emerged with the most consistent correlations with age and histologic changes and inversely correlated with cell replication.

  6. p16 (INK4a) has clinicopathological and prognostic impact on oropharynx and larynx squamous cell carcinoma

    PubMed Central

    Silva, S.D.; Nonogaki, S.; Soares, F.A.; Kowalski, L.P.

    2012-01-01

    CDKN2A encodes proteins such as p16 (INK4a), which negatively regulate the cell-cycle. Molecular genetic studies have revealed that deletions in CDKN2A occur frequently in cancer. Although p16 (INK4a) may be involved in tumor progression, the clinical impact and prognostic implications in head and neck squamous cell carcinoma (HNSCC) are controversial. The objective of this study was to evaluate the frequency of the immunohistochemical expression of p16 (INK4a) in 40 oropharynx and 35 larynx from HNSCC patients treated in a single institution and followed-up at least for 10 years in order to explore potential associations with clinicopathological outcomes and prognostic implications. Forty cases (53.3%) were positive for p16 (INK4a) and this expression was more intense in non-smoking patients (P = 0.050), whose tumors showed negative vascular embolization (P = 0.018), negative lymphatic permeation (P = 0.002), and clear surgical margins (P = 0.050). Importantly, on the basis of negative p16 (INK4a) expression, it was possible to predict a probability of lower survival (P = 0.055) as well as tumors presenting lymph node metastasis (P = 0.050) and capsular rupture (P = 0.0010). Furthermore, increased risk of recurrence was observed in tumors presenting capsular rupture (P = 0.0083). Taken together, the alteration in p16 (INK4a) appears to be a common event in patients with oropharynx and larynx squamous cell carcinoma and the negative expression of this protein correlated with poor prognosis. PMID:22948376

  7. γ-Tocotrienol prevents cell cycle arrest in aged human fibroblast cells through p16(INK4a) pathway.

    PubMed

    Zainuddin, Azalina; Chua, Kien-Hui; Tan, Jen-Kit; Jaafar, Faizul; Makpol, Suzana

    2017-02-01

    Human diploid fibroblasts (HDFs) proliferation in culture has been used as a model of aging at the cellular level. Growth arrest is one of the most important mechanisms responsible for replicative senescence. Recent researches have been focusing on the function of vitamin E in modulating cellular signaling and gene expression. Therefore, the aim of this study was to elucidate the effect of palm γ-tocotrienol (vitamin E) in modulating cellular aging through p16(INK4a) pathway in HDF cells. Primary culture of senescent HDFs was incubated with 70 μM of palm γ-tocotrienol for 24 hours. Silencing of p16(INK4a) was carried out by siRNA transfection. RNA was extracted from the different treatment groups and gene expression analysis was carried out by real-time reverse transcription polymerase chain reaction. Proteins that were regulated by p16(INK4a) were determined by western blot technique. The finding of this study showed that p16(INK4a) mRNA was overexpressed in senescent HDFs, and hypophosphorylated-pRb and cyclin D1 protein expressions were increased (p < 0.05). However, downregulation of p16(INK4a) and hypophosphorylated-pRb and cyclin D1 protein expressions (p < 0.05) by γ-tocotrienol led to modulation of the cell cycle regulation during cellular aging. In conclusion, senescent HDFs showed change in biological process specifically in cell cycle regulation with elevated expression of genes and proteins which may contribute to cell cycle arrest. Palm γ-tocotrienol may delay cellular senescence of HDFs by regulating cell cycle through downregulation of p16(INK4a) and hypophosphorylated-pRb and cyclin D1 protein expressions.

  8. Association between P16INK4a promoter methylation and HNSCC: a meta-analysis of 21 published studies.

    PubMed

    Shi, Hao; Chen, Xiong; Lu, Cheng; Gu, Changmei; Jiang, Hongwei; Meng, RuiWei; Niu, Xun; Huang, Yangxin; Lu, Meixia

    2015-01-01

    The p16INK4a is an important tumor suppressor gene (TSG) and aberrant methylation of promoter is known to be a major inactivation mechanism of the tumor suppressor and tumor-related genes. Aberrant TSG methylation was considered an important epigenetic silencing mechanism in the progression of head and neck squamous cell carcinoma (HNSCC). However, some studies have reported differences in the methylation frequencies of P16INK4a promoter between cancer and the corresponding control group. Therefore, we conducted a meta-analysis to better identify the association. PubMed, Ovid, ISI Web of Science, and EMBASE were searched to identify eligible studies to evaluate the association of p16INK4a promoter methylation and HNSCC. Odds ratio (ORs) and 95% confidence intervals (95%CI) were calculated to evaluate the strength of association between p16INK4a promoter methylation and HNSCC. A total of twenty-one studies with 1155 cases and 1017 controls were included in the meta-analysis. The frequencies of p16INK4a promoter methylation in the cancer group were significantly higher than those in the control group (cancer group: median: 46.67%, range = 7.84%-95.12%; control group: median: 18.37%, range = 0-83.33%; respectively). The pooled odds ratio was 3.37 (95%CI = 2.32-4.90) in the cancer group versus the corresponding control group under the random-effects model. This meta-analysis of 21 published studies identified that aberrant methylation of p16INK4a promoter was found to be significantly associated with HNSCC.

  9. Disruptive cell cycle regulation involving epigenetic downregulation of Cdkn2a (p16(Ink4a)) in early-stage liver tumor-promotion facilitating liver cell regeneration in rats.

    PubMed

    Tsuchiya, Takuma; Wang, Liyun; Yafune, Atsunori; Kimura, Masayuki; Ohishi, Takumi; Suzuki, Kazuhiko; Mitsumori, Kunitoshi; Shibutani, Makoto

    2012-09-28

    Cell cycle aberration was immunohistochemically examined in relation to preneoplastic liver cell foci expressing glutathione S-transferase placental form (GST-P) at early stages of tumor-promotion in rats with thioacetamide (TAA), a hepatocarcinogen facilitating liver cell regeneration. Immunoexpression of p16(Ink4a) following exposure to other hepatocarcinogens/promoters and its DNA methylation status were also analyzed during early and late tumor-promotion stages. GST-P(+) liver cell foci increased cell proliferation and decreased apoptosis when compared with surrounding liver cells. In concordance with GST-P(+) foci, checkpoint proteins at G(1)/S (p21(Cip1), p27(Kip1) and p16(Ink4a)) and G(2)/M (phospho-checkpoint kinase 1, Cdc25c and phospho-Wee1) were either up- or downregulated. Cellular distribution within GST-P(+) foci was either increased or decreased with proteins related to G(2)-M phase or DNA damage (topoisomerase IIα, phospho-histone H2AX, phospho-histone H3 and Cdc2). In particular, p16(Ink4a) typically downregulated in GST-P(+) foci and regenerative nodules at early tumor-promotion stage with hepatocarcinogens facilitating liver cell regeneration and in neoplastic lesions at late tumor-promotion stage with hepatocarcinogens/promoters irrespective of regenerating potential. Hypermethylation at exon 2 of Cdkn2a was detected at both early- and late-stages. Thus, diverse disruptive expression of G(1)/S and G(2)/M proteins, which allows for clonal selection of GST-P(+) foci, results in the acquisition of multiple aberrant phenotypes to disrupt checkpoint function. Moreover, increased DNA-damage responses within GST-P(+) foci may be the signature of genetic alterations. Intraexonic hypermethylation may be responsible for p16(Ink4a)-downregulation, which facilitates cell cycle progression in early preneoplastic lesions produced by repeated cell regeneration and late-stage neoplastic lesions irrespective of the carcinogenic mechanism. Copyright © 2012

  10. p16INK4a Expression and Immunologic Aging in Chronic HIV Infection

    PubMed Central

    M. Milush, Jeffrey; Cunha-Neto, Edecio; G. Kallas, Esper; Kalil, Jorge; D. Passero, Luiz Felipe; W. Hunt, Peter; G. Deeks, Steven; F. Nixon, Douglas; SenGupta, Devi

    2016-01-01

    Chronic HIV infection is characterized by increased immune activation and immunosenescence. p16 INK4a (p16) is a member of the cyclin-dependent kinase antagonist family that inhibits cellular proliferation, and its protein expression increases during normal chronological aging. However, some infectious diseases can increase the expression of this anti-proliferative protein, potentially accelerating immunological aging and dysfunction. In order to investigate the immunological aging in HIV patients, p16 protein expression was evaluated by flow cytometry, in T cell subsets in a cohort of chronically HIV-infected patients on and off ART as well as age-matched healthy controls. Results showed that untreated HIV-infected subjects exhibited increased per-cell p16 protein expression that was discordant with chronological aging. ART restored p16 protein expression to levels comparable with HIV-negative subjects in the CD4 compartment, but not in CD8 T cells, which can be an indicative of an irreversible activation/exhaustion status on these cells. Additionally, the frequency of activated CD4+ and CD8+ T cells was positively correlated with p16 expression in CD4+ and CD8+ T cells in untreated subjects. In contrast to healthy controls, untreated HIV-infected individuals had increased p16 levels within the effector memory (TEM) subset, indicating a possible role for this marker in impaired clonal expansion during antiviral effector function. Taken together, these data demonstrate that chronic HIV infection is associated with elevated expression of the cellular aging marker p16 in T cells. ART restored normal p16 levels in the CD4+ T cell compartment, indicating that use of therapy can be of fundamental importance to normal cell cycling and maintaining immune homeostasis. PMID:27861555

  11. p16INK4a suppresses BRCA1-deficient mammary tumorigenesis

    PubMed Central

    Scott, Alexandria; Bai, Feng; Chan, Ho Lam; Liu, Shiqin; Ma, Jinshan; Slingerland, Joyce M; Robbins, David J.; Capobianco, Anthony J.; Pei, Xin-Hai

    2016-01-01

    Senescence prevents the proliferation of genomically damaged, but otherwise replication competent cells at risk of neoplastic transformation. p16INK4A (p16), an inhibitor of CDK4 and CDK6, plays a critical role in controlling cellular senescence in multiple organs. Functional inactivation of p16 by gene mutation and promoter methylation is frequently detected in human breast cancers. However, deleting p16 in mice or targeting DNA methylation within the murine p16 promoter does not result in mammary tumorigenesis. How loss of p16 contributes to mammary tumorigenesis in vivo is not fully understood. In this article, we reported that disruption of Brca1 in the mammary epithelium resulted in premature senescence that was rescued by p16 loss. We found that p16 loss transformed Brca1-deficient mammary epithelial cells and induced mammary tumors, though p16 loss alone was not sufficient to induce mammary tumorigenesis. We demonstrated that loss of both p16 and Brca1 led to metastatic, basal-like, mammary tumors with the induction of EMT and an enrichment of tumor initiating cells. We discovered that promoter methylation silenced p16 expression in most of the tumors developed in mice heterozygous for p16 and lacking Brca1. These data not only identified the function of p16 in suppressing BRCA1-deficient mammary tumorigenesis, but also revealed a collaborative effect of genetic mutation of p16 and epigenetic silencing of its transcription in promoting tumorigenesis. To the best of our knowledge, this is the first genetic evidence directly showing that p16 which is frequently deleted and inactivated in human breast cancers, collaborates with Brca1 controlling mammary tumorigenesis. PMID:27811360

  12. Cyclin-dependent kinase inhibitor p16(INK4a) and telomerase may co-modulate endothelial progenitor cells senescence.

    PubMed

    Yang, De-Guang; Liu, Ling; Zheng, Xiao-Yan

    2008-04-01

    Endothelial cells (ECs) damage is an initial and pivotal step in the formation of atherosclerosis. Endothelial progenitor cells (EPCs), which have been considered as the precursor of ECs, can migrate and home to the site of injured ECs to divide into mature ECs and keep the integrity of the endothelial monolayer. It has been shown that the number and function of EPCs are negatively correlated with various atherosclerotic risk factors. This finding may be explained partly by accelerated senescence of EPCs induced by telomere attrition or shortening owning to oxidative stress and accumulative ROS. However, elevated telomerase activity which extends the telomere cannot lead to cellular immortal in the presence of the cyclin-dependent kinase inhibitor p16(INK4a). Researchers have the opinion that senescence is the balance between the regeneration and cancer. High expression of phosphorylated p16(INK4a), which is caused by oxidative stress and accumulative ROS, can prevent tumor cells from unlimited division and becoming malignant ones by accelerating premalignant cells premature senescence. It has been demonstrated that the expression of p16(INK4a) increases remarkably with age due to oxidative stress and accumulative ROS in some stem and progenitor cells, and regulates these cells age-dependent senescence. It is observed that telomeres shortening exists in these cells. Therefore, it can be hypothesized that p16(INK4a), together with telomerase, may co-modulate EPCs senescence.

  13. Telomere dysfunction and inactivation of the p16(INK4a)/Rb pathway in pyothorax-associated lymphoma.

    PubMed

    Tresnasari, Kristianti; Takakuwa, Tetsuya; Ham, Maria Francisca; Rahadiani, Nur; Nakajima, Hiroo; Aozasa, Katsuyuki

    2007-07-01

    Previous studies have indicated that genome instability is involved in the lymphomagenesis of pyothorax-associated lymphoma (PAL), which develops in patients with a long-standing history of pyothorax. One of the well-known causes of genome instability is telomere dysfunction. In the present study, the condition of telomeres was analyzed in the cell lines and clinical samples from PAL. Telomere length (TL) in PAL cell lines was extremely short (<4.5 kbp). TL in tumor samples was broad in range, and shorter than that in the peripheral blood leukocytes from the matched patients. Three of five PAL cell lines showed frequent loss of telomere signals (telomere erosion); however, telomerase activity in PAL cell lines was similar to that in Burkitt lymphoma cell lines. Rb expression was detected in three PAL cell lines and four of 15 clinical samples, respectively. Rb protein expressed in three PAL cell lines was heavily phosphorylated, indicating that function of Rb protein was suppressed. p16(INK4a) expression was not detected in either cell lines or clinical samples. The promoter region in p16(INK4a) was heavily methylated in all cell lines as well as the clinical samples. Inactivation of the p16(INK4a)/Rb pathway may allow continuous cell division and critical telomere shortening, which induce genome instability, finally leading to malignant transformation. Taken together, telomere dysfunction and inactivation of the p16(INK4a)/Rb pathway might play a role for PAL development.

  14. Inactivation of p16INK4a in primary tumors and cell lines of head and neck squamous cell carcinoma.

    PubMed

    Kim, H S; Chung, W B; Hong, S H; Kim, J A; Na, S Y; Jang, H J; Sohn, Y K; Kim, J W

    2000-10-31

    Inactivation of the p16INK4a gene by mutation and deletion is common in head and neck squamous cell carcinoma (HNSCC). The present study demonstrates that hypermethylation of the 5' CpG islands can serve as an alternative mechanism for the inactivation of the p16INK4a gene in this tumor. We studied 11 HNSCC cell lines and 17 oral squamous cell carcinoma (OSCC) primary tumors for p16INK4a gene status by protein/mRNA and DNA genetic/epigenetic analyses to determine the incidence of its inactivation. Our study indicates that: (1) inactivation of p16 protein is frequent in HNSCC cell lines (6/11, 54.5%) and OSCC primary tumors (15/17, 88.2%), (2) inactivation of p16INK4a protein is commonly associated with the presence of gene alteration such as mutation, homozygous deletion and especially aberrant methylation, and (3) genomic sequencing of bisulfite-modified DNA shows that the carcinoma develops a heterogeneous pattern of hypermethylation.

  15. Chemotherapy and Stem Cell Transplantation Increase p16(INK4a) Expression, a Biomarker of T-cell Aging.

    PubMed

    Wood, William A; Krishnamurthy, Janakiraman; Mitin, Natalia; Torrice, Chad; Parker, Joel S; Snavely, Anna C; Shea, Thomas C; Serody, Jonathan S; Sharpless, Norman E

    2016-09-01

    The expression of markers of cellular senescence increases exponentially in multiple tissues with aging. Age-related physiological changes may contribute to adverse outcomes in cancer survivors. To investigate the impact of high dose chemotherapy and stem cell transplantation on senescence markers in vivo, we collected blood and clinical data from a cohort of 63 patients undergoing hematopoietic cell transplantation. The expression of p16(INK4a), a well-established senescence marker, was determined in T-cells before and 6months after transplant. RNA sequencing was performed on paired samples from 8 patients pre- and post-cancer therapy. In patients undergoing allogeneic transplant, higher pre-transplant p16(INK4a) expression was associated with a greater number of prior cycles of chemotherapy received (p=0.003), prior autologous transplantation (p=0.01) and prior exposure to alkylating agents (p=0.01). Transplantation was associated with a marked increase in p16(INK4a) expression 6months following transplantation. Patients receiving autologous transplant experienced a larger increase in p16(INK4a) expression (3.1-fold increase, p=0.002) than allogeneic transplant recipients (1.9-fold increase, p=0.0004). RNA sequencing of T-cells pre- and post- autologous transplant or cytotoxic chemotherapy demonstrated increased expression of transcripts associated with cellular senescence and physiological aging. Cytotoxic chemotherapy, especially alkylating agents, and stem cell transplantation strongly accelerate expression of a biomarker of molecular aging in T-cells.

  16. Senescence Mediated by p16INK4a Impedes Reprogramming of Human Corneal Endothelial Cells into Neural Crest Progenitors

    PubMed Central

    Lu, Wen-Juan; Tseng, Scheffer C. G.; Chen, Shuangling; Tighe, Sean; Zhang, Yuan; Liu, Xin; Chen, Szu-Yu; Su, Chen-Wei; Zhu, Ying-Ting

    2016-01-01

    Human corneal endothelial cells (HCECs) have limited proliferative capacity due to “contact-inhibition” at G1 phase. Such contact-inhibition can be delayed from Day 21 to Day 42 by switching EGF-containing SHEM to LIF/bFGF-containing MESCM through transient activation of LIF-JAK1-STAT3 signaling that delays eventual nuclear translocation of p16INK4a. Using the latter system, we have reported a novel tissue engineering technique by implementing 5 weekly knockdowns with p120 catenin (p120) and Kaiso siRNAs since Day 7 to achieve effective expansion of HCEC monolayers to a transplantable size with a normal HCEC density, through reprogramming of HCECs into neural crest progenitors by activating p120-Kaiso-RhoA-ROCK-canonical BMP signaling. Herein, we noted that a single knockdown with p120-Kaiso siRNAs at Day 42 failed to achieve such reprogramming when contact inhibition transitioned to senescence with nuclear translocation of p16INK4a. In contrast, 5 weekly knockdowns with p120-Kaiso siRNAs since Day 7 precluded senescence mediated by p16INK4a by inducing nuclear translocation of Bmi1 because of sustained activation of JAK2-STAT3 signaling downstream of p120-Kaiso-RhoA-ROCK signaling. STAT3 or Bmi1 siRNA impeded nuclear exclusion of p16INK4a and suppressed the reprogramming induced by p120-Kaiso siRNAs, suggesting that another important engineering strategy of HCEC lies in prevention of senescence mediated by nuclear translocation of p16INK4a. PMID:27739458

  17. Induction of p16INK4A mediated by beta-catenin in a TCF4-independent manner: implications for alterations in p16INK4A and pRb expression during trans-differentiation of endometrial carcinoma cells.

    PubMed

    Saegusa, Makoto; Hashimura, Miki; Kuwata, Takeshi; Hamano, Mieko; Okayasu, Isao

    2006-11-15

    Excessive beta-catenin is considered to contribute to tumor progression by inducing transcription of cell cycle-related genes such as cyclin D1 and c-myc. In contrast, our recent studies demonstrated that beta-catenin could inhibit cell proliferation through activation of p14(ARF)/p53/p21(WAF1) pathway during trans-differentiation toward morular phenotype of endometrial carcinoma (Em Ca) cells. Here, we focused on associations with alterations in p16(INK4A) and pRb expression during this process. In clinical cases, p16(INK4A) immunoreactivity was found to frequently overlap with nuclear beta-catenin accumulation in small-sized morules and surrounding glandular carcinomas (Sur-Ca), demonstrating a significant positive correlation (r = 0.447, p < 0.0001) overall, while the immunoreactions showed stepwise decrease in enlarged morules, despite persistent accumulation of beta-catenin and p21(WAF1) in nuclei. Immunoreactivity for both total pRb and its phosphorylated form was apparently decreased in all morules as compared to Sur-Ca lesions, with a significantly positive correlation. In cell lines, transcriptional activation of p16(INK) (4A) promoter by active form beta-catenin, as well as p21(WAF1), occurred through the region from -385 to -280 bp relative to the translation start site, in a TCF4-independent manner. Moreover, cell proliferation was accompanied with phosphorylation of pRb and increased p16(INK4A) expression, while its inhibition by serum starvation caused decreased expression of total pRb but not p16(INK4A), resulting in high relative amounts of the latter. These findings indicate that induction of p16(INK4A) mediated by nuclear beta-catenin and p21(WAF1), along with loss of pRb expression, may be important for initial steps during trans-differentiation of Em Ca cells. In addition, its down-regulation is associated with progression of lesions.

  18. MOZ-mediated repression of p16(INK) (4) (a) is critical for the self-renewal of neural and hematopoietic stem cells.

    PubMed

    Perez-Campo, Flor M; Costa, Guilherme; Lie-A-Ling, Michael; Stifani, Stefano; Kouskoff, Valerie; Lacaud, Georges

    2014-06-01

    Although inhibition of p16(INK4a) expression is critical to preserve the proliferative capacity of stem cells, the molecular mechanisms responsible for silencing p16(INK4a) expression remain poorly characterized. Here, we show that the histone acetyltransferase (HAT) monocytic leukemia zinc finger protein (MOZ) controls the proliferation of both hematopoietic and neural stem cells by modulating the transcriptional repression of p16(INK4a) . In the absence of the HAT activity of MOZ, expression of p16(INK4a) is upregulated in progenitor and stem cells, inducing an early entrance into replicative senescence. Genetic deletion of p16(INK4a) reverses the proliferative defect in both Moz(HAT) (-) (/) (-) hematopoietic and neural progenitors. Our results suggest a critical requirement for MOZ HAT activity to silence p16(INK4a) expression and to protect stem cells from early entrance into replicative senescence. © 2013 AlphaMed Press.

  19. Biomarkers for cervical cancer screening: the role of p16(INK4a) to highlight transforming HPV infections.

    PubMed

    von Knebel Doeberitz, Magnus; Reuschenbach, Miriam; Schmidt, Dietmar; Bergeron, Christine

    2012-04-01

    Biomarkers indicating the initiation of neoplastic transformation processes in human papillomavirus (HPV)-infected epithelial cells are moving into the focus of cancer prevention research, particularly for anogenital cancer, including cancer of the uterine cervix. Based on the in-depth understanding of the molecular events leading to neoplastic transformation of HPV-infected human cells, the cyclin-dependent kinase inhibitor p16(INK4a) turned out to be substantially overexpressed in virtually all HPV-transformed cells. This finding opened novel avenues in diagnostic histopathology to substantially improve the diagnostic accuracy of cervical cancer and its precursor lesions. Furthermore, it provides a novel technical platform to substantially improve the accuracy of cytology-based cancer early-detection programs. Here, we review the molecular background and the current evidence for the clinical utility of the p16(INK4a) biomarker for HPV-related cancers, and cervical cancer prevention in particular.

  20. Implications of Genetic and Epigenetic Alterations of CDKN2A (p16(INK4a)) in Cancer.

    PubMed

    Zhao, Ran; Choi, Bu Young; Lee, Mee-Hyun; Bode, Ann M; Dong, Zigang

    2016-06-01

    Aberrant gene silencing is highly associated with altered cell cycle regulation during carcinogenesis. In particular, silencing of the CDKN2A tumor suppressor gene, which encodes the p16(INK4a) protein, has a causal link with several different types of cancers. The p16(INK4a) protein plays an executional role in cell cycle and senescence through the regulation of the cyclin-dependent kinase (CDK) 4/6 and cyclin D complexes. Several genetic and epigenetic aberrations of CDKN2A lead to enhanced tumorigenesis and metastasis with recurrence of cancer and poor prognosis. In these cases, the restoration of genetic and epigenetic reactivation of CDKN2A is a practical approach for the prevention and therapy of cancer. This review highlights the genetic status of CDKN2A as a prognostic and predictive biomarker in various cancers. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Role of p16/INK4a and Ki-67 as specific biomarkers for cervical intraepithelial neoplasia: An institutional study

    PubMed Central

    Hebbar, Ankitha; Murthy, Venkataramappa Srinivasa

    2017-01-01

    BACKGROUND: P16/INK4a and Ki-67 have emerged as important biomarkers for the detection of high-risk human papilloma virus (HR-HPV) associated dysplastic changes in the cervical biopsy samples. The increasing inter- and intra-observer variability in the diagnosis of dysplastic lesions and immature squamous metaplasia on histopathology has led to the advent of these biomarkers. This study was taken up with an aim to study their role in increasing the diagnostic accuracy in equivocal cases on histopathology. MATERIALS AND METHODS: Fifty cervical biopsy specimens were stained with p16/INK4a and Ki-67 consisting of 10 cases each of cervical intraepithelial neoplasia (CIN I/II/III) along with five cases of squamous metaplasia. Histopathological diagnosis was considered as the gold standard. Statistical analysis was done by kappa statistics, and P value was calculated. RESULTS: The sensitivity and specificity of p16/INK4a and Ki-67 were 76.2%, 87.5%, 90.5%, and 87.5%, respectively. The overall agreement of both the immunostains with histopathological diagnosis was statistically significant (P < 0.05) and the diagnostic accuracy improved when both the stains were used in conjunction. CONCLUSION: Ki-67 and p16/INK4a can be used as complimentary tests in differentiating dysplastic and nondysplastic lesions and help in confirming the histopathological diagnosis. They aid in recognition of dysplasias caused by HR-HPV, which have higher tendency to progress to neoplasia. However, further research is advocated before the widespread use of these markers for screening of dysplasias. PMID:28367025

  2. The lncRNA MIR31HG regulates p16(INK4A) expression to modulate senescence.

    PubMed

    Montes, Marta; Nielsen, Morten M; Maglieri, Giulia; Jacobsen, Anders; Højfeldt, Jonas; Agrawal-Singh, Shuchi; Hansen, Klaus; Helin, Kristian; van de Werken, Harmen J G; Pedersen, Jakob S; Lund, Anders H

    2015-04-24

    Oncogene-induced senescence (OIS) can occur in response to oncogenic insults and is considered an important tumour suppressor mechanism. Here we identify the lncRNA MIR31HG as upregulated in OIS and find that knockdown of MIR31HG promotes a strong p16(INK4A)-dependent senescence phenotype. Under normal conditions, MIR31HG is found in both nucleus and cytoplasm, but following B-RAF expression MIR31HG is located mainly in the cytoplasm. We show that MIR31HG interacts with both INK4A and MIR31HG genomic regions and with Polycomb group (PcG) proteins, and that MIR31HG is required for PcG-mediated repression of the INK4A locus. We further identify a functional enhancer, located between MIR31HG and INK4A, which becomes activated during OIS and interacts with the MIR31HG promoter. Data from melanoma patients show a negative correlation between MIR31HG and p16(INK4A) expression levels, suggesting a role for this transcript in cancer. Hence, our data provide a new lncRNA-mediated regulatory mechanism for the tumour suppressor p16(INK4A).

  3. Suppression of p53 and p21CIP1/WAF1 reduces arsenite-induced aneuploidy

    PubMed Central

    Salazar, Ana María; Miller, Heather L.; McNeely, Samuel C.; Sordo, Monserrat; Ostrosky-Wegman, Patricia; States, J. Christopher

    2009-01-01

    Aneuploidy and extensive chromosomal rearrangements are common in human tumors. The role of DNA damage response proteins p53 and p21CIP1/WAF1 in aneugenesis and clastogenesis was investigated in telomerase immortalized diploid human fibroblasts using siRNA suppression of p53 and p21CIP1/WAF1. Cells were exposed to the environmental carcinogen sodium arsenite (15 and 20 µM), and the induction of micronuclei (MN) was evaluated in binucleated cells using the cytokinesis-block assay. To determine whether MN resulted from missegregation of chromosomes or from chromosomal fragments, we used a fluorescent in situ hybridization with a centromeric DNA probe. Micronuclei were predominantly of clastogenic origin in control cells regardless of p53 or p21CIP1/WAF1 expression. MN with centromere signals in cells transfected with NSC siRNA or Mock increased 30% after arsenite exposure, indicating that arsenite induced aneuploidy in the tGM24 cells. Although suppression of p53 increased the fraction of arsenite-treated cells with MN, it caused a decrease in the fraction of with centeromeric DNA. Suppression of p21CIP1/WAF1 like p53 suppression decreased the fraction of with centromeric DNA. Our results suggest that cells lacking normal p53 function cannot become aneuploid because they die by mitotic arrest-associated apoptosis, whereas cells with normal p53 function that are able to exit from mitotic arrest can become aneuploid. Furthermore our current results support this role for p21CIP1/WAF1. Since suppression of p21CIP1/WAF1 caused a decrease in aneuploidy induced by arsenite suggesting that p21CIP1/WAF1 plays a role in mitotic exit. PMID:20000476

  4. Increased expression of senescence-associated cell cycle inhibitor p16INK4a in deteriorating renal transplants and diseased native kidney.

    PubMed

    Melk, Anette; Schmidt, Bernhard M W; Vongwiwatana, Attapong; Rayner, David C; Halloran, Philip F

    2005-06-01

    Some features of kidney transplants with dysfunction overlap the lesions of aging, such as tubular atrophy and interstitial fibrosis (TA/IF) without major glomerular abnormalities. Somatic cell limitations could contribute to deterioration in aging and disease states. Since expression of p16(INK4a), a cell cycle inhibitor associated with somatic cell senescence in vitro, is induced in aged kidney, we studied whether kidneys with dysfunction and TA/IF manifested increased p16(INK4a) expression. We performed p16(INK4a) immunostaining on transplanted kidneys and native kidneys with chronic renal diseases. At implantation, transplants manifested little TA/IF, and nuclear p16(INK4a) immunostaining was consistent with age. However, transplants biopsied for abnormal function displaying TA/IF showed strong nuclear and cytoplasmic p16(INK4a) staining, beyond the amount predicted for age. Both atrophic and non-atrophic nephrons displayed increased p16(INK4a), suggesting that it was not simply a feature of atrophy. Epithelial p16(INK4a) staining was not increased in transplants with good function, but was increased in diseased native kidneys. The finding of increased p16(INK4a) expression in renal transplants and diseased kidneys with TA/IF and impaired function supports the concept that some cell senescence changes that accompany aging are also induced by injury and disease stresses. Thus, aging, injury and disease may share common pathways involving somatic cell senescence.

  5. Hepatitis B virus X protein promotes hypermethylation of p16(INK4A) promoter through upregulation of DNA methyltransferases in hepatocarcinogenesis.

    PubMed

    Zhu, Ya-Zhen; Zhu, Rong; Shi, Lian-Guo; Mao, Yi; Zheng, Guang-Juan; Chen, Qi; Zhu, Hong-Guang

    2010-12-01

    The hepatitis B virus×protein (HBx) has been implicated as a potential trigger of the epigenetic deregulation of some genes, but the underlying mechanism remains unknown. The aim of this study is to identify underlying mechanisms involved in HBx-mediated epigenetic modification in the process of HBx induced p16(INK4A) promoter hypermethylation. Liver cell lines were stably transfected with HBx-expressing vector. The methylation status of p16(INK4A) was examined by methyl-specific polymerase chain reaction (MSP) and bisulfite sequencing. Reverse transcription and real-time polymerase chain reaction (real-time RT-PCR), Western blot and immunohistochemistry were used to analyze the expression of HBx, HBx-mediated DNA methylation abnormalities and p16(INK4A). Some cases of HCC and corresponding noncancerous liver tissues were studied. HBx up-regulates DNMT1 and DNMT3A expression in both mRNA level and protein level, and HBx represses p16(INK4A) expression through inducing hypermethylation of p16(INK4A) promoter. Moreover, HBx induces hypermethylation of p16(INK4A) promoter through DNMT1 and DNMT3A. Regulation of DNMT1 and DNMT3A by HBx promoted hypermethylation of p16(INK4A) promoter region. HBx-DNMTs-p16(INK4A) promoter hypermethylation may suggest a mechanism for tumorigenesis during hepatocarcinogenesis. Copyright © 2010 Elsevier Inc. All rights reserved.

  6. 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

  7. Tumor suppressor p16(INK4a) inhibits cancer cell growth by downregulating eEF1A2 through a direct interaction.

    PubMed

    Lee, Mee-Hyun; Choi, Bu Young; Cho, Yong-Yeon; Lee, Sung-Young; Huang, Zunnan; Kundu, Joydeb Kumar; Kim, Myoung Ok; Kim, Dong Joon; Bode, Ann M; Surh, Young-Joon; Dong, Zigang

    2013-04-15

    The tumor suppressor protein p16(INK4a) is a member of the INK4 family of cyclin-dependent kinase (Cdk) inhibitors, which are involved in the regulation of the eukaryotic cell cycle. However, the mechanisms underlying the anti-proliferative effects of p16(INK4a) have not been fully elucidated. Using yeast two-hybrid screening, we identified the eukaryotic elongation factor (eEF)1A2 as a novel interacting partner of p16(INK4a). eEF1A2 is thought to function as an oncogene in cancers. The p16(INK4a) protein interacted with all but the D2 (250-327 aa) domain of eEF1A2. Ectopic expression of p16(INK4a) decreased the expression of eEF1A2 and inhibited cancer cell growth. Furthermore, suppression of protein synthesis by expression of p16(INK4a) ex vivo was verified by luciferase reporter activity. Microinjection of p16(INK4a) mRNA into the cytoplasm of Xenopus embryos suppressed the luciferase mRNA translation, whereas the combination of p16(INK4a) and morpholino-eEF1A2 resulted in a further reduction in translational activity. We conclude that the interaction of p16(INK4a) with eEF1A2, and subsequent downregulation of the expression and function of eEF1A2 is a novel mechanism explaining the anti-proliferative effects of p16(INK4a).

  8. Loss of p16(INK4a) is associated with reduced patient survival in soft tissue tumours, and indicates a senescence barrier.

    PubMed

    Knösel, Thomas; Altendorf-Hofmann, Annelore; Lindner, Lars; Issels, Rolf; Hermeking, Heiko; Schuebbe, Gesa; Gibis, Sebastian; Siemens, Helge; Kampmann, Eric; Kirchner, Thomas

    2014-07-01

    p16(INK4a) is an important factor in carcinogenesis, and its expression is linked to oncogene-induced senescence. Very recently it was shown that upregulation and downregulation of p16 indicates a senescence barrier in the serrated route of colorectal cancer. However, in soft tissue sarcoma (STS), the senescence mechanism is still not understood. In this study, we analysed a well characterised cohort of STS for p16(INK4a) expression and correlated the results with clinicopathological parameters including survival. Tissue microarrays (TMA) of 183 soft tissue and bone tumours were analysed immunohistochemically. Furthermore, mRNA expression of p16(INK4a) was evaluated in four sarcoma cell lines, and a demethylation test was performed by treatment with 5-aza-2'-deoxycytide. On protein level, expression of p16(INK4a) was observed in undifferentiated pleomorphic sarcoma (UPS) in 69.1%, leiomyosarcoma in 85.7%, synovial sarcoma in 77.8%, liposarcoma in 88.9%, angiosarcoma in 60.9% and MPNST in 22.2%. Loss of p16(INK4a) was observed in high grade sarcomas and showed a significant correlation with reduced patient survival (p=0.032). On DNA level, one out of four sarcoma cell lines exhibited a methylated p16(INK4a) promoter analysed by methylation-specific PCR. p16(INK4a) mRNA and protein expression was restored after demethylation using 5-aza-2'-deoxycytide. Upregulation of p16(INK4a) might be associated with the induction of senescence and indicates a senescence barrier. Downregulation of p16(INK4a) is found in malignant progression, and is significantly correlated with reduced patient survival. Downregulation of p16(INK4a) may be explained by DNA-hypermethylation in sarcoma cells. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  9. p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis

    PubMed Central

    2014-01-01

    Introduction Recent evidence suggests that tissue accumulation of senescent p16INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16INK4a in the OA-induced SASP and its regulation by microRNAs (miRs). Methods We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis. Results p16INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16INK4a. Accordingly, p16INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis. Conclusions We disclosed herein a new role of the senescence marker p16INK4a and its regulation by miR-24 during OA and terminal chondrogenesis. PMID:24572376

  10. [Targeting p16(INK4a) by therapeutic vaccination : Concept and status of clinical investigations in HPV-associated head and neck cancers].

    PubMed

    Reuschenbach, M

    2015-02-01

    Up to 70% of oropharyngeal cancers are attributable to human papillomavirus (HPV) infection; however, a therapy specific for patients with HPV-associated cancers is currently not available. Overexpression of the viral oncogenes E6 and E7 results in cellular alterations that represent interesting targets for novel therapies. One consequence of E6/E7 overexpression is strong expression of the cellular protein p16(INK4a). The elimination of p16(INK4a)-expressing tumor cells by the immune system could be achieved through a therapeutic p16(INK4a) vaccine. The current article provides an overview of HPV-associated head and neck cancers and the associated p16(INK4a) expression. Based on this overview, the concept and status of the clinical investigation of therapeutic p16(INK4a) vaccination is described. In addition to discussing published literature, a clinical study is described. In this phase I/IIa study, patients with advanced HPV-associated p16(INK4a)-expressing tumors were vaccinated with a p16(INK4a) peptide. HPV-associated head and neck cancers continuously display strong overexpression of the cellular protein p16(INK4a). Vaccination with p16(INK4a) could represent a novel therapy for patients with HPV-associated carcinomas. Further studies will evaluate the clinical efficacy of therapeutic p16(INK4a) vaccination. Combinations with other immunotherapeutic approaches are interesting considering the modulating role of the immune system, particularly in HPV-associated tumors.

  11. The tumour suppressor CDKN2A/p16(INK4a) regulates adipogenesis and bone marrow-dependent development of perivascular adipose tissue.

    PubMed

    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

    2017-09-01

    The genomic CDKN2A/B locus, encoding p16(INK4a) 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. p16(INK4a) is a cell cycle regulator and tumour suppressor. Whether it plays a role in adipose tissue formation is unknown. p16(INK4a) knock-down in 3T3/L1 preadipocytes or p16(INK4a) deficiency in mouse embryonic fibroblasts enhanced adipogenesis, suggesting a role for p16(INK4a) in adipose tissue formation. p16(INK4a)-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 p16(INK4a)-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 p16(INK4a)-deficient bone marrow had more epicardial adipose tissue compared to controls when fed a high-fat diet. In humans, p16(INK4a) 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 p16(INK4a) modulates epicardial adipose tissue development, providing a potential mechanistic link between the genetic association of the CDKN2A/B locus and cardiovascular disease risk.

  12. Downregulation of p16(ink4a) inhibits cell proliferation and induces G1 cell cycle arrest in cervical cancer cells.

    PubMed

    Zhang, Chu-Yue; Bao, Wei; Wang, Li-Hua

    2014-06-01

    Studies have suggested that p16(ink4a) may be a surrogate biomarker for the diagnosis of cervical cancer; however, the function of p16(ink4a) in human cervical cancer cells remains largely unknown. Therefore, in this study, we aimed to investigate the role of p16(ink4a) in human cervical cancer cells. Immunocytochemistry was used to examine invasive squamous cell carcinoma and its precancerous lesions. p16(ink4a)-siRNA was transfected into SiHa and HeLa cells to deplete its expression. The cellular levels of p16(ink4a) mRNA and protein were detected by qRT-PCR and western blot analysis. Proliferation rates were assessed by methyl thiazolyl tetrazolium (MTT) and plate colony formation assays. Cellular migration and invasion ability were assessed by a wound healing assay and Transwell assay. Cellular apoptosis and the cell cycle were measured by flow cytometry. The protein levels of retinoblastoma (Rb), phosphorylated Rb (phospho-Rb), cyclin D1 and caspase-3 were determined by western blot analysis. The results revealed that p16(ink4a) was overexpressed in the cervical cancer and precancerous lesions (P<0.05). The downregulation of p16(ink4a) in the SiHa and HeLa cells inhibited their proliferation, migration and invasion. In the SiHa cells, p16(ink4a)-siRNA also induced G1 cell cycle arrest and apoptosis. Western blot analysis revealed that the downregulation of p16(ink4a) in the SiHa cells markedly induced caspase-3 activation and decreased cyclin D1 expression. These data suggest that the overexpression of p16(ink4a) appears to be useful in monitoring cervical precancerous lesions, which supports that the hypothesis that p16(ink4a) is a surrogate biomarker for the diagnosis of cervical cancer. The therapeutic targeting of overexpressed p16(ink4a) in the p16(ink4a)-cyclin-Rb pathway may be a useful strategy in the treatment of cervical cancer.

  13. Novel frameshift mutation in the p16/INK4A tumor suppressor gene in canine breast cancer alters expression from the p16/INK4A/p14ARF locus.

    PubMed

    Lutful Kabir, Farruk M; Agarwal, Payal; Deinnocentes, Patricia; Zaman, Jishan; Bird, Allison Church; Bird, R Curtis

    2013-01-01

    The INK4 family of cyclin-dependent kinase inhibitors (CKI) encode important cell cycle regulators that tightly control cell cycle during G1 to S phase. These related genes are considered tumor suppressors as loss of function contributes to the malignant phenotype. Expression of CKIs p16, p14ARF, or p15 were defective in six different canine mammary tumor (CMT) cell lines compared to normal thoracic canine fibroblasts. This suggests CKI defects are frequently responsible for neoplastic transformation in canine mammary carcinomas. p16 and p14ARF are two alternatively spliced products derived from the canine p16/INK4A/p14ARF gene locus. Despite omissions in the published p16 transcript and canine genome and the presence of GC-rich repeats, we determined the complete coding sequence of canine p16 revealing a deletion and frameshift mutation in p16 exon 1α in CMT28 cells. In addition, we determined canine p14ARF mRNA and protein sequences. Mapping of these mutations uncovered important aspects of p16 and p14ARF expression and defects in CMT28 cells shifting the p16 reading frame into p14ARF making a fusion protein that was predicted to be truncated, unstable and devoid of structural and functional integrity. This data describes an important neoplastic mechanism in the p16/INK4A/p14ARF locus in a spontaneous canine model of breast cancer. Copyright © 2012 Wiley Periodicals, Inc.

  14. Methylation of CpG islands of p16(INK4a) and cyclinD1 overexpression associated with progression of intraductal proliferative lesions of the breast.

    PubMed

    Liu, Tieju; Niu, Yun; Feng, Yumei; Niu, Ruifang; Yu, Yong; Lv, Ajuan; Yang, Yi

    2008-11-01

    P16(INK4a) is a tumor suppressor gene frequently inactivated by aberrant promoter hypermethylation. In this study, p16(INK4a) methylation was evaluated in intraductal proliferative lesions of the breast, using real-time quantitative polymerase chain reaction (MethyLight) and methylation-sensitive restriction endonuclease polymerase chain reaction. Immunohistochemistry was performed to compare and validate the methylation analysis. P16(INK4a) methylation associated with oncogene cyclinD1 expression, detected through the use of in situ hybridization and immunohistochemistry, was likewise characterized. P16(INK4a) methylation displayed varying significance among different types of intraductal proliferative lesions. Both the positive rate and the median quantitative methylation value increased with the evolution of intraductal proliferative lesions through the use of quantitative and qualitative assays. P16(INK4a) methylation was positively correlated to cyclinD1 overexpression. This study demonstrated that p16(INK4a) methylation served as the silencing mechanism of p16(INK4a) protein expression and played a crucial role in the intraductal proliferative lesions' progression. In the differential diagnosis of intraductal proliferative lesions, quantitative DNA methylation analysis of p16(INK4a) by MethyLight may be used as a surrogate, especially to distinguish atypical ductal hyperplasia from usual ductal hyperplasia and low-grade ductal carcinoma in situ. Furthermore, this study discovered that flat epithelial atypia do not share similar molecular profiles of p16(INK4a) epigenetic modification with atypical ductal hyperplasia and low-grade ductal carcinoma in situ.

  15. Up and downregulation of p16(Ink4a) expression in BRAF-mutated polyps/adenomas indicates a senescence barrier in the serrated route to colon cancer.

    PubMed

    Kriegl, Lydia; Neumann, Jens; Vieth, Michael; Greten, Florian R; Reu, Simone; Jung, Andreas; Kirchner, Thomas

    2011-07-01

    P16(Ink4a) is an important factor in carcinogenesis and its expression can be linked to oncogene-induced senescence. Oncogene-induced senescence is characterized by growth arrest and occurs as a consequence of oncogene activation due to KRAS or BRAF mutation. It has been shown that the induction of p16(Ink4a) in premalignant lesions and its loss during malignant transformation is an important mechanism in the carcinogenesis of several tumours. Loss of p16(Ink4a) is often caused by CDKN2A promoter hypermethylation. This mechanism of gene silencing is associated with the CpG island methylator phenotype (CIMP) in colorectal carcinomas, which is characterized by widespread promoter methylation. In particular, colorectal carcinomas with BRAF mutations have been shown to be strongly associated with CIMP. Also, BRAF mutations are strongly correlated with the serrated route to colorectal cancer. In this study, we investigated p16(Ink4a) expression and promoter methylation in BRAF-mutated serrated lesions of the colon. P16(Ink4a) expression was found to be upregulated in premalignant lesions and was lost in invasive serrated carcinomas. P16(Ink4a) expression and Ki67 expression were mutually exclusive, indicating that p16(Ink4a) acts as cell cycle inhibitor. Additionally, progression of malignant transformation in serrated lesions was accompanied by increasing methylation of the CDKN2A promoter. Therefore, our data provide evidence for oncogene-induced senescence in the serrated route to colorectal cancer with BRAF mutation and upregulation of p16(Ink4a) expression appears to be a useful indicator of induction of senescence. Loss of p16(Ink4a) expression occurs during malignant transformation and is caused mainly by aberrant methylation of the CDKN2A promoter.

  16. Relationships among folate, alcohol consumption, gene variants in one-carbon metabolism and p16 INK4a methylation and expression in healthy breast tissues

    PubMed Central

    Llanos, Adana A.; Dumitrescu, Ramona G.; Brasky, Theodore M.; Liu, Zhenhua; Mason, Joel B.; Marian, Catalin; Makambi, Kepher H.; Spear, Scott L.; Kallakury, Bhaskar V.S.; Freudenheim, Jo L.; Shields, Peter G.

    2015-01-01

    p16 INK4a is a tumor suppressor gene, frequently hypermethylated in breast cancer; this epigenetic silencing of p16 INK4a occurs early in carcinogenesis. The risk factors and functional consequences of p16 INK4a methylation are unknown. Alcohol consumption, a breast cancer risk factor, impedes folate metabolism and may thereby alter gene methylation since folate plays a pivotal role in DNA methylation. In a cross-sectional study of 138 women with no history of breast cancer who underwent reduction mammoplasty, we studied breast cancer risk factors, plasma and breast folate concentrations, variation in one-carbon metabolism genes, p16 INK4a promoter methylation and P16 protein expression. Logistic regression was used to estimate multivariable-adjusted odds ratios (OR) and 95% confidence intervals (CI). p16 INK4a methylation was negatively correlated with P16 expression (r = −0.28; P = 0.002). Alcohol consumption was associated with lower breast folate (P = 0.03), higher p16 INK4a promoter methylation (P = 0.007) and less P16 expression (P = 0.002). Higher breast folate concentrations were associated with lower p16 INK4a promoter methylation (P = 0.06). Genetic variation in MTRR (rs1801394) and MTHFD1 (rs1950902) was associated with higher p16 INK4a promoter methylation (OR = 2.66, 95% CI: 1.11–6.42 and OR = 2.72, 95% CI: 1.12–6.66, respectively), whereas variation in TYMS (rs502396) was associated with less P16 protein expression (OR = 0.22, 95% CI: 0.05–0.99). Given that this is the first study to indicate that alcohol consumption, breast folate and variation in one-carbon metabolism genes are associated with p16 INK4a promoter methylation and P16 protein expression in healthy tissues; these findings require replication. PMID:25344837

  17. HPV-associated p16 INK4A expression and response to therapy and survival in selected head and neck cancers.

    PubMed

    Kanyilmaz, Gul; Ekinci, Ozgur; Muge, Akmansu; Celik, Sevinc; Ozturk, Furkan

    2015-01-01

    Development of squamous cell cancer of head and neck (SCCHN) is associated with human papillomavirus (HPV) infection, which in turn is closely related with expression of p16 INK4A. Loss of p16 INK4A expression by deletion, mutation, or hypermethylation is common in SCCHN. We here evaluated p16 INK4A as a prognostic marker of treatment response and survival in our SCCHN patients with laryngeal, hypopharyngeal or nasopharyngeal cancers. 131 patients diagnosed with SCCHN between January 2,2006 and July 17, 2010 were examined for p16 INK4A. The median age was 60 years (15-82 years). Fifty one patients were stage I-II and 80 were stage III-IV. Immunohistochemical expression of p16 INK4A was analyzed in pretreatment paraffin-embedded tumor blocks. The influence of p16 INK4A status on disease-free survival, and overall survival after treatment was evaluated. P16 INK4A positivity was found in 58 patients (44%). Tumor-positivity for p16INK4A was correlated with improved disease free survival (70.1 months vs 59 months) and improved overall survival (2, 3 and 5-year values; 77% vs 72%, 70% vs 63% and, 63% vs 55%; respectively). On multivariate analysis, stage was determined as independent prognostic factor for disease-free survival. Stage was the major prognostic factor on treatment response and survival in our patients. P16 INK4A status predicts better outcome in laryngeal, hypopharyngeal or nasopharyngeal cancer cases treated with surgery plus adjuvant radiochemotherapy as well as with definitive radiation therapy and/or chemotherapy.

  18. Detection of p16INK4a promoter methylation status in non-small cell lung cancer by a fluorescence polarization assay.

    PubMed

    Song, Zujun; Zhou, Rongbin; Li, Ding; Chen, Yanan; Liang, Ping; Liu, Wenchao; Zhang, Ju

    2011-09-01

    The detection of the p16INK4a promoter methylation status has a good value for the prognosis, early detection, and individualized management of patients with non-small cell lung cancer. A novel method detecting the p16INK4a promoter methylation status of primary carcinoma tissue samples by a fluorescence polarization assay was developed in this research. A pair of general primers was used to amplify a 305-basepair fragment in the promoter region of p16INK4a. Two probes specific for either methylated p16INK4a or unmethylated p16INK4a DNA labeled with either tetramethyl 6-carboxyrhodamine or 6-carboxy-fluorescein hybridized, respectively, with their target amplicons, and the hybridization increased the fluorescence polarization values. The p16INK4a promoter methylation status was determined by the analysis of the fluorescence polarization values. One hundred and twenty-nine non-small cell lung cancer samples were analyzed in parallel with a fluorescence polarization assay and a gel-based methylation-specific polymerase chain reaction (PCR) assay. There was no significant difference between the results of the p16INK4a promoter methylation status obtained with the fluorescence polarization assay and the results obtained with the gel-based methylation-specific PCR assay. The minimum detection level of the fluorescence polarization assay was 25 copies/μL. The fluorescence polarization assay allowed the semiautomated detection of the methylated p16INK4a and unmethylated p16INK4a promoters directly in the solution with 1 PCR cycle, and it was much simpler than methylation-specific PCR and methylation-specific multiplex ligation-dependent probe amplification assays.

  19. Piwil2 expressed in various stages of cervical neoplasia is a potential complementary marker for p16INK4a

    PubMed Central

    He, Gang; Chen, Li; Ye, Yin; Xiao, Yi; Hua, Keding; Jarjoura, David; Nakano, Toru; Barsky, Sanford H; Shen, Rulong; Gao, Jian-Xin

    2010-01-01

    Generally, cancers may undergo the developmental stages of benign proliferation, precancer and invasive cancer. Identification of biomarkers that are expressed throughout the developmental stages will facilitate detection, prevention and therapy of cancer. Piwil2, a member of AGO/PIWI family of proteins, has been suggested to be associated with tumor development. Here we reported that piwil2 can be detected by immunohistochemistry (IHC) in various stages of human cervical squamous cell carcinomas and adenocarcinomas. Interestingly, piwil2 was also detected in some metaplastic epithelial cells as well as histologically “normal” appearing tissues adjacent to malignant lesions. While all the premalignant and malignant lesions expressed varying levels of piwil2, p16INK4a (p16), a surrogate indicator of high-risk human papillomavirus (HR-HPV) infection, was detected in only 84.62% of the specimens. In Papanicolaou (Pap) test, piwil2 was also detected in atypical glandular cells (AGC), low-grade (LSIL) and high-grade squamous intraepithelial lesions (HSIL), whereas p16 was not always concomitantly detected in the same specimens. The results suggest that piwil2 might play important roles throughout the process of cervical cancer development and have the potential to be used as a complementary marker for p16INK4a. It is worth further study to improve the sensitivity and specificity of current screening methods for cervical cancers. PMID:20407605

  20. 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

  1. p16INK4a, a Senescence Marker, Influences Tenofovir/Emtricitabine Metabolite Disposition in HIV‐Infected Subjects

    PubMed Central

    Collins, JW; Cottrell, ML; Trezza, CR; Prince, HMA; Sykes, C; Torrice, C; White, N; Malone, S; Wang, R; Patterson, KB; Sharpless, NE; Forrest, A

    2016-01-01

    The goal of this study was to explore the relationships between tenofovir (TFV) and emtricitabine (FTC) disposition and markers of biologic aging, such as the frailty phenotype and p16INK4a gene expression. Chronologic age is often explored in population pharmacokinetic (PK) analyses, and can be uninformative in capturing the impact of aging on physiology, particularly in human immunodeficiency virus (HIV)‐infected patients. Ninety‐one HIV‐infected participants provided samples to quantify plasma concentrations of TFV/FTC, as well as peripheral blood mononuclear cell (PBMC) samples for intracellular metabolite concentrations; 12 participants provided 11 samples, and 79 participants provided 4 samples, over a dosing interval. Nonlinear mixed effects modeling of TFV/FTC and their metabolites suggests a relationship between TFV/FTC metabolite clearance (CL) from PBMCs and the expression of p16INK4a, a marker of cellular senescence. This novel approach to quantifying the influence of aging on PKs provides rationale for further work investigating the relationships between senescence and nucleoside phosphorylation and transport. PMID:28019088

  2. Natural History of Meningioma Development in Mice Reveals: A Synergy of Nf2 and p16Ink4a Mutations

    PubMed Central

    Kalamarides, Michel; Stemmer-Rachamimov, Anat O; Takahashi, Masaya; Han, Zhi-Yan; Chareyre, Fabrice; Niwa-Kawakita, Michiko; Black, Peter M; Carroll, Rona S; Giovannini, Marco

    2008-01-01

    Meningiomas account for approximately 30% of all primary central nervous system tumors and are found in half of neurofibromatosis type 2 patients often causing significant morbidity. Although most meningiomas are benign, 10% are classified as atypical or anaplastic, displaying aggressive clinical behavior. Biallelic inactivation of the neurofibromatosis 2 (NF2) tumor suppressor is associated with meningioma formation in all NF2 patients and 60% of sporadic meningiomas. Deletion of the p16INK4a/p14ARF locus is found in both benign and malignant meningiomas, while mutation of the p53 tumor suppressor gene is uncommon. Previously, we inactivated Nf2 in homozygous conditional knockout mice by adenoviral Cre delivery and showed that Nf2 loss in arachnoid cells is rate-limiting for meningioma formation. Here, we report that additional nullizygosity for p16Ink4a increases the frequency of meningioma and meningothelial proliferation in these mice without modifying the tumor grade. In addition, by using magnetic resonance imaging (MRI) to screen a large cohort of mutant mice, we were able to detect meningothelial proliferation and meningioma development opening the way to future studies in which therapeutic interventions can be tested as preclinical assessment of their potential clinical application. PMID:17924978

  3. Up-regulation of expression and lack of 5' CpG island hypermethylation of p16 INK4a in HPV-positive cervical carcinomas

    PubMed Central

    Ivanova, Tatiana A; Golovina, Daria A; Zavalishina, Larisa E; Volgareva, Galina M; Katargin, Alexey N; Andreeva, Yulia Y; Frank, Georgy A; Kisseljov, Fjodor L; Kisseljova, Natalia P

    2007-01-01

    Background High risk type human papilloma viruses (HR-HPV) induce carcinomas of the uterine cervix by expressing viral oncogenes E6 and E7. Oncogene E7 of HR-HPV disrupts the pRb/E2F interaction, which negatively regulates the S phase entry. Expression of tumor suppressor p16ink4a drastically increases in majority of HR-HPV associated carcinomas due to removal of pRb repression. The p16ink4a overexpression is an indicator of an aberrant expression of viral oncogenes and may serve as a marker for early diagnostic of cervical cancer. On the other hand, in 25–57% of cervical carcinomas hypermethylation of the p16 INK4a promoter has been demonstrated using a methylation-specific PCR, MSP. To evaluate a potential usage of the p16 INK4a 5' CpG island hypermethylation as an indicator of tumor cell along with p16ink4a overexpression, we analyzed the methylation status of p16 INK4a in cervical carcinomas Methods Methylation status of p16 INK4a was analyzed by MSP and by bisulfite-modified DNA sequencing. The expression of p16ink4a was analyzed by RT-PCR and by immunohistochemical technique. Results The extensive methylation within p16 INK4a 5' CpG island was not detected either in 13 primary cervical carcinomas or in 5 cancer cell lines by bisulfite-modified DNA sequencing (including those that were positive by MSP in our hands). The number and distribution of rare partially methylated CpG sites did not differ considerably in tumors and adjacent normal tissues. The levels of the p16 INK4a mRNA were increased in carcinomas compared to the normal tissues independently of the number of partially methylated CpGs within 5'CpG island. The transcriptional activation of p16 INK4a was accompanied by p16ink4a cytoplasmic immunoreactivity in the majority of tumor cells and presence of a varied number of the p16 positive nuclei in different tumors. Conclusion Hypermethylaion of the p16INK4a 5' CpG island is not a frequent event in HR-HPV-positive cervical carcinomas and cannot be an

  4. Both p16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse.

    PubMed

    Bardeesy, Nabeel; Aguirre, Andrew J; Chu, Gerald C; Cheng, Kuang-Hung; Lopez, Lyle V; Hezel, Aram F; Feng, Bin; Brennan, Cameron; Weissleder, Ralph; Mahmood, Umar; Hanahan, Douglas; Redston, Mark S; Chin, Lynda; Depinho, Ronald A

    2006-04-11

    Activating KRAS mutations and p16(Ink4a) inactivation are near universal events in human pancreatic ductal adenocarcinoma (PDAC). In mouse models, Kras(G12D) initiates formation of premalignant pancreatic ductal lesions, and loss of either Ink4a/Arf (p16(Ink4a)/p19(Arf)) or p53 enables their malignant progression. As recent mouse modeling studies have suggested a less prominent role for p16(Ink4a) in constraining malignant progression, we sought to assess the pathological and genomic impact of inactivation of p16(Ink4a), p19(Arf), and/or p53 in the Kras(G12D) model. Rapidly progressive PDAC was observed in the setting of homozygous deletion of either p53 or p16(Ink4a), the latter with intact germ-line p53 and p19(Arf) sequences. Additionally, Kras(G12D) in the context of heterozygosity either for p53 plus p16(Ink4a) or for p16(Ink4a)/p19(Arf) produced PDAC with longer latency and greater propensity for distant metastases relative to mice with homozygous deletion of p53 or p16(Ink4a)/p19(Arf). Tumors from the double-heterozygous cohorts showed frequent p16(Ink4a) inactivation and loss of either p53 or p19(Arf). Different genotypes were associated with specific histopathologic characteristics, most notably a trend toward less differentiated features in the homozygous p16(Ink4a)/p19(Arf) mutant model. High-resolution genomic analysis revealed that the tumor suppressor genotype influenced the specific genomic patterns of these tumors and showed overlap in regional chromosomal alterations between murine and human PDAC. Collectively, our results establish that disruptions of p16(Ink4a) and the p19(ARF)-p53 circuit play critical and cooperative roles in PDAC progression, with specific tumor suppressor genotypes provocatively influencing the tumor biological phenotypes and genomic profiles of the resultant tumors.

  5. p16INK4A immunohistochemical staining and predictive value for progression of cervical intraepithelial neoplasia grade 1: a prospective study in China.

    PubMed

    Liao, Guang-Dong; Sellors, John W; Sun, Hai-Kui; Zhang, Xun; Bao, Yan-Ping; Jeronimo, Jose; Chen, Wen; Zhao, Fang-Hui; Song, Yan; Cao, Zhi; Zhang, Shao-Kai; Xi, Ming-Rong; Qiao, You-Lin

    2014-04-01

    p16(INK4A) is strongly expressed in tissues diagnosed as cervical intraepithelial neoplasia (CIN) and cancer in women infected with human papillomavirus (HPV), but few prospective studies have evaluated p16(INK4A) as a marker for the risk of low-grade CIN (CIN1) progression. We investigated the prevalence of p16(INK4A) immunostaining by CIN grade and whether overexpression of p16(INK4A) in CIN1 predicts future risk for high-grade CIN in Chinese women. 6,557 Chinese women aged 30-49 years were screened from 2003 to 2005 using cytology and carcinogenic HPV test. Colposcopy was performed on women with any abnormal result. p16(INK4A) Immunostaining was performed on biopsies from all women with CIN1, as well as randomly selected women with normal or CIN grade 2 and worse (CIN2+) biopsies. Women with CIN1 were followed up without treatment. Colposcopy was performed on all untreated women at a 2-year interval. The prevalence of p16(INK4A) staining was 2.7%, 42.7%, 75.5%, 79.6% and 100% among women with normal, CIN1, 2, 3 and cancer biopsies, respectively (p < 0.001). HPV positivity was strongly associated with p16(INK4A) staining [odds ratios (OR) = 12.8; 95% confidence intervals (CI): 5.2-31.6]. p16(INK4A) staining of CIN1 biopsies at baseline was associated with an increased risk of finding high-grade CIN over 2 years of follow-up (OR = 1.43; 95% CI: 0.52-3.91). The two-year cumulative incidence of CIN2+ for p16(INK4A) positive women was higher at 10.71% than for p16(INK4A) negative women at 1.30% (crude RR = 8.25, 95% CI: 1.02-66.62). p16(INK4A) overexpression is strongly associated with grade of CIN and risk of progression to high-grade CIN in women with low-grade lesions.

  6. p16INK4a immunohistochemistry in cervical biopsy specimens: A systematic review and meta-analysis of the interobserver agreement.

    PubMed

    Reuschenbach, Miriam; Wentzensen, Nicolas; Dijkstra, Maaike G; von Knebel Doeberitz, Magnus; Arbyn, Marc

    2014-12-01

    The interpretation of cervical biopsy specimens guides management of women with suspected cervical cancer precursors. However, morphologic evaluation is subjective and has low interobserver agreement. Addition of p16(INK4a) immunohistochemistry may improve interpretation. We performed a systematic review and meta-analysis of published data on interobserver agreement of p16(INK4a) positivity using p16(INK4a) immunohistochemistry and of cervical intraepithelial neoplasia grade 2 (CIN2+) and CIN grade 3 (CIN3+) classification using H&E morphology in conjunction with p16(INK4a) in comparison with H&E morphology alone. The literature search revealed five eligible articles. The results show strong agreement of pathologists' interpretation of cervical biopsy specimens as p16(INK4a) positive or negative (pooled κ = 0.90; 95% confidence interval [CI], 0.88-0.92) and significantly higher agreement for a CIN2+ diagnosis with H&E morphology in conjunction with p16(INK4a) (κ = 0.73; 95% CI, 0.67-0.79) compared with H&E morphology alone (κ = 0.41; 95% CI, 0.17-0.65). Also, a slightly higher agreement for CIN3+ can be observed (κ = 0.66; 95% CI, 0.39-0.94 for H&E morphology in conjunction with p16(INK4a) and κ = 0.61; 95% CI, 0.44-0.78 for H&E morphology alone), but this difference was not statistically significant. The published literature indicates improved interobserver agreement of the diagnosis of CIN2+ with the conjunctive use of H&E morphology with p16(INK4a) immunohistochemistry compared with H&E morphology alone. Copyright© by the American Society for Clinical Pathology.

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

    PubMed

    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

    2016-08-01

    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. © The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Utility of p16(ink4a) immunocytochemistry in liquid-based cytology specimens from women treated for high-grade squamous intraepithelial lesions.

    PubMed

    Carydis, Vasiliki Bessie; Walker, Todd; Wing, Anthony; Colgan, Terence J

    2007-01-01

    To examine whether p16(ink4a) immunocytochemical (ICC) expression detected intraepithelial disease in liquid-based cytology (LBC) specimens from women with high-grade squamous intraepithelial lesions (HSIL), whose specimen was labeled negative for intraepithelial lesion or malignany (NILM). Residual LBC specimens from women treated for HSIL (n = 21), whose LBC test was interpreted as NILM including marked benign inflammatory changes (BCC) were used. The control (n = 25) consisted of residual LBC specimens from women with documented HSIL. ICC for p16p(16k4a) was performed on a second ThinPrep (ThinPrep 2000, Cylyl Corporation, Boxborough, Massachusetts, U.S.A.) preparation; the percentage ofpositive cells and intensity of immunostaining were recorded. Standard LBC preparations for p16(ink4a) ICC-positive and ICC-negative control cases were reviewed. Twenty-four of 25 (96%) of the HSIL control group were ICC p16(ink4a) positive. In the NILM/BCC group, 2 of 21 with adequate LBC residua were ICC p16(ink4a) positive; on review both were reclassified as epithelial abnormality--1 HSIL and 1 atypical squamous cells cannot exclude HSIL. In both, subsequent colposcopic biopsy yielded HSIL. p16(ink4a) ICC positivity on NILM/BCC LBC residua from patients with HSIL may identify cases that merit cytologic review and possible reclassification. The utility of p16(ink4a) ICC in this situation requires further study.

  9. The clinical impact of using p16(INK4a) immunochemistry in cervical histopathology and cytology: an update of recent developments.

    PubMed

    Bergeron, Christine; Ronco, Guglielmo; Reuschenbach, Miriam; Wentzensen, Nicolas; Arbyn, Marc; Stoler, Mark; von Knebel Doeberitz, Magnus

    2015-06-15

    Cervical cancer screening test performance has been hampered by either lack of sensitivity of Pap cytology or lack of specificity of Human Papillomavirus (HPV) testing. This uncertainty can lead to unnecessary referral and treatment, which is disturbing for patients and increases costs for health care providers. The identification of p16(INK4a) as a marker for neoplastic transformation of cervical squamous epithelial cells by HPVs allows the identification of HPV-transformed cells in histopathology or cytopathology specimens. Diagnostic studies have demonstrated that the use of p16(INK4a) immunohistochemistry substantially improves the reproducibility and diagnostic accuracy of histopathologic diagnoses. p16(INK4a) cytology has substantially higher sensitivity for detection of cervical precancer in comparison to conventional Pap tests. Compared to HPV DNA tests, immunochemical detection of p16(INK4a) -stained cells demonstrates a significantly improved specificity with remarkably good sensitivity. About 15 years after the initial observation that p16(INK4a) is overexpressed in HPV-transformed cells we review the accumulated clinical evidence suggesting that p16(INK4a) can serve as a useful biomarker in the routine diagnostic work up of patients with HPV infections and associated lesions of the female anogenital tract. © 2014 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

  10. Expressions of survivin, P16(INK4a), COX-2, and Ki-67 in cervical cancer progression reveal the potential clinical application.

    PubMed

    Zhou, W Q; Sheng, Q Y; Sheng, Y H; Hou, W J; Xu, G X; Wu, Y M; Lu, H

    2015-01-01

    To explore the significance of survivin, P16(INK4a), COX-2, and Ki-67 expressions for prediction of cervical cancer progression. A retrospective study was performed in 129 cases including 24 squamous carcinoma of the cervix (SCC), 70 cervical intraepithelial neoplasias (CIN), 15 cervical condyloma acuminatum (CCA), ten chronic cervicitis (CC), and ten normal cervix (NC). Protein expressions were evaluated using immunohistochemistry. Survivin, P16(INK4a); COX-2, and Ki-67 were highly expressed in SCC and CIN compared with others. Their expression rates were gradually increased in CIN I, CIN II, CIN III, and SCC groups, showing 72.00%, 88.00%, 90.00%, and 95.83% for P16(INK4a), 68.00%, 84.00%, 95.00% and 100.00% for COX-2, 76.00%, 96.00%, 100.00%, and 100.00 for Ki-67, respectively. There were significant correlations between survivin and P16(INK4a), COX-2, Ki-67, as well as P16(INK4a) and Ki-67. Survivin, P16(INK4a), COX-2 and Ki-67 play critical roles for development and progression of cervical cancer.

  11. Human papillomavirus, p16INK4A, and Ki-67 in relation to clinicopathological variables and survival in primary carcinoma of the vagina

    PubMed Central

    Hellman, K; Lindquist, D; Ranhem, C; Wilander, E; Andersson, S

    2014-01-01

    Background: This study aimed to determine human papillomavirus (HPV) status and to investigate p16INK4A and Ki-67 expression and their correlation with clinical parameters and survival in women with primary carcinoma of the vagina (PCV). Methods: The presence of HPV DNA was evaluated by PCR. Genotyping was performed by Luminex in 68 short-term (⩽2 years) and long-term (⩾8 years) PCV survivors. p16INK4A and Ki-67 expression was evaluated by immunohistochemistry. Results: Human papillomavirus DNA was detected in 43% of patients, the majority (63%) of whom were HPV16 positive. High p16INK4A expression was significantly correlated with low histopathological grade (P=0.004), HPV positivity (P=0.032), and long-term survival (P=0.045). High Ki-67 expression was negatively correlated with histopathological grade (P<0.001) and tumour size (P=0.047). There was an association between HPV positivity and low histopathological grade, but not between HPV positivity and survival. Conclusion: High p16INK4A expression was associated with long-term survival, but the only independent predictors for survival were tumour size and histopathological grade. Our results indicate that p16INK4A and Ki-67 expression might be useful in tumour grading, and that it might be possible to use p16INK4A expression as a marker for HPV positivity, but this has to be further elucidated. PMID:24525695

  12. Activation of the BRCA1/Chk1/p53/p21(Cip1/Waf1) pathway by nitric oxide and cell cycle arrest in human neuroblastoma NB69 cells.

    PubMed

    Van de Wouwer, Marlies; Couzinié, Célia; Serrano-Palero, Miguel; González-Fernández, Oscar; Galmés-Varela, Clara; Menéndez-Antolí, Paula; Grau, Laura; Villalobo, Antonio

    2012-03-31

    Nitric oxide (NO) works as a bi-modal effector of cell proliferation, inducing either the increase or decrease of cell growth when cells are exposed, respectively, to low or high NO concentrations. To get further insight into the action of NO, we tested the effect of short- and long-lived NO donors on the control of the cell cycle in human neuroblastoma NB69 cells. We demonstrated that long-time exposure of cells to NO not only decreased the expression and/or the phosphorylation of elements involved in the control of the G(1)/S transition, such as the transcriptional repressor pRb and cyclin D1, but also down-regulated systems controlling the S and G(2)/M phases, such as the phosphorylation of Cdk1(cdc2) and the expression of cyclins A and B1. Increasing concentrations of NO also induced a biphasic effect on the expression of cyclins D1, A and B1, while this effect was less pronounced for cyclin E expression, but the levels of mRNAs of those cyclins changed in a distinct and complex manner. NO also changed the phosphorylation pattern of cyclin E and decreased the levels of phospho-cyclins D1 and B1. Moreover, NO decreased the expression of the Cdk inhibitors p16(Ink4a) and p19(Ink4d), without affecting p27(Kip1). In contrast, NO induced a biphasic effect on p21(Cip1/Waf1) expression. The BRCA1/Chk1/p53 pathway mediated the upregulation of p21(Cip1/Waf1). We also demonstrated that the NO-mediated up-regulation of p21(Cip1/Waf1) was inversely correlated with the activation status of the p38MAPK pathway. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. 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.

  14. 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.

  15. Suppressing P16(Ink4a) and P14(ARF) pathways overcomes apoptosis in individualized human embryonic stem cells.

    PubMed

    Wang, Wenqian; Zhu, Yanling; Huang, Ke; Shan, Yongli; Du, Juan; Dong, Xiaoya; Ma, Ping; Wu, Penafei; Zhang, Jian; Huang, Wenhao; Zhang, Tian; Liao, Baojian; Yao, Deyang; Pan, Guangjin; Liu, Jiajun

    2017-03-01

    Dissociation-induced apoptosis is a striking phenomenon in human embryonic stem cells (hESCs), but not in naive mouse ESCs. Rho-associated kinase-dependent actin-myosin hyperactivation is an underlying mechanism that triggers apoptosis in dissociated hESCs; however, in this study, we show that the Ink4A-ARF-mediated senescence pathway is another mechanism to cause apoptosis in individualized hESCs. We show that P16(INK4A) and P14(ARF) are immediately induced in hESCs upon dissociation, but not in mouse ESCs. Overexpression of BMI1, a suppressor for Ink4A-ARF, greatly promotes survival and cloning efficiency of individualized hESCs mechanistically via direct binding the H3K27me3-marked Ink4A-ARF locus. Forced expression of BMI1 in hESCs does not reduce the actin-myosin activation that is triggered by dissociation, which indicates it is an independent pathway for hESC survival. Furthermore, dual inhibition of both Ink4A-ARF and actin-myosin hyperactivation enables successful passaging of hESCs via gelatin, a nonbioactive matrix. In sum, we provide an additional mechanism that underlies cell death in individualized hESCs that might help to fully understand the differential cell characteristics between naive and primed ESCs.-Wang, W., Zhu, Y., Huang, K., Shan, Y., Du, J., Dong, X., Ma, P., Wu, P., Zhang, J., Huang, W., Zhang, T., Liao, B., Yao, D., Pan, G., Liu, J. Suppressing P16(Ink4a) and P14(ARF) pathways overcomes apoptosis in individualized human embryonic stem cells.

  16. Aurora B Overexpression Causes Aneuploidy and p21Cip1 Repression during Tumor Development.

    PubMed

    González-Loyola, Alejandra; Fernández-Miranda, Gonzalo; Trakala, Marianna; Partida, David; Samejima, Kumiko; Ogawa, Hiromi; Cañamero, Marta; de Martino, Alba; Martínez-Ramírez, Ángel; de Cárcer, Guillermo; Pérez de Castro, Ignacio; Earnshaw, William C; Malumbres, Marcos

    2015-10-01

    Aurora kinase B, one of the three members of the mammalian Aurora kinase family, is the catalytic component of the chromosomal passenger complex, an essential regulator of chromosome segregation in mitosis. Aurora B is overexpressed in human tumors although whether this kinase may function as an oncogene in vivo is not established. Here, we report a new mouse model in which expression of the endogenous Aurkb locus can be induced in vitro and in vivo. Overexpression of Aurora B in cultured cells induces defective chromosome segregation and aneuploidy. Long-term overexpression of Aurora B in vivo results in aneuploidy and the development of multiple spontaneous tumors in adult mice, including a high incidence of lymphomas. Overexpression of Aurora B also results in a reduced DNA damage response and decreased levels of the p53 target p21(Cip1) in vitro and in vivo, in line with an inverse correlation between Aurora B and p21(Cip1) expression in human leukemias. Thus, overexpression of Aurora B may contribute to tumor formation not only by inducing chromosomal instability but also by suppressing the function of the cell cycle inhibitor p21(Cip1).

  17. Aurora B Overexpression Causes Aneuploidy and p21Cip1 Repression during Tumor Development

    PubMed Central

    González-Loyola, Alejandra; Fernández-Miranda, Gonzalo; Trakala, Marianna; Partida, David; Samejima, Kumiko; Ogawa, Hiromi; Cañamero, Marta; de Martino, Alba; Martínez-Ramírez, Ángel; de Cárcer, Guillermo; Pérez de Castro, Ignacio; Earnshaw, William C.

    2015-01-01

    Aurora kinase B, one of the three members of the mammalian Aurora kinase family, is the catalytic component of the chromosomal passenger complex, an essential regulator of chromosome segregation in mitosis. Aurora B is overexpressed in human tumors although whether this kinase may function as an oncogene in vivo is not established. Here, we report a new mouse model in which expression of the endogenous Aurkb locus can be induced in vitro and in vivo. Overexpression of Aurora B in cultured cells induces defective chromosome segregation and aneuploidy. Long-term overexpression of Aurora B in vivo results in aneuploidy and the development of multiple spontaneous tumors in adult mice, including a high incidence of lymphomas. Overexpression of Aurora B also results in a reduced DNA damage response and decreased levels of the p53 target p21Cip1 in vitro and in vivo, in line with an inverse correlation between Aurora B and p21Cip1 expression in human leukemias. Thus, overexpression of Aurora B may contribute to tumor formation not only by inducing chromosomal instability but also by suppressing the function of the cell cycle inhibitor p21Cip1. PMID:26240282

  18. p16INK4A, p53, EGFR expression and KRAS mutation status in squamous cell cancers of the anus: correlation with outcomes following chemo-radiotherapy.

    PubMed

    Gilbert, Duncan C; Williams, Anthony; Allan, Kimberley; Stokoe, Joanna; Jackson, Tim; Linsdall, Suzanne; Bailey, Charles Mh; Summers, Jeff

    2013-10-01

    Squamous cell carcinomas of the anal canal are associated with infection with Human Papilloma Viruses (HPVs). Chemo-radiotherapy (CRT) gives 70% 3-year relapse-free survival. Improved predictive markers and therapeutic options are required. Tumours from 153 patients treated with radical chemo-radiotherapy (50.4 Gy in 28# with concurrent Mitomycin and 5-Fluorouracil between 2004 and 2009) were retrieved and immunohistochemistry performed for p16(INK4A), p53 and EGFR and correlated with outcome. Primary and relapsed samples were analysed for mutations in KRAS. 137/153 (89.5%) stained moderately or strongly for p16(INK4A). p16(INK4A) correlated strongly with outcome. 37/137 patients demonstrating moderate/strong p16(INK4A) expression relapsed (27.0%), as opposed to 10/16 (62.5%) with absent/weak staining (log rank test p<0.001). p16 and p53 expression were inversely correlated. p16(INK4A) negative tumours were more frequent in men. p16(INK4A) negative patients had significantly worse overall survival (p<0.001). No mutations in KRAS were identified in primary tumours or relapses following treatment. p16(INK4A) is strongly associated with relapse in SCC of the anus and identifies patients with very poor rates of relapse-free and overall survival. Primary and recurrent anal cancer expresses wild type KRAS, unaffected by treatment, supporting trials targeting EGFR in poor risk/recurrent anal cancer. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. 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

  20. Co-Regulation of p16INK4a and Migratory Genes in Culture Conditions that Lead to Premature Senescence in Human Keratinocytes

    PubMed Central

    Darbro, Benjamin W.; Schneider, Galen B.; Klingelhutz, Aloysius J.

    2007-01-01

    Cellular stasis, also known as telomere-independent senescence, prevents many epithelial cells from becoming immortalized by telomerase alone. As human keratinocytes age in culture, protein levels of the tumor suppressor p16INK4a continue to increase, resulting in growth arrest independent of telomere length. Differences in culture conditions have been shown to modulate both p16INK4a expression and replicative capacity of human keratinocytes; however, the mechanism of p16INK4a induction under these conditions is unknown. Using multiple primary keratinocyte cell strains, we verified a delay in p16INK4a induction and an extended lifespan of human keratinocytes when grown in co-culture with post-mitotic fibroblast feeder cells as compared with keratinocytes grown on tissue culture plastic alone. Evaluation of gene expression levels in the two culture conditions by microarray analysis, and subsequent validation, demonstrated that keratinocytes cultured on plastic alone had significantly increased expression of many genes involved in keratinocyte migration and reduced expression levels of genes involved in keratinocyte differentiation. Higher levels of p16INK4a expression were present in cells that also displayed increased amounts of autophosphorylated focal adhesion kinase and urokinase plaminogen activator receptor (uPAR), both markers of keratinocyte migration. Furthermore, when tyrosine phosphorylation or urokinase-type plasminogen activator (uPA)/uPAR function was inhibited, both keratinocyte migration and p16INK4a expression were reduced. Our results indicate that keratinocytes cultured in the absence of feeder cells exhibit a migratory phenotype and suggest that p16INK4a is selectively induced under these conditions by a mechanism involving tyrosine kinase activity and the urokinase plasminogen activation system. PMID:16117791

  1. p16(INK4a) immunostaining in cytological and histological specimens from the uterine cervix: a systematic review and meta-analysis.

    PubMed

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

    2009-05-01

    P16(INK4a) 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. We conducted a systematic review of all studies that evaluated the use of p16(INK4a) in cytological or histological specimens from the uterine cervix. We also estimated the mean proportion of samples that were positive for p16(INK4a) in cytology and histology, stratified by the grade of the lesion. Sixty-one studies were included. The proportion of cervical smears overexpressing p16(INK4a) 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 p16(INK4a) compared to 68% of CIN2 (95% CI: 44-92%) and 82% of CIN3 (95% CI: 72-92%). Although there is good evidence that p16(INK4a) 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 p16(INK4a) staining and the biomarker needs to be assessed in various clinical settings addressing specific clinical questions.

  2. p16(INK4a) overexpression is not linked to oncogenic human papillomaviruses in patients with high-grade urothelial cancer cells.

    PubMed

    Piaton, Eric; Casalegno, Jean-Sébastien; Advenier, Anne-Sophie; Decaussin-Petrucci, Myriam; Mege-Lechevallier, Florence; Ruffion, Alain; Mekki, Yahia

    2014-10-01

    p16(INK4a) Is overexpressed in almost all precancerous and carcinomatous lesions of the uterine cervix, secondary to interference between high-risk human papillomaviruses (hr-HPVs) and the retinoblastoma gene product. Overexpression of p16(INK4a) has also been identified in patients with high-grade urothelial lesions, both cytologically and histologically. However, the etiological role of HPV has not been documented except in inverted papillomas, low-grade bladder tumors, and younger patients. We therefore attempted to verify if HPV DNA was detectable in p16(INK4a) -positive urothelial tumors. A total of 90 urinary cytology samples (33 negative/low-grade cases and 57 high-grade proliferations) were analyzed for p16(INK4a) and HPV DNA. HPV genotyping was performed by polymerase chain reaction using a low-density DNA microarray enabling the detection of 35 HPVs. A reasoned approach combining tissue genotyping and in situ hybridization (ISH) for hr-HPVs was used in patients with urinary HPV. Low-risk HPV (HPV-84) and hr-HPVs (HPV-16, -31, and -70) were detected. The prevalence of hr-HPVs in the urine was low: 5 of 82 patients (6.1%) and only 4 of 50 patients (8.0%) with high-grade urothelial malignancy. p16(INK4a) overexpression was noted in 49 high-grade samples (85.9%). In patients with p16(INK4a) -positive tumor cells and hr-HPV in the urine, HPV genotyping and ISH for hr-HPVs were negative in matched tissue sections. Our study shows a low prevalence of hr-HPVs in the urine of patients with high-grade urothelial malignancy. In those, p16(INK4a) overexpression occurs in the absence of demonstrable HPV DNA in the tissue sections, contrary to what is noted in gynecopathology. © 2014 American Cancer Society.

  3. 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.

  4. Different roles for p16(INK) (4a) -Rb pathway and INK4a/ARF methylation between adenocarcinomas of gastric cardia and distal stomach.

    PubMed

    Xue, Liying; Ouyang, Qin; Li, Jie; Meng, Xinxing; Li, Yuehong; Xing, Lingxiao; Wang, Junling; Yan, Xia; Zhang, Xianghong

    2014-01-01

    The incidence of distal gastric adenocarcinoma has significantly decreased, but gastric cardia adenocarcinoma has been on the rise. Cardia adenocarcinoma might be a specific entity distinct from the carcinoma of the rest stomach. The aim was to explore putative differences in p16(INK) (4a) -retinoblastoma (Rb) pathway and INK4a/ARF methylation between gastric cardia and distal adenocarcinomas. Ninety-six cardia adenocarcinomas and 79 distal samples were analyzed for comparing p16(INK) (4a) -Rb expressions, INK4a/ARF deletion, and methylation using immunohistochemistry, polymerase chain reaction, and methylation-specific polymerase chain reaction. The expression of p16(INK) (4a) in cardia adenocarcinoma (43.2%) was significantly lower than in distal cases (75.0%, P < 0.05). As well, cardia adenocarcinoma showed lower expression of p14(ARF) compared with distal cases (34.1% vs 57.5%, P < 0.05). The incidence of p16(INK) (4a) deletion was 20.5% and 15.0%, while p14(ARF) deletion was 18.2% and 10.0% in cardia and distal adenocarcinomas, respectively, showing no significant differences between two entities. However, the incidences of p14(ARF) and p16(INK) (4a) methylation in cardia adenocarcinoma were significantly higher than in distal samples (p14(ARF) : 61.5% vs 43.6%; p16(INK) (4a) : 73.1% vs 51.3%, P < 0.05). INK4a/ARF methylations were more prevalent in poorly differentiated cardia carcinoma compared with poorly differentiated distal cases. There were differences in p16(INK) (4a) -Rb immunotypes and INK4a/ARF methylation between two entities, indicating that cardia adenocarcinoma may be different in cell proliferation, differentiation, and gene biomarkers compared with distal gastric adenocarcinoma. © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  5. Prognostic value of p16(INK4a) as a marker of clinical evolution in patients with cervical intraepithelial neoplasia grade 3 (CIN 3) treated by cervical conization.

    PubMed

    Cardoso, Fernanda Araujo; Campaner, Adriana Bittencourt; Silva, Maria Antonieta Longo Galvão

    2014-03-01

    The aim of this study was to assess the prognostic value of p16(INK4a) as a marker of post-conization relapse in patients treated for cervical intraepithelial neoplasia grade 3 (CIN 3). A retrospective study of 76 women with CIN 3 diagnoses, treated at the Hospital of Santa Casa de Misericórdia of São Paulo (Brazil) between January 2003 and September 2004, was performed. The study samples were obtained from cervical conization procedures, where paraffin blocks containing areas with the greatest amount of neoplastic tissue were selected. Immunohistochemical techniques were used on individual paraffin blocks for each case to detect p16(INK4a) protein expression. The p16(INK4a) cell counts were performed in 10 different high-amplification fields (400x) by light microscopy and total cell count expressed as number of cells per mm(2) . Patients involved in this study were followed up at the colposcopy outpatient unit for at least 48 months after cervical conization. The correlation of p16(INK4a) values with post-conization evolution in the patients (disease relapse or disease free) was determined. A significantly higher count of cells expressing p16(INK4a) was found in those patients with disease relapse during follow-up (p < 0.001). The variables age, number of gestations, and births correlated positively with number of cells expressing p16(INK4a) cells (p < 0.001; p = 0.001; 0.009, respectively). No correlation was found for the variables menopause, hormonal contraception, or smoking (p = 0.369, 0.425 and 0.853, respectively). p16(INK4a) can be considered a biomarker of cervical intraepithelial neoplasia grade 3 cases presenting high risk of relapse or evolution to invasive carcinoma. © 2013 APMIS. Published by John Wiley & Sons Ltd.

  6. p16 INK4a and Ki67 expression in normal, dysplastic and neoplastic uterine cervical epithelium and human papillomavirus (HPV) infection.

    PubMed

    Calil, L N; Edelweiss, M I A; Meurer, L; Igansi, C N; Bozzetti, M C

    2014-08-01

    Cellular cycle proteins like the p16(INK4a) and the Ki67 proliferation nuclear antigen have been used as oncogenicity cellular markers. The E6 and E7 oncoproteins interact with tumor suppressor genes p53 and pRb, culminating with the p16(INK4a) overexpression. The objective of this study was to evaluate the presence of HPV-DNA in 174 cervical biopsies and correlate the different histological grades with the p16(INK4a) and Ki67 immunohistochemical expression (IHC). A cross-sectional study that enrolled a total of 174 women who underwent uterine cervical biopsies between February 2003 and December 2006, in southern Brazil, was performed. Cervical smear samples were analyzed for the presence of HPV-DNA through polymerase chain reaction (PCR), and biopsy samples were examined for p16(INK4A) and Ki67 expression through IHC techniques. The presence of HPV-DNA was observed in 89% of the tested patients, among which 52% were positive for high-risk (HR) viral types [16, 18 and 31]. Regarding p16(INK4a), an expression of 69% was observed, being expressed in 100% of the high-grade squamous lesions (HSIL) and HR-HPV-DNA positives. Ki67 expression was associated with the lesion grade, being more expressive in the most severe lesions (p<0.001). p16(INK4A) and Ki67 markers coexpression was present in 86% of the samples (p<0.001), being 100% among those positive to HR-HPV-DNA with HSIL (p<0.001). The results suggest an association between the presence of HR-HPV infection and the p16(INK4a) and Ki67 expression and which is even stronger among women with HSIL. Copyright © 2014 Elsevier GmbH. All rights reserved.

  7. Diagnostic accuracy of p16(INK4a) immunohistochemistry in oropharyngeal squamous cell carcinomas: A systematic review and meta-analysis.

    PubMed

    Prigge, Elena-Sophie; Arbyn, Marc; von Knebel Doeberitz, Magnus; Reuschenbach, Miriam

    2017-03-01

    The accurate diagnosis of human papillomavirus (HPV) causality in oropharyngeal squamous cell carcinomas (OPSCC) is likely to influence therapeutic decisions in affected patients in the near future. We conducted a systematic review and meta-analysis to determine the diagnostic accuracy of p16(INK4a) immunohistochemistry (IHC) to identify HPV-induced OPSCC. We identified all studies that performed p16(INK4a) IHC (index test) and HPV E6/E7 mRNA detection using an amplification-based method (gold standard to indicate a transforming relevance of HPV) in OPSCC. Testing with one or more comparator tests (HPV DNA PCR, HPV DNA in situ hybridization (ISH) and p16(INK4a) IHC/HPV DNA PCR combined testing) was an optional criterion for inclusion. Among 1,636 retrieved studies 24 fulfilled the inclusion criteria. The pooled sensitivity of p16(INK4a) IHC, HPV DNA PCR, HPV DNA ISH and p16(INK4a) IHC/HPV DNA PCR combined testing was 94% (95%-confidence interval (CI) 91-97%), 98% (CI 94-100%), 85% (CI 76-92%) and 93% (CI 87-97%), respectively. The pooled specificity was 83% (CI 78-88%), 84% (CI 74-92%), 88% (CI 78-96%) and 96% (CI 89-100%), respectively. p16(INK4a) IHC/HPV DNA PCR combined testing was as sensitive as either p16(INK4a) IHC or HPV DNA PCR alone but significantly more specific than either separate test. In conclusion, p16(INK4a) IHC is highly sensitive but moderately specific to diagnose HPV-transformed OPSCC when used as a single test. Combined p16(INK4a) IHC and HPV DNA PCR testing significantly enhances specificity while maintaining high sensitivity. This diagnostic test combination thus represents an attractive testing strategy for the reliable diagnosis of HPV-induced OPSCC in the clinical setting and may constitute an inclusion criterion for future therapeutic trials. © 2016 UICC.

  8. [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.

  9. 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

  10. Transcriptional factor HBP1 targets P16(INK4A), upregulating its expression and consequently is involved in Ras-induced premature senescence.

    PubMed

    Li, H; Wang, W; Liu, X; Paulson, K E; Yee, A S; Zhang, X

    2010-09-09

    Oncogene-mediated premature senescence has emerged as a potential tumor-suppressive mechanism in early cancer transitions. Many studies showed that Ras and p38 mitogen-activated protein kinase (MAPK) participate in premature senescence. Our previous work indicated that the HMG box-containing protein 1 (HBP1) transcription factor is involved in Ras- and p38 MAPK-induced premature senescence, but the mechanism of which has not yet been identified. Here, we showed that the p16(INK4A) cyclin-dependent kinase inhibitor is a novel target of HBP1 participating in Ras-induced premature senescence. The promoter of the p16(INK4A) gene contains an HBP1-binding site at position -426 to -433 bp from the transcriptional start site. HBP1 regulates the expression of the endogenous p16(INK4A) gene through direct sequence-specific binding. With HBP1 expression and the subsequent increase of p16(INK4A) gene expression, Ras induces premature senescence in primary cells. The data suggest a model in which Ras and p38 MAPK signaling engage HBP1 and p16(INK4A) to trigger premature senescence. In addition, we report that HBP1 knockdown is also required for Ras-induced transformation. All the data indicate that the mechanism of HBP1-mediated transcriptional regulation is important for not only premature senescence but also tumorigenesis.

  11. Human Papillomavirus Genotyping and p16(INK4a) Expression in Cervical Lesions: A Combined Test to Avoid Cervical Cancer Progression.

    PubMed

    Zouheir, Yassine; Fechtali, Taoufiq; Elgnaoui, Nadia

    2016-06-01

    Cervical cancer is a major public health problem in Morocco. The cervical cancer has a long precancerous period that provides an opportunity for the screening and treatment. Improving screening tests is a priority goal for the early diagnosis of cervical cancer. This study was conducted to evaluate the combination of p16(INK4a) protein expression, human papillomavirus (HPV) typing, and histopathology for the identification of cervical lesions with high risk to progress to cervical cancer among Moroccan women. A total of 96 cervical biopsies were included in this study. Signal amplification in situ hybridization with biotinylated probes was used to detect HPV. Immunohistochemistry was used to evaluate the expression of p16(INK4a) protein. HPV DNA was detected in 74.0% of the biopsies (71/96). Of the seventy-one positive HPV cases, we detected 67.6% (48/71) of high risk (HR)-HPV (HPV 16 and 18), 24% of low risk-HPV (HPV 6 and 11), 1.4% intermediate risk-HPV (HPV 31, 33, and 35), and 7% coinfections (HPV 6/11 and 16/18). Overexpression of p16(INK4a) protein was observed in 72.9% (70/96) of the biopsies. In addition, p16(INK4a) protein detection was closely correlated with recovery of HR HPV. Our result showed that p16(INK4a) expression level is correlated with HR-HPV status.

  12. Cloning and characterization of a novel p21(Cip1/Waf1)-interacting zinc finger protein, ciz1.

    PubMed

    Mitsui, K; Matsumoto, A; Ohtsuka, S; Ohtsubo, M; Yoshimura, A

    1999-10-22

    p21(Cip1/Waf1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. Here, we report a novel p21(Cip1/Waf1)-interacting protein, Ciz1 (for Cip1 interacting zinc finger protein), which contains polyglutamine repeats and glutamine-rich region in the N-terminus as well as three zinc-finger motifs and one MH3 (matrin 3-homologous domain 3) in the C-terminal region. Ciz1 bound to the N-terminal, the CDK2-interacting part of p21(Cip1/Waf1), and the interaction was disrupted by the overexpression of CDK2. A region of about 150 amino acids containing the first zinc-finger motif in Ciz1 was the binding site for p21(Cip1/Waf1). When Ciz1 and p21(Cip1/Waf1) were individually overexpressed in U2-OS cells, they mostly localized in the nucleus. However, coexpression of Ciz1 induced cytoplasmic distribution of p21(Cip1/Waf1). These data indicate that Ciz1 is a unique nuclear protein that regulates the cellular localization of p21(Cip1/Waf1).

  13. p16(INK4a)/CDKN2 expression and its relationship with oral squamous cell carcinoma is our current knowledge enough?

    PubMed

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

    2011-07-28

    Oral squamous cell carcinomas (OSCC) are the most common malignancy of the oral cavity and their multistep development requires the accumulation of multiple genetic and epigenetic alterations. Inactivation of p16(INK4a), encoded by the CDKN2 gene has been widely associated with this type of tumors. The purpose of this review is to elucidate the relationship between p16(INK4a) expression and the different clinical and pathological aspects of OSCC, analyze the variation in results between studies, detailing the described genetic/epigenetic alterations that result in gene silencing and the relationship between p16(INK4a) and HPV infection. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed Central

    Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

    2016-01-01

    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. PMID:27465215

  15. BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence

    PubMed Central

    Hayashi, Yohei; Hsiao, Edward C.; Sami, Salma; Lancero, Mariselle; Schlieve, Christopher R.; Nguyen, Trieu; Yano, Koyori; Nagahashi, Ayako; Ikeya, Makoto; Matsumoto, Yoshihisa; Nishimura, Ken; Fukuda, Aya; Hisatake, Koji; Tomoda, Kiichiro; Asaka, Isao; Toguchida, Junya; Conklin, Bruce R.; Yamanaka, Shinya

    2016-01-01

    Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes. PMID:27794120

  16. Reversible cell cycle inhibition and premature aging features imposed by conditional expression of p16Ink4a

    PubMed Central

    Boquoi, Amelie; Arora, Sanjeevani; Chen, Tina; Litwin, Sam; Koh, James; Enders, Greg H

    2015-01-01

    The cyclin-dependent kinase (Cdk) inhibitor p16Ink4a (p16) is a canonical mediator of cellular senescence and accumulates in aging tissues, where it constrains proliferation of some progenitor cells. However, whether p16 induction in tissues is sufficient to inhibit cell proliferation, mediate senescence, and/or impose aging features has remained unclear. To address these issues, we generated transgenic mice that permit conditional p16 expression. Broad induction at weaning inhibited proliferation of intestinal transit-amplifying and Lgr5+ stem cells and rapidly imposed features of aging, including hair loss, skin wrinkling, reduced body weight and subcutaneous fat, an increased myeloid fraction in peripheral blood, poor dentition, and cataracts. Aging features were observed with multiple combinations of p16 transgenes and transactivators and were largely abrogated by a germline Cdk4 R24C mutation, confirming that they reflect Cdk inhibition. Senescence markers were not found, and de-induction of p16, even after weeks of sustained expression, allowed rapid recovery of intestinal cell proliferation and reversal of aging features in most mice. These results suggest that p16-mediated inhibition of Cdk activity is sufficient to inhibit cell proliferation and impose aging features in somatic tissues of mammals and that at least some of these aging features are reversible. PMID:25481981

  17. Influence of human papillomavirus and p16(INK4a) on treatment outcome of patients with anal cancer.

    PubMed

    Koerber, Stefan Alexander; Schoneweg, Clara; Slynko, Alla; Krug, David; Haefner, Matthias F; Herfarth, Klaus; Debus, Juergen; Sterzing, Florian; von Knebel Doeberitz, Magnus; Prigge, Elena-Sophie; Reuschenbach, Miriam

    2014-12-01

    The purpose of this study was to evaluate HPV-DNA and p16(INK4a) (p16) expression as prognostic markers for outcome in patients with anal cancer. From January 2000 to December 2011 a cohort of 105 anal cancer patients was treated with definitive chemoradiation at our institution. Tumor biopsies from 90 patients were analyzed for HPV-DNA by polymerase chain reaction and for p16 expression by immunohistochemistry. Median follow-up was 48.6months (range 2.8-169.1months). HPV-DNA or p16-expression was found in 75 anal cancers each (83.3%), concordance was detectable in 70 tumors (77.8%). Significantly improved overall survival (OS) [77.1% vs. 51.4%, p=0.005], progression-free survival (PFS) [64.0% vs. 35.0%, p<0.001] and improved local control [81.0% vs. 55.9%, p=0.023] was found for concomitant HPV- and p16-positive anal carcinomas (cHPPAC) in univariate analysis. Multivariate analysis showed better OS [p=0.015] and PFS [p=0.002] for cHPPAC. The combination of HPV-DNA and p16 can be used as an independent prognostic parameter in anal cancer patients. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  18. Impact of p16(INK4a) immunohistochemistry staining on interobserver agreement on the diagnosis of cervical intraepithelial neoplasia.

    PubMed

    Pacchiarotti, Alberto; Galeotti, Simona; Bellardini, Paola; Chini, Francesco; Collina, Guido; Dalla Palma, Paolo; Ghiringhello, Bruno; Maccallini, Vincenzo; Musolino, Fabio; Negri, Giovanni; Pisa, Roberto; Sabatucci, Ilaria; Giorgi Rossi, Paolo

    2014-03-01

    This study aimed to compare the interobserver Cohen κ on H&E staining and on H&E plus p16(INK4a) staining of all cervical biopsy specimens in a population-based screening program. All the colposcopy-guided biopsies generated by the routine screening of 23,258 women aged 25 to 64 years were stained with H&E and H&E plus p16. Biopsy specimens were reviewed by six external experts. The four diagnoses were available in 441 cases. The interobserver κ values were 0.52 (95% confidence interval [CI], 0.45-0.58) and 0.48 (95% CI, 0.42-0.56) with H&E and H&E + p16, respectively, when using a five-group classification (normal, CIN 1, CIN 2, CIN 3, and cancer); adopting a two-group classification (≤CIN 1 and ≥CIN 2), the values were 0.75 (95% CI, 0.66-0.82) and 0.70 (95% CI, 0.61-0.79), respectively. The use of p16 on all cervical biopsy specimens in a screening program showed virtually no effect on reproducibility of the histologic diagnosis.

  19. 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.

  20. 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

  1. Cigarette Smoke Extract Changes Expression of Endothelial Nitric Oxide Synthase (eNOS) and p16(INK4a) and is Related to Endothelial Progenitor Cell Dysfunction.

    PubMed

    He, Zhihui; Chen, Yan; Hou, Can; He, Wenfang; Chen, Ping

    2017-07-02

    BACKGROUND Endothelial dysfunction is an important pathophysiologic feature in many smoke-related diseases. Endothelial progenitor cells (EPCs) are the precursors of endothelial cells and play a fundamental role in the maintenance of endothelial integrity and function. Endothelial nitric oxide synthase (eNOS) is the dominant NOS isoform in the vasculature and plays a central role in the maintenance of endothelial homeostasis. p16(INK4a) is a cyclin-dependent kinase inhibitor and could be regarded as a major dominant senescence gene. The present study aimed to determine whether the expression of eNOS and p16(INK4a) in EPCs is related to EPCs function and the possible epigenetic mechanism, if any. MATERIAL AND METHODS We investigated EPCs capacity for proliferation, adhesion, and secretion, and the expression of eNOS and p16(INK4a) in EPCs which were altered by cigarette smoke extract (CSE) in vitro. Furthermore, Decitabine (Dec), an agent of demethylation, was used to examine whether it could alter the changes induced by CSE. RESULTS The present study demonstrated that EPCs altered by CSE in vitro displayed decreased capacities of proliferation, adhesion, and secretion, which was accompanied by decreased eNOS expression and increased p16(INK4a) expression in EPCs. Furthermore, Dec could alleviate the changes in the expression of eNOS and p16(INK4a), and protect against the EPCs dysfunction caused by CSE. CONCLUSIONS The decreased eNOS expression and increased p16(INK4a) expression was associated with dysfunction of EPCs caused by CSE. The mechanism of methylation, one of the most common epigenetic mechanism, may be involved in the EPCs dysfunction caused by CSE.

  2. Human papillomavirus shows highly variable prevalence in esophageal squamous cell carcinoma and no significant correlation to p16INK4a overexpression: a systematic review.

    PubMed

    Michaelsen, Sanne Høxbroe; Larsen, Christian Grønhøj; von Buchwald, Christian

    2014-06-01

    This review investigates the role of p16(INK4a) as a marker of transcriptionally active human papillomavirus (HPV) in esophageal squamous cell carcinoma (ESCC) and the regional prevalence of HPV in ESCC. PubMed, EMBASE, and the Cochrane Library were systematically searched with the purpose of identifying all studies published between January 1980 and July 2013 reporting both HPV and p16 results in a minimum of five human ESCC specimens. Twelve studies were identified, providing data on a total of 1383 ESCC specimens collected between 1987 and 2009 from 10 different countries. HPV DNA was detected in 12.0% (n = 161) of 1347 specimens, and p16(INK4a) was detected in 33.9% (n = 209) of 617 specimens. The HPV presence varied from 0% to 70% among the studies. The prevalence of p16(INK4a) overexpression in HPV-positive and HPV-negative specimens demonstrated no statistically significant difference, neither for the combined data (p = 0.7507) nor for any individual study, and detection of p16(INK4a) overexpression did not affect the odds of tumors being HPV positive (odds ratio = 1.0666 with 95% confidence interval 0.7040-1.6157). In a pooled analysis, the sensitivity of p16(INK4a) overexpression as a marker of HPV DNA presence was 0.35, the specificity 0.67, and the positive predictive value 0.25. This systematic review reports great regional variation in the prevalence of HPV in ESCC and suggests that p16(INK4a) is not a reliable marker of HPV status in ESCC.

  3. Differential expression of p16(INK4A) and cyclin D1 in benign and malignant salivary gland tumors: a study of 44 Cases.

    PubMed

    Jour, G; West, K; Ghali, V; Shank, D; Ephrem, G; Wenig, B M

    2013-09-01

    Salivary gland tumors (SGT) are a heterogeneous group of lesions. There is conflicting data concerning the molecular events involving the tumour suppressor retinoblastoma protein (pRb) pathway in these tumors. Few studies examined the alterations in components of the Rb pathway by immunohistochemical (IHC) methods in benign and malignant SGTs. Furthermore, recent evidence implicates human papillomavirus (HPV) in mucoepidermoid carcinoma (MEC) carcinogenesis. The purpose of our study is to examine p16(INK4A) and cyclin D1 expression in a variety of benign and malignant salivary gland tumors, and to investigate p16(INK4A) expression as a surrogate marker for HPV infection in MEC. Our series includes 30 malignant tumors [14 MEC, 6 acinic cell carcinomas (ACC), 5 polymorphous low grade adenocarcinomas (PLGA), 5 (AdCC)] and 14 benign tumors (4 benign cysts, 5 Warthin tumors and 5 pleomorphic adenomas (PA). All cases were tested by IHC for p16(INK4A) and cyclin D1. Testing for HPV wide spectrum (HPV-WS) was performed by in situ hybridization in all MEC cases. Staining intensity was recorded semi quantitatively (on a scale from 0 to 4+). Fisher's exact test and Pearson X2 test with a p < 0.05 were used. Cyclin D1 and p16(INK4A) are expressed similarly in malignant and benign tumors (p = 0.146 and p = 0.543, respectively). None of the MEC cases showed nuclear reactivity for HPV-WS. Statistical analysis showed positive correlation between cyclin D1 and p16(INK4A) expression. Our findings suggest that p16(INK4A) overexpression is likely secondary to cyclin D1 gene upregulation or amplification. Further molecular studies are warranted.

  4. p16(INK4A) induces senescence and inhibits EMT through microRNA-141/microRNA-146b-5p-dependent repression of AUF1.

    PubMed

    Al-Khalaf, Huda H; Aboussekhra, Abdelilah

    2017-03-01

    Senescence and epithelial-to-mesenchymal transition (EMT) processes are under the control of common tumor suppressor proteins, EMT transcription factors, and microRNAs. However, the molecular mechanisms that coordinate the functional link between senescence and EMT are still elusive. We have shown here that p16(INK4A) -related induction of senescence is mediated through miR-141 and miR-146b-5p. These two microRNAs are up-regulated in aging human fibroblast and epithelial cells. Furthermore, miR-141 and miR146b-5p trigger cell cycle arrest at G1 phase and induce senescence in primary human fibroblasts and breast cancer cells in the presence and absence of p16(INK4A) . Like p16(INK4A) -induced senescence, miR-141/miR146b-5p-related senescence is not associated with secretory phenotype, and is mediated through the RNA binding protein AUF1. We have further demonstrated that p16(INK4A) and its downstream miRNA targets inhibit EMT through suppressing the EMT inducer ZEB1 in an AUF1-dependent manner. Indeed, AUF1 binds the mRNA of this gene leading to increase in its level. These results indicate that p16(INK4A) controls both senescence and EMT through repressing EMT-related transcription factor via miR-141/miR146b-5p and their target AUF1. This sheds more light on the molecular basis of the tumor suppressive functions of p16(INK4A) , which represses both the proliferative and the migratory/invasive capacities of cells. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. p16(INK4a) is a marker of good prognosis for primary invasive penile squamous cell carcinoma: a multi-institutional study.

    PubMed

    Gunia, Sven; Erbersdobler, Andreas; Hakenberg, Oliver W; Koch, Stefan; May, Matthias

    2012-03-01

    We assessed the prognostic role of p16(INK4a) expression in penile cancer with respect to cancer specific survival. Based on a multi-institutional collaboration wax embedded tissues from 92 surgically treated patients, including 27 with total and 65 with partial penectomy, were retrospectively evaluated. After a central histopathological review by 1 pathologist a tissue microarray was constructed for p16(INK4a) immunostaining. Two independent pathologists evaluated p16(INK4a) expression, which was correlated with cancer specific survival. The κ statistic was used to assess interobserver variability. Univariate and multivariate Cox proportional hazards analysis was applied to assess the independent effects of prognostic factors on cancer specific survival during a median postoperative followup of 32 months (IQR 6-66). The κ statistic revealed excellent interobserver agreement (κ 0.934, p <0.001). Two and 5-year cancer specific survival rates for the entire study cohort were 86% and 74%, respectively. The 2 and 5-year rates for patients without and with p16(INK4a) expression differed significantly (73% and 57% vs 95% and 85%, respectively, p = 0.011). Univariate analysis revealed p16(INK4a) expression as a significant prognostic factor with respect to cancer specific survival (p = 0.018). Multivariate analysis identified koilocytosis (HR 0.24, 95% CI 0.07-0.83, p = 0.024), p16(INK4a) expression (HR 0.44, 95% CI 0.23-0.84, p = 0.013), and histological stage (HR 3.54, 95% CI 1.88-6.67, p <0.001) and grade (HR 2.47, 95% CI 1.00-6.09, p = 0.049) as independent prognostic factors for cancer specific survival. Results show that p16(INK4a) seems to be a prognostic parameter for primary invasive penile cancer with excellent interobserver reproducibility. At pathology laboratories without antibodies against p16(INK4a) conventional histological determination of koilocytosis by the pathologist also appears to provide important prognostic information for cancer specific

  6. p16INK4a immunocytochemistry versus human papillomavirus testing for triage of women with minor cytologic abnormalities: a systematic review and meta-analysis.

    PubMed

    Roelens, Jolien; Reuschenbach, Miriam; von Knebel Doeberitz, Magnus; Wentzensen, Nicolas; Bergeron, Christine; Arbyn, Marc

    2012-10-25

    The best method for identifying women who have minor cervical lesions that require diagnostic workup remains unclear. The authors of this report performed a meta-analysis to assess the accuracy of cyclin-dependent kinase inhibitor 2A (p16(INK4a)) immunocytochemistry compared with high-risk human papillomavirus DNA testing with Hybrid Capture 2 (HC2) to detect grade 2 or greater cervical intraepithelial neoplasia (CIN2+) and CIN3+ among women who had cervical cytology indicating atypical squamous cells of undetermined significance (ASC-US) or low-grade cervical lesions (LSIL). A literature search was performed in 3 electronic databases to identify studies that were eligible for this meta-analysis. Seventeen studies were included in the meta-analysis. The pooled sensitivity of p16(INK4a) to detect CIN2+ was 83.2% (95% confidence interval [CI], 76.8%-88.2%) and 83.8% (95% CI, 73.5%-90.6%) in ASC-US and LSIL cervical cytology, respectively, and the pooled specificities were 71% (95% CI, 65%-76.4%) and 65.7% (95% CI, 54.2%-75.6%), respectively. Eight studies provided both HC2 and p16(INK4a) triage data. p16(INK4a) and HC2 had similar sensitivity, and p16(INK4a) has significantly higher specificity in the triage of women with ASC-US (relative sensitivity, 0.95 [95% CI, 0.89-1.01]; relative specificity, 1.82 [95% CI, 1.57-2.12]). In the triage of LSIL, p16(INK4a) had significantly lower sensitivity but higher specificity compared with HC2 (relative sensitivity, 0.87 [95% CI, 0.81-0.94]; relative specificity, 2.74 [95% CI, 1.99-3.76]). The published literature indicated the improved accuracy of p16(INK4a) compared with HC2 testing in the triage of women with ASC-US. In LSIL triage, p16(INK4a) was more specific but less sensitive. Copyright © 2012 American Cancer Society.

  7. Sp1 regulates Raf/MEK/ERK-induced p21(CIP1) transcription in TP53-mutated cancer cells.

    PubMed

    Karkhanis, Mansi; Park, Jong-In

    2015-03-01

    We previously reported that the upregulation of mortalin, an Hsp70 family chaperone, is important for B-Raf(V600E) tumor cells to bypass p21(CIP1) expression, which is activated as a tumor-suppressive mechanism in response to aberrant MEK/ERK activation (Wu et al., 2013). Interestingly, mortalin depletion induced p21(CIP1) transcription not only in wild-type TP53 but also in TP53-mutated B-Raf(V600E) cancer cells, suggesting the presence of an additional mechanism for p21(CIP1) regulation. In the present study, using luciferase reporter truncation analysis in a TP53-mutated B-Raf(V600E) cancer cell line, SK-MEL28, we identified a proximal p21(CIP1) promoter region responsive to mortalin depletion. Interestingly, when Sp1-like cis-elements in this promoter region were mutagenized, the p21(CIP1) promoter luciferase reporter was no longer responsive to mortalin depletion. Consistent with this, our ChIP analysis revealed that mortalin knockdown could induce Sp1 binding to p21(CIP1) promoter in a MEK/ERK-dependent manner. Moreover, RNA interference of Sp1 substantially attenuated p21(CIP1) expression induced by mortalin depletion in SK-MEL28 cells. Consistent with this observation in SK-MEL28 cells, Sp1 was necessary for the tamoxifen-regulated ∆Raf-1:ER to induce p21(CIP1) transcription in U251 cells, in which TP53 is mutated. However, in contrast, Sp1 was not necessary for ∆Raf-1:ER to induce p21(CIP1) transcription in LNCaP cells, in which TP53 is wild type. These data suggest that Sp1 may address TP53-independent p21(CIP1) transcription in Raf/MEK/ERK-activated cancer cells and that its requirement in Raf/MEK/ERK-induced p21(CIP1) transcription is subject to TP53 status.

  8. Loss of p16INK4A stimulates aberrant mitochondrial biogenesis through a CDK4/Rb-independent pathway

    PubMed Central

    Li, Chelsea; Liu, Tong; Rutter, Jared; Grossman, Douglas

    2017-01-01

    The tumor suppressor p16INK4A (p16) inhibits cell cycle progression through the CDK4/Rb pathway. We have previously shown that p16 regulates cellular oxidative stress, independent of its role in cell cycle control. We investigated whether loss of p16 had a direct impact on the mitochondria. We found that p16-null primary mouse fibroblasts (PMFs) displayed increased mitochondrial mass and expression of mitochondrial respiratory subunit proteins compared to wild-type (WT) PMFs. These findings in p16-null PMFs were associated with increased expression of the mitochondrial biogenesis transcription factors PRC and TFAM. On the other hand, p16-deficient PMFs demonstrated reduced mitochondrial respiration capacity consistent with electron microscopy findings showing that mitochondria in p16-deficient PMFs have abnormal morphology. Consistent with increased mitochondrial mass and reduced respiratory capacity, p16-deficient PMFs generated increased mitochondrial superoxide. One biological consequence of elevated ROS in p16-deficient PMFs was enhanced migration, which was reduced by the ROS scavenger N-acetylcysteine. Finally, p16-deficient PMFs displayed increased mitochondrial membrane potential, which was also required for their enhanced migration. The mitochondrial and migration phenotype was restored in p16-deficient PMFs by forced expression of p16. Similarly, over-expression of p16 in human melanocytes and A375 melanoma cells led to decreased expression of some mitochondrial respiratory proteins, enhanced respiration, and decreased migration. Inhibition of Rb phosphorylation in melanocytes and melanoma cells, either by addition of chemical CDK4 inhibitors or RNAi-mediated knockdown of CDK4, did not mimic the effects of p16 loss. These results suggest that p16 regulates mitochondrial biogenesis and function, which is independent of the canonical CDK4/Rb pathway. PMID:28915557

  9. Stathmin 1 and p16INK4A are sensitive adjunct biomarkers for serous tubal intraepithelial carcinoma

    PubMed Central

    Novak, Marián; Lester, Jenny; Karst, Alison M.; Parkash, Vinita; Hirsch, Michelle S.; Crum, Christopher P.; Karlan, Beth Y.

    2015-01-01

    Objective To credential Stathmin 1 (STMN1) and p16INK4A (p16) as adjunct markers for the diagnosis of serous tubal intraepithelial carcinoma (STIC), and to compare STMN1 and p16 expression in p53-positive and p53-negative STIC and invasive high-grade serous carcinoma (HGSC). Methods Immunohistochemistry (IHC) was used to examine STMN1 and p16 expression in fallopian tube specimens (n=31) containing p53-positive and p53-negative STICs, invasive HGSCs, and morphologically normal FTE (fallopian tube epithelium). STMN1 and p16 expression was scored semiquantitatively by four individuals. The semiquantitative scores were dichotomized, and reported as positive or negative. Pooled siRNA was used to knockdown p53 in a panel of cell lines derived from immortalized FTE and HGSC. Results STMN1 and p16 were expressed in the majority of p53-positive and p53-negative STICs and concomitant invasive HGSCs, but only scattered positive cells were positive in morphologically normal FTE. Both proteins were expressed consistently across multiple STICs from the same patient and in concomitant invasive HGSC. Knockdown of p53 in immortalized FTE cells and in four HGSC-derived cell lines expressing different missense p53 mutations did not affect STMN1 protein levels. Conclusions This study demonstrates that STMN1 and p16 are sensitive and specific adjunct biomarkers that, when used with p53 and Ki-67, improve the diagnostic accuracy of STIC. The addition of STMN1 and p16 helps to compensate for practical limitations of p53 and Ki-67 that complicate the diagnosis in up to one third of STICs. PMID:26206555

  10. Methylation of tumor suppressor genes p16(INK4a), p27(Kip1) and E-cadherin in carcinogenesis.

    PubMed

    Auerkari, Elza Ibrahim

    2006-01-01

    Not only genomic mutations but also abnormal epigenetic methylation can significantly contribute to gene silencing and carcinogenesis. Methylation is particularly often observed in the CpG islands of the promoter regions in the regulatory genes. However, there are considerable differences in the incidence of methylation e.g. in the tumor suppressor genes, so that aberrant methylation of p16(INK4a) is relatively frequently observed in tumors, p27(Kip1) methylation is rare, and the incidence of E-cadherin methylation occurs at an intermediate rate. Although true genomic defects are generally much less common than methylation, parallel tendencies for both are often observed, probably reflecting the different levels of evolutionary advantage for tumor cells from inactivation of different genes. This also suggests that loss of p27 expression could be more a consequence of carcinogenesis, while lost p16 expression is a true oncogenic event. Due to the role of p27 in maintaining cellular quiescence, however, loss of its expression can still be a useful partial indicator of the aggressiveness of cancer. Loss of E-cadherin or its catenin partners of cellular adhesion will result in increasing invasiveness and metastatic potential of neoplastic cells but, because of several alternative routes to the same effect, incidence of lost expression for one component gene like E-cadherin does not need to be very high. Similarly, there must be a relatively high number of genes with modest or low incidence of aberrant silencing by methylation, to reflect multiple alternatives for the multistep process of carcinogenesis. Nevertheless, methylation of different genes also shows characteristic differences between different cancer and tumor types, and the epigenetic methylation patterns therefore have considerable diagnostic and prognostic potential. Realising this potential requires efficient methods for profiling the status of methylation. Such profiling methods have only recently become

  11. Meta-analysis demonstrates no association between p16 (ink4a) promoter methylation and epithelial ovarian cancer.

    PubMed

    Jiang, Yi; Yan, Fangrong; Liang, Li; Wan, Yicong; Liu, Jinsong; Cheng, Wenjun

    2017-03-01

    p16 (INK4A) (p16) functions as a tumor suppressor gene in various malignancies. Aberrant p16 methylation has been proposed to be essential in ovarian carcinogenesis. However, it is unclear whether p16 can be used as a diagnostic marker owing to the small sample sizes in previous studies. To determine whether p16 promoter methylation is associated with epithelial ovarian cancer and can thus be used as a diagnostic marker, we performed a meta-analysis of published studies. The following databases were searched using a systematic search method: PubMed, Web of Science, EMBASE, and Chinese National Knowledge Infrastructure. We used a random-effects model to analyze the relative risk (RR); we also evaluated between-study heterogeneity, subgroup heterogeneity, and publication bias. Our meta-analysis included eight eligible studies, with 428 ovarian cancers and 278 normal tissue samples and benign neoplasms. p16 promoter methylation was identified in 5.4 to 43.2% (median 27.86%) of ovarian cancers and 0 to 37.5% (median 15.8%) of normal tissue and benign neoplasms indicating that no significant association exists between p16 promoter methylation and epithelial ovarian cancer. However, the pooled results also showed that the RR was 1.52 (95% CI 0.80-2.87) in the ovarian cancer cases versus the corresponding normal and benign cases under the random-effects model. Between-study heterogeneity was determined through a sensitivity analysis; the I (2) value did not change when one study was excluded. Our study showed that p16 promoter methylation cannot be used to differentiate benign from malignant epithelial ovarian tumors. Therefore, p16 promoter methylation cannot be used as a marker for diagnosing the early stage of ovarian cancer.

  12. Loss of p16(INK4A) stimulates aberrant mitochondrial biogenesis through a CDK4/Rb-independent pathway.

    PubMed

    Tyagi, Ethika; Liu, Bin; Li, Chelsea; Liu, Tong; Rutter, Jared; Grossman, Douglas

    2017-08-22

    The tumor suppressor p16INK4A (p16) inhibits cell cycle progression through the CDK4/Rb pathway. We have previously shown that p16 regulates cellular oxidative stress, independent of its role in cell cycle control. We investigated whether loss of p16 had a direct impact on the mitochondria. We found that p16-null primary mouse fibroblasts (PMFs) displayed increased mitochondrial mass and expression of mitochondrial respiratory subunit proteins compared to wild-type (WT) PMFs. These findings in p16-null PMFs were associated with increased expression of the mitochondrial biogenesis transcription factors PRC and TFAM. On the other hand, p16-deficient PMFs demonstrated reduced mitochondrial respiration capacity consistent with electron microscopy findings showing that mitochondria in p16-deficient PMFs have abnormal morphology. Consistent with increased mitochondrial mass and reduced respiratory capacity, p16-deficient PMFs generated increased mitochondrial superoxide. One biological consequence of elevated ROS in p16-deficient PMFs was enhanced migration, which was reduced by the ROS scavenger N-acetylcysteine. Finally, p16-deficient PMFs displayed increased mitochondrial membrane potential, which was also required for their enhanced migration. The mitochondrial and migration phenotype was restored in p16-deficient PMFs by forced expression of p16. Similarly, over-expression of p16 in human melanocytes and A375 melanoma cells led to decreased expression of some mitochondrial respiratory proteins, enhanced respiration, and decreased migration. Inhibition of Rb phosphorylation in melanocytes and melanoma cells, either by addition of chemical CDK4 inhibitors or RNAi-mediated knockdown of CDK4, did not mimic the effects of p16 loss. These results suggest that p16 regulates mitochondrial biogenesis and function, which is independent of the canonical CDK4/Rb pathway.

  13. Inverse association of p16 INK4a and p14 ARF methylation of the CDKN2a locus in different Gleason scores of prostate cancer.

    PubMed

    Verdoodt, B; Sommerer, F; Palisaar, R-J; Noldus, J; Vogt, M; Nambiar, S; Tannapfel, A; Mirmohammadsadegh, A; Neid, M

    2011-12-01

    Promoter hypermethylation is an important epigenetic mechanism in the regulation of several key modulators of prostate carcinoma progression. Recent studies suggest that the polycomb-group (PcG) protein BMI1 may have an impact on epigenetic regulation of several targets, including the CDKN2a locus. In this study, we investigated the association of BMI1 expression, promoter methylation of CDKN2a (p16(INK4a) and p14(ARF)) and TMS1 with pathological variables (Gleason score, TNM stage, perineural invasion) in prostate cancer (PCa). Methylation of p16(INK4a) and p14(ARF) revealed an inverse association with Gleason score 7b and Gleason score 6. No significant association could be demonstrated for BMI1 -overexpression and promoter methylation of p16(INK4a), p14(ARF) and TMS1 as well as pT category. Our data suggest that the CDKN2a locus is a switch in PCa with methylation of p16(INK4a) being a marker for more aggressive tumours of Gleason score 7b, but no association with BMI overexpression was observed.

  14. LncRNA PANDAR regulates the G1/S transition of breast cancer cells by suppressing p16(INK4A) expression.

    PubMed

    Sang, Yi; Tang, Jianjun; Li, Siwei; Li, Liping; Tang, XiaoFeng; Cheng, Chun; Luo, Yanqin; Qian, Xia; Deng, Liang-Ming; Liu, Lijuan; Lv, Xiao-Bin

    2016-03-01

    It has been reported that lncRNA PANDAR (promoter of CDKN1A antisense DNA damage-activated RNA) is induced as a result of DNA damage, and it regulates the reparation of DNA damage. In this study, we investigated the role of lncRNA PANDAR in the progression of breast cancer and found that PANDAR was up-regulated in breast cancer tissues and cell lines. The knockdown of PANDAR suppresses G1/S transition of breast cancer cells. We demonstrated mechanistically that the regulation of G1/S transition by PANDAR was partly due to the transcriptional modulation of p16(INK4A). Moreover, we showed that PANDAR impacted p16(INK4A) expression by regulating the recruitment Bmi1 to p16(INK4A) promoter. To our knowledge, this is the first study which showed the functional roles and mechanisms of PANDAR in regulating the progression of breast cancer. The PANDAR/Bmi1/p16(INK4A) axis could serve as novel targets for breast cancer therapy.

  15. CDKN2A (p14(ARF)/p16(INK4a)) and ATM promoter methylation in patients with impalpable breast lesions.

    PubMed

    Delmonico, Lucas; Moreira, Aline dos Santos; Franco, Marco Felipe; Esteves, Eliane Barbosa; Scherrer, Luciano; Gallo, Claúdia Vitória de Moura; do Nascimento, Cristina Moreira; Ornellas, Maria Helena Faria; de Azevedo, Carolina Maria; Alves, Gilda

    2015-10-01

    Early detection of breast cancer increases the chances of cure, but the reliable identification of impalpable lesions is still a challenge. In spite of the advances in breast cancer detection, the molecular basis of impalpable lesions and the corresponding circulating biomarkers are not well understood. Impalpable lesions, classified by radiologists according to the Breast Imaging Reporting and Data System in the categories 3 and 4, can be either benign or malignant (slow growing or aggressive). In this article, we report the DNA methylation pattern in CDKN2A (p14(ARF)/p16(INK4a)) and in ATM gene promoters from 62 impalpable lesions, 39 peripheral blood samples, and 39 saliva samples, assessed by methylation-specific polymerase chain reaction method. ATM showed the greatest percentage of methylation in DNA from lesions (benign and malignant), blood (even with p16(INK4a)), and saliva, followed by p16(INK4a) and p14(ARF). Among the malignant cases, ATM promoter was the most hypermethylated in lesion DNA and in blood and saliva DNAs, and p14(ARF), the least. The highest percentage of p16(INK4a) methylation was found in the blood. Finally, our data are relevant because they were obtained using impalpable breast lesions from patients who were carefully recruited in 2 public hospitals of Rio de Janeiro. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Hepatitis B virus X protein induces hypermethylation of p16(INK4A) promoter via DNA methyltransferases in the early stage of HBV-associated hepatocarcinogenesis.

    PubMed

    Zhu, Y-Z; Zhu, R; Fan, J; Pan, Q; Li, H; Chen, Q; Zhu, H-G

    2010-02-01

    The aim of the present study was to authenticate the involvement of DNA methyltransferases (DNMTs) and methyl-CpG binding domain protein 2 (MBD2) in the process of HBx induced p16(INK4A) promoter hypermethylation in HBV-related hepatocellular carcinoma (HCC) and their corresponding noncancerous liver tissues. Eighty-eight fresh tissue specimens of surgically resected HBV-associated HCC and their corresponding noncancerous liver tissues were studied. The methylation status of the p16(INK4A) promoter was determined by methylation-specific polymerase chain reaction (MSP). Reverse transcription and real-time polymerase chain reaction (RT-PCR) showed the expression of DNMTs, MBD2 and HBx. Western blot and immunohistochemistry were used for the protein analysis of HBx, DNMT1, DNMT3A and P16. Tissue HBV-DNA levels were determined by RT-PCR. HBV genotype was examined by nested PCR and restriction fragment length polymorphism (RFLP). In the corresponding noncancerous liver tissues, higher HBx expression was associated with the hypermethylation of the p16(INK4A) promoter. HBx was positively correlated with the DNMT1 and DNMT3A at both the mRNA and protein level. Furthermore, HBx, DNMT1 and DNMT3A protein expression were negatively correlated with p16 protein expression. In HCC tissues, HBx was positively correlated with DNMT1 and DNMT3A at both mRNA and protein level, but HBx expression did not correlate with hypermethylation of the p16(INK4A) promoter or p16 protein expression. The methylation status of the p16(INK4A) promoter did not correlate with clinicopathological characteristics. DNMT1 and DNMT3A may play important roles in the process of HBx inducing hypermethylation of the p16(INK4A) promoter in the early stages of HBV-associated HCC. HBx-DNMTs-p16(INK4A) promoter hypermethylation may constitute a mechanism for tumorigenesis during HBV-associated hepatocarcinogenesis.

  17. Over-expression of Id-1 induces cell proliferation in hepatocellular carcinoma through inactivation of p16INK4a/RB pathway.

    PubMed

    Lee, Terence Kin-Wah; Man, Kwan; Ling, Ming-Tat; Wang, Xiang-Hong; Wong, Yong-Chuan; Lo, Chung-Mau; Poon, Ronnie Tung-Ping; Ng, Irene Oi-Lin; Fan, Sheung-Tat

    2003-11-01

    Inhibitors of differentiation and DNA binding-1 (Id-1) have been demonstrated to oppose Ets-mediated activation of p16INK4a. As p16INK4a protein is inactivated in hepatocellular carcinoma (HCC), we aimed to investigate the role of Id-1 in regulating p16INK4a expression during the development of HCC in HCC patients and direct ectopic Id-1 introduction into the PLC/PRF/5 HCC cell line. Sixty-two HCC samples were recruited for evaluation of Id-1 and proliferating cell nuclear antigen (PCNA) protein expression. The messenger RNA (mRNA) expression of Id-1 and p16INK4a was detected by quantitative reverse transcription-polymerase chain reaction. For in vitro Id-1 transfection, five Id-1 transfected clones were isolated and the effect of ectopic Id-1 introduction was investigated by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, flow cytometry, immunostaining and western blot. Our results showed that Id-1 was over-expressed in HCC specimens both at mRNA and protein levels. Over-expression of Id-1 protein was correlated with PCNA (r = 0.334, P = 0.033). HCC samples showing low Id-1 protein expression had a lower Id-1 mRNA level (340.2 versus 1467%, P = 0.039) and higher p16INK4a expression (195 versus -78.6%, P = 0.039) than samples with high Id-1 protein expression. In the PLC/PRF/5 HCC cell line study, ectopic Id-1 expression resulted in proliferation of HCC cells and an increased percentage of S phase cells and PCNA expression. The results showed that over-expression of Id-1 induces cell proliferation in HCC through inactivation of p16INK4a/retinoblastoma pathway. In conclusion, the results provided an insight for the understanding of the role of Id-1 in functional inactivation of p16INK4a in HCC.

  18. Prevalence of human papillomavirus16 DNA and p16(INK4a) protein in oral squamous cell carcinoma: A systematic review and meta-analysis.

    PubMed

    Smitha, T; Mohan, C V; Hemavathy, S

    2017-01-01

    Indian patients with oral squamous cell carcinoma (OSCC) are etiologically associated with the use of tobacco and alcohol; yet, a proportion of tumors that may harbor human papillomavirus (HPV) infections cannot be neglected. The following meta-analysis was conducted to address the association of p16(INK4a) and HPV DNA with OSCC. In addition, the study also provides the updated prevalence of HPV-induced OSCC. Literature survey was performed using databases such as PubMed with the help of the following keywords - "HPV infection," "oral squamous cell carcinoma," "p16(INK4a)," "HPV DNA," "E6," "E7," "L1," "L2" and "LCR." Proportion method was performed to derive the forest plot using MedCalc statistical software version 16.4.3. Among 145 research articles, 33 articles were selected for further analysis, in which 13 articles were related to HPV DNA detection in tissues, 11 articles detected the overexpression of p16(INK4a) and nine articles reported the detection of both HPV DNA and p16(INK4a) expression. Meta-analysis revealed significant heterogeneity (P < 0.0001) among the articles. Overall, the study consisted of 3339 patients with OSCC, among which 559 patients were diagnosed with the presence of HPV16 DNA with a random proportion of 20.1% at 95% confidence interval (CI) (13.9-27.1, P < 0.0001). Overexpression of p16(INK4a) protein was observed in 709 patients with a random proportion of 25.4% at 95% CI (14.3-38.3, P < 0.0001). HPV DNA and expression of p16(INK4a) was suggested as gold standard for the detection of HPV infection in many cases of cancers. Frequency of HPV infection is significantly higher in patients with OSCC as identified through the detection of HPV DNA and p16(INK4a) expression. Even though the association of HPV infection has been established in head and neck cancer, this review could further the establishment of molecular level interaction of HPV in patients with oral cancer.

  19. Use of p16-INK4A overexpression to increase the specificity of human papillomavirus testing: a nested substudy of the NTCC randomised controlled trial.

    PubMed

    Carozzi, Francesca; Confortini, Massimo; Dalla Palma, Paolo; Del Mistro, Annarosa; Gillio-Tos, Anna; De Marco, Laura; Giorgi-Rossi, Paolo; Pontenani, Giovanni; Rosso, Stefano; Sani, Cristina; Sintoni, Catia; Segnan, Nereo; Zorzi, Manuel; Cuzick, Jack; Rizzolo, Raffaella; Ronco, Guglielmo

    2008-10-01

    Human-papillomavirus (HPV) testing is more sensitive, but less specific, than conventional cytology for detecting high-grade cervical intraepithelial neoplasia (CIN). We assessed whether HPV testing with triage by p16-INK4A overexpression can increase specificity while maintaining sensitivity. HPV-positive women were enrolled between June 10, 2003, and Dec 31, 2004 in a multicentre randomised controlled trial, which compared stand-alone HPV testing by Hybrid Capture 2 (experimental group) with conventional cytology, were referred for colposcopy. In seven of nine centres, cytospin preparations from these women were tested for p16-INK4A overexpression by immunostaining. The sensitivity and specificity for CIN grade 2 or more, determined at blind review of histology, were calculated for these women. We also estimated the relative sensitivity and relative referral to colposcopy that would have been obtained by HPV testing with p16-INK4A triage compared with conventional cytology. This trial is registered as a Standard Randomised Controlled Trial, number ISRCTN81678807. 24 661 women were randomly assigned to the experimental group. 1137 women (74% of those undergoing colposcopy in relevant centres), including 50 with CIN2 and 42 with CIN3 or cancer, had valid p16-INK4A immunostaining. For the endpoint of CIN2+, sensitivity and specificity of p16-INK4A (deemed positive with any number of stained cells-except endocervical, metaplastic, and atrophic cells if morphologically normal) in HPV-positive women of any age were 88% (81 of 92; 95% CI 80-94) and 61% (633 of 1045; 57-64), respectively. In the 35-60-year age group, the relative sensitivity of HPV testing and p16-INK4A triage versus conventional cytology for CIN2+ was 1.53 (95% CI 1.15-2.02) and relative referral was 1.08 (0.96-1.21). In the 25-34-year age group, relative sensitivity was 3.01 (1.82-5.17) and relative referral was 1.15 (0.96-1.37). In the latter age group, when 5% or more stained cells were deemed

  20. [Expression and significance of P16INK4A and PTEN in high-risk human papillomavirus-related cervical cancer].

    PubMed

    Zhao, Xian-Lan; Cheng, Shu-Xia; Kong, Xiang-Dong

    2007-05-01

    High-risk human papillomavirus (HR-HPV) is the most important etiologic factor for cervical cancer. Recent studies have revealed that abnormal expression of tumor suppressor gene P16INK4A is closely associated with HR-HPV infection during carcinogenesis of cervical epithelium. Tumor suppressor gene PTEN is also involved in cervical tumorigenesis. This study was to investigate the correlations of HR-HPV infection to P16INK4A and PTEN expression and its clinical significance in the carcinogenesis of cervical epithelium. The expression of P16INK4A and PTEN in 30 specimens of normal cervical tissues, 11 specimens of cancer in situ (CIS), and 24 specimens of invasive cervical carcinoma (ICC) was detected by SP immunohistochemistry; 13 types of HR-HPV DNA in these cases were detected by Hybrid Capture 2 (HC-2) assay. The positive rates of HR-HPV and P16INK4A were significantly higher in ICC and CIS than in normal tissues (91.7% and 90.9% vs. 30.0%, P<0.001; 87.5% and 81.8% vs. 6.7%, P<0.001). Both HR-HPV DNA and P16INK4A overexpression (moderate or strong expression) were observed simultaneously in 21 specimens of ICC and 9 specimens of CIS; they were simultaneously negative in 20 specimens of normal cervical tissues and 1 specimen of CIS and 2 specimens of ICC. Overexpression of P16INK4A was positively correlated to HR-HPV infection in cervical cancer (rs = 0.690, P<0.001). PTEN was moderately or strongly expressed in 26 specimens of normal cervical tissues. The positive rate of PTEN was significantly lower in ICC and CIS than in normal cervical tissues (37.5% and 36.4% vs. 83.3%, P<0.01). No obvious relationship between PTEN and HR-HPV was found (rs = -0.174, P = 0.167). P16INK4A is overexpressed in HR-HPV-infected cervical cancer, but its tumor suppressor action might be inhibited. In contrast, the functional down-regulation of PTEN contributes to cervical tumorigenesis through HR-HPV-independent mechanism.

  1. AN UPWARD TREND IN DNA P16INK4A METHYLATION PATTERN AND HIGH RISK HPV INFECTION ACCORDING TO THE SEVERITY OF THE CERVICAL LESION

    PubMed Central

    Carestiato, Fernanda Nahoum; Afonso, Larissa Alves; Moysés, Natalia; Almeida, Gutemberg Leão; Velarde, Luis Guillermo Coca; Cavalcanti, Silvia Maria Baeta

    2013-01-01

    SUMMARY High-risk human papillomavirus (hr-HPV) infection is necessary but not sufficient for cervical cancer development. Recently, P16INK4A gene silencing through hypermethylation has been proposed as an important cofactor in cervical carcinogenesis due to its tumor suppressor function. We aimed to investigate P16INK4A methylation status in normal and neoplastic epithelia and evaluate an association with HPV infection and genotype. This cross-sectional study was performed with 141 cervical samples from patients attending Hospital Moncorvo Filho, Rio de Janeiro. HPV detection and genotyping were performed through PCR and P16INK4A methylation by nested-methylation specific PCR (MSP). HPV frequency was 62.4% (88/141). The most common HPV were HPV16 (37%), HPV18 (16.3%) and HPV33/45(15.2%). An upward trend was observed concerning P16INK4A methylation and lesion degree: normal epithelia (10.7%), low grade lesions (22.9%), high grade (57.1%) and carcinoma (93.1%) (p < 0.0001). A multivariate analysis was performed to evaluate an association between methylation, age, tobacco exposure, HPV infection and genotyping. A correlation was found concerning methylation with HPV infection (p < 0.0001), hr-HPV (p = 0.01), HSIL (p < 0.0007) and malignant lesions (p < 0.0001). Since viral infection and epigenetic alterations are related to cervical carcinoma, we suggest that P16INK4A methylation profile maybe thoroughly investigated as a biomarker to identify patients at risk of cancer. PMID:24037287

  2. Human papillomavirus infection in Bowen disease: negative p53 expression, not p16(INK4a) overexpression, is correlated with human papillomavirus-associated Bowen disease.

    PubMed

    Murao, Kazutoshi; Yoshioka, Rika; Kubo, Yoshiaki

    2014-10-01

    Genital Bowen disease (BD) has been linked to the high-risk types of human papillomavirus (HPV) infection. Recently, it has been recognized that HPV also can be associated with extragenital BD. HPV oncoproteins E6 and E7 interfere with the function of p53 and pRb, respectively, leading carcinogenesis. p16(INK4a) overexpression induced by inactivation of pRb is recognized as a surrogate marker for HPV-associated cervical cancer. In this study, we examined the presence of HPV DNA in 142 BD lesions by polymerase chain reaction (PCR), and determined the type of HPV by PCR restriction fragment length polymorphism or direct DNA sequencing. HPV DNA was detected in 66.7% of genital BD and 8.3% of extragenital BD. The types of HPV detected were HPV types 6, 16, 33, 52, 56, 58 and 59. We also investigated the expression of p16(INK4a) , pRb and p53 by immunohistochemistry. Positive expression was detected in 88.6% for p16(INK4a) , 25.2% for pRb, and 63.8% for p53. There was no significant difference in p16(INK4a) and pRb expression between HPV-positive and -negative BD. However, a strong correlation of HPV positivity with p53 negativity was found. A total of 66.7% of HPV-positive BD showed no p53 expression, whereas the corresponding rate was 32.8% of HPV-negative BD. This study demonstrated that HPV can participate in the development of BD, not only in the genital lesion, but also in extragenital lesion. p16(INK) (4a) overexpression is not a marker for HPV infection in BD. Instead, negative p53 expression is correlated with HPV-associated BD.

  3. EGFR, p16INK4a and E-cadherin immuno-histochemistry and EGFR point mutations analyses in invasive cervical cancer specimens from Moroccan women.

    PubMed

    El Hamdani, W; Amrani, M; Attaleb, M; Laantri, N; Ennaji, M M; Khyatti, M; El Mzibri, M

    2010-09-11

    The involvement of human papillomavirus in the development of cervical cancer has been firmly established. However, better management of cervical cancer rests on good diagnosis and an effective therapy. In this study we evaluated the frequency of point mutations in epidermal growth factor receptor (EGFR) for future use of tyrosine kinase inhibitors in clinical treatment and to assess the use of EGFR, p16INK4a and E-cadherin as biomarkers in cervical cancer diagnosis with immunohistochemistry. Fifty-three patient specimens of cervical cancer were analysed for HPV infection, for EGFR mutations in exons 18 through 21, and for expression of EGFR, p16INK4a and E-cadherin by immunostaining. Results showed that 79.24% of the cases (42/53) are HPV positive and the HPV types more closely associated with risk are HPV 16 and 18. In all 53 analysed specimens, any mutation affecting the EGFR kinase domain in exons 18 through 21 was observed. Expressions of EGFR, p16INK4a and E-cadherin were detected in 88,67% (47/53), 92,45% (49/53) and 79,24% (42/53) of analysed specimens respectively. Thus, EGFR, p16INK4a and E-cadherin would be excellent tools for IHC analysis during the cervical cancer development. EGFR and p16INK4a can be used for early diagnosis and E-cadherin for cancer progression and cell migration. However, treatment of cervical cancer with TKIs may not be effective and the identification of other EGFR inhibitors is needed.

  4. Bmi1 Loss in the Organ of Corti Results in p16ink4a Upregulation and Reduced Cell Proliferation of Otic Progenitors In Vitro

    PubMed Central

    Dos Santos, Aurélie; Avci, Hasan X.; Löwenheim, Hubert; Müller, Marcus

    2016-01-01

    The mature mammalian organ of Corti does not regenerate spontaneously after injury, mainly due to the absence of cell proliferation and the depletion of otic progenitors with age. The polycomb gene B lymphoma Mo-MLV insertion region 1 homolog (Bmi1) promotes proliferation and cell cycle progression in several stem cell populations. The cell cycle inhibitor p16ink4a has been previously identified as a downstream target of Bmi1. In this study, we show that Bmi1 is expressed in the developing inner ear. In the organ of Corti, Bmi1 expression is temporally regulated during embryonic and postnatal development. In contrast, p16ink4a expression is not detectable during the same period. Bmi1-deficient mice were used to investigate the role of Bmi1 in cochlear development and otosphere generation. In the absence of Bmi1, the postnatal organ of Corti displayed normal morphology at least until the end of the first postnatal week, suggesting that Bmi1 is not required for the embryonic or early postnatal development of the organ of Corti. However, Bmi1 loss resulted in the reduced sphere-forming capacity of the organ of Corti, accompanied by the decreased cell proliferation of otic progenitors in otosphere cultures. This reduced proliferative capacity was associated with the upregulation of p16ink4a in vitro. Viral vector-mediated overexpression of p16ink4a in wildtype otosphere cultures significantly reduced the number of generated otospheres in vitro. The findings strongly suggest a role for Bmi1 as a promoter of cell proliferation in otic progenitor cells, potentially through the repression of p16ink4a. PMID:27755610

  5. p16( INK4a) positively regulates cyclin D1 and E2F1 through negative control of AUF1.

    PubMed

    Al-Khalaf, Huda H; Colak, Dilek; Al-Saif, Maher; Al-Bakheet, Albandary; Hendrayani, Siti-Faujiah; Al-Yousef, Nujoud; Kaya, Namik; Khabar, Khalid S; Aboussekhra, Abdelilah

    2011-01-01

    The cyclin-D/CDK4,6/p16(INK4a)/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined. We have shown here that the tumor suppressor p16(INK4a) protein positively controls the expression of cyclin D1 and E2F1 in both human and mouse cells. p16(INK4a) stabilizes the mRNAs of the corresponding genes through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs. Furthermore, AUF1 down-regulation increased the expression levels of these genes, while concurrent silencing of AUF1 and p16(INK4a), using specific siRNAs, restored normal expression of both cyclinD1 and E2F1. Besides, we have shown the presence of functional AU-rich elements in the E2F1 3'UTR, which contributed to p16/AUF1-mediated regulation of E2F1 post-transcriptional events in vivo. Importantly, genome-wide gene expression microarray analysis revealed the presence of a large number of genes differentially expressed in a p16(INK4a) -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis. These findings show that the cyclin-dependent kinase inhibitor p16( INK4a) is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.

  6. Correlation of p16(INK4A) expression and HPV copy number with cellular FTIR spectroscopic signatures of cervical cancer cells.

    PubMed

    Ostrowska, Kamila M; Garcia, Amaya; Meade, Aidan D; Malkin, Alison; Okewumi, Ifeoluwapo; O'Leary, John J; Martin, Cara; Byrne, Hugh J; Lyng, Fiona M

    2011-04-07

    Cervical cancer, a potentially preventable disease, has its main aetiology in infection by high risk human papillomavirus (HR-HPV). Approaches to improving cervical cancer screening and diagnostic methodologies include molecular biological analysis, targeting of biomarker proteins, but also exploration and implementation of new techniques such as vibrational spectroscopy. This study correlates the biomarker protein p16(INK4A) expression levels dependent on HPV copy number with the infrared absorption spectral signatures of the cervical cancer cell lines, HPV negative C33A, HPV-16 positive SiHa and CaSki and HPV-18 positive HeLa. Confocal fluorescence microscopy demonstrated that p16(INK4A) is expressed in all investigated cell lines in both nuclear and cytoplasmic regions, although predominantly in the cytoplasm. Flow cytometry was used to quantify the p16(INK4A) expression levels and demonstrated a correlation, albeit nonlinear, between the reported number of integrated HPV copies and p16(INK4A) expression levels. CaSki cells were found to have the highest level of expression, HeLa intermediate levels, and SiHa and C33A the lowest levels. FTIR spectra revealed differences in nucleic acid, lipid and protein signatures between the cell lines with varying HPV copy number. Peak intensities exhibited increasing tendency in nucleic acid levels and decreasing tendency in lipid levels with increasing HPV copy number, and although they were found to be nonlinearly correlated with the HPV copy number, their dependence on p16(INK4A) levels was found to be close to linear. Principal Component Analysis (PCA) of the infrared absorption spectra revealed differences between nuclear and cytoplasmic spectroscopic signatures for all cell lines, and furthermore clearly differentiated the groups of spectra representing each cell line. Finally, Partial Least Squares (PLS) analysis was employed to construct a model which can predict the p16(INK4A) expression level based on a spectral

  7. Polyamine-modulated c-Myc expression in normal intestinal epithelial cells regulates p21Cip1 transcription through a proximal promoter region

    PubMed Central

    Liu, Lan; Guo, Xin; Rao, Jaladanki N.; Zou, Tongtong; Marasa, Bernard S.; Chen, Jie; Greenspon, Jose; Casero, Robert A.; Wang, Jian-Ying

    2006-01-01

    Maintenance of intestinal mucosal epithelial integrity requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest and apoptosis. Our previous studies have shown that polyamines are essential for expression of the c-myc gene and that polyamine-induced c-Myc plays a critical role in stimulation of normal IEC (intestinal epithelial cell) proliferation, but the exact downstream targets of induced c-Myc are still unclear. The p21Cip1 protein is a major player in cell cycle control, which is primarily regulated at the transcriptional level. The current study was designed to determine whether induced c-Myc stimulates normal IEC proliferation by repressing p21Cip1 transcription following up-regulation of polyamines. Overexpression of the ODC (ornithine decarboxylase) gene increased levels of cellular polyamines, induced c-Myc expression and inhibited p21Cip1 transcription, as indicated by repression of p21Cip1 promoter activity and a decrease in p21Cip1 protein levels. In contrast, depletion of cellular polyamines by inhibiting ODC enzyme activity with α-difluoromethylornithine decreased c-Myc, but increased p21Cip1 transcription. Ectopic expression of wild-type c-myc not only inhibited basal levels of p21Cip1 transcription in control cells, but also prevented increased p21Cip1 in polyamine-deficient cells. Experiments using different p21Cip1 promoter mutants showed that transcriptional repression of p21Cip1 by c-Myc was mediated through Miz-1- and Sp1-binding sites within the proximal region of the p21Cip1 promoter in normal IECs. These findings confirm that p21Cip1 is one of the direct mediators of induced c-Myc following increased polyamines and that p21Cip1 repression by c-Myc is implicated in stimulation of normal IEC proliferation. PMID:16706751

  8. p16INK4a hypermethylation and p53, p16 and MDM2 protein expression in esophageal squamous cell carcinoma.

    PubMed

    Taghavi, Noushin; Biramijamal, Firouzeh; Sotoudeh, Masoud; Khademi, Hooman; Malekzadeh, Reza; Moaven, Omeed; Memar, Bahram; A'rabi, Azadeh; Abbaszadegan, Mohammad Reza

    2010-04-13

    Tumor suppressor genes p53 and p16INK4a and the proto-oncogene MDM2 are considered to be essential G1 cell cycle regulatory genes whose loss of function is associated with ESCC carcinogenesis. We assessed the aberrant methylation of the p16 gene and its impact on p16INK4a protein expression and correlations with p53 and MDM2 protein expressions in patients with ESCC in the Golestan province of northeastern Iran in which ESCC has the highest incidence of cancer, well above the world average. Cancerous tissues and the adjacent normal tissue obtained from 50 ESCC patients were assessed with Methylation-Specific-PCR to examine the methylation status of p16. The expression of p16, p53 and MDM2 proteins was detected by immunohistochemical staining. Abnormal expression of p16 and p53, but not MDM2, was significantly higher in the tumoral tissue. p53 was concomitantly accumulated in ESCC tumor along with MDM2 overexpression and p16 negative expression. Aberrant methylation of the p16INK4a gene was detected in 31/50 (62%) of esophageal tumor samples, while two of the adjacent normal mucosa were methylated (P < 0.001). p16INK4a aberrant methylation was significantly associated with decreased p16 protein expression (P = 0.033), as well as the overexpression of p53 (P = 0.020). p16 hypermethylation is the principal mechanism of p16 protein underexpression and plays an important role in ESCC development. It is associated with p53 protein overexpression and may influence the accumulation of abnormally expressed proteins in p53-MDM2 and p16-Rb pathways, suggesting a possible cross-talk of the involved pathways in ESCC development.

  9. p16INK4a hypermethylation and p53, p16 and MDM2 protein expression in Esophageal Squamous Cell Carcinoma

    PubMed Central

    2010-01-01

    Background Tumor suppressor genes p53 and p16INK4a and the proto-oncogene MDM2 are considered to be essential G1 cell cycle regulatory genes whose loss of function is associated with ESCC carcinogenesis. We assessed the aberrant methylation of the p16 gene and its impact on p16INK4a protein expression and correlations with p53 and MDM2 protein expressions in patients with ESCC in the Golestan province of northeastern Iran in which ESCC has the highest incidence of cancer, well above the world average. Methods Cancerous tissues and the adjacent normal tissue obtained from 50 ESCC patients were assessed with Methylation-Specific-PCR to examine the methylation status of p16. The expression of p16, p53 and MDM2 proteins was detected by immunohistochemical staining. Results Abnormal expression of p16 and p53, but not MDM2, was significantly higher in the tumoral tissue. p53 was concomitantly accumulated in ESCC tumor along with MDM2 overexpression and p16 negative expression. Aberrant methylation of the p16INK4a gene was detected in 31/50 (62%) of esophageal tumor samples, while two of the adjacent normal mucosa were methylated (P < 0.001). p16INK4a aberrant methylation was significantly associated with decreased p16 protein expression (P = 0.033), as well as the overexpression of p53 (P = 0.020). Conclusions p16 hypermethylation is the principal mechanism of p16 protein underexpression and plays an important role in ESCC development. It is associated with p53 protein overexpression and may influence the accumulation of abnormally expressed proteins in p53-MDM2 and p16-Rb pathways, suggesting a possible cross-talk of the involved pathways in ESCC development. PMID:20388212

  10. [Detection of human papilloma virus (HPV) in liquid-based cervical samples. Correlation with protein p16INK4a expression].

    PubMed

    Toro de Méndez, Morelva; Ferrández Izquierdo, Antonio

    2011-03-01

    The liquid-based cervical cytology improves the quality of the sample and the residual sample could be used efficiently to carry out complementary tests, such as the detection of HPV DNA and the immunocytochemical biomarkers study. The purpose of this study was to correlate the presence of HPV and immunoexpression of p16INK4a in liquid-based cervical samples to examine the utility of these new tools in the detection of cervical cancer. The included patients (n = 67) presented an abnormal cytology or previous cervical pathology. The HPV detection and genotyping were carried out with PCR-SPF10/LiPA (INNOLiPA Extra Amp) and for p16INK4a immunodetection was used antibody clone E6H4. The conventional cytology provided the same cytologic interpretations that those of liquid-based cytology. The overall HPV prevalence was 43.3% (29/67). HPV16 was the most frequent viral type (31.03%) and 48.3% of the cases were infected with multiple HPV types. p16INK4a immunoexpression was observed in 35.8% of liquid-based cytological samples and this was significantly (p < 0.020) associated to the HPV presence. These results support the evidence that the implementation of new technologies in the daily routine of the laboratory, contribute significantly in the early detection of cervical cancer and provide important data to help in the patient's efficient management. The combined use of HPV detection and p16INK4a expression could be used for evaluation of patients with more risk to develop significant cervical lesions.

  11. Hepatitis B virus X protein overcomes stress-induced premature senescence by repressing p16(INK4a) expression via DNA methylation.

    PubMed

    Kim, Ye-Jin; Jung, Jin Kyu; Lee, Sun Young; Jang, Kyung Lib

    2010-02-28

    Cellular senescence is an important tumor suppression process under diverse oncogenic conditions, entering a state of irreversible growth arrest to prevent damaged cells from undergoing aberrant proliferation. Developing a means of evading senescence thus seems to be a fundamental task that all cancer cells should solve early on. Here, we show that an oncogenic X protein of hepatitis B virus (HBx) overcomes cellular senescence provoked by a universal premature senescence inducer, H(2)O(2), in human hepatoma cells, as demonstrated by impaired induction of senescence-associated biomarkers, including morphological change, G(1) arrest, and beta-galactosidase activity, in the presence of HBx. HBx induced DNA hypermethylation of p16(INK4a) promoter and subsequently interfered action of transcription factors like Ets1 and Ets2 activated by H(2)O(2) through the p38(MAPK) pathway, resulting in inhibition of its transcription. Down-regulation of p16(INK4a) expression by HBx subsequently led to activation of G(1)-CDKs, phosphorylation of Rb, activation of E2F1, and finally evasion from G(1) arrest induced by H(2)O(2). Levels of another senescence regulator, p21(waf1), however, were not affected by HBx under our senescence-inducing conditions. In addition, the potentials of HBx to inactivate Rb and subsequently inhibit cellular senescence almost completely disappeared when levels of p16(INK4a) were recovered either by exogenous complementation or inhibition of the promoter hypermethylation. To our knowledge, our present study represents the first report that an oncogenic virus evades cellular senescence through epigenetic down-regulation of p16(INK4a) expression. 2009 Elsevier Ireland Ltd. All rights reserved.

  12. The p16(INK4A)/pRb pathway and telomerase activity define a subgroup of Ph+ adult Acute Lymphoblastic Leukemia associated with inferior outcome.

    PubMed

    Chien, Wei W; Catallo, Régine; Chebel, Amel; Baranger, Laurence; Thomas, Xavier; Béné, Marie-Christine; Gerland, Luc M; Schmidt, Aline; Beldjord, Kheira; Klein, Nathalie; Escoffre-Barbe, Martine; Leguay, Thibaut; Huguet, Françoise; Larosa, Fabrice; Hayette, Sandrine; Plesa, Adriana; Ifrah, Norbert; Dombret, Hervé; Salles, Gilles; Chassevent, Agnès; Ffrench, Martine

    2015-04-01

    Adult Acute Lymphoblastic Leukemia (ALL) therapies have been improved by pediatric-like approaches. However, treatment failures and relapses are common and new markers are needed to identify patients with poor prognosis in prospective trials. The p16(INK4A)/CDK4-6/pRb pathway and telomerase activity, which are implicated in cell activation and aging, were analyzed to identify new prognostic markers. Proteins of the p16(INK4A)/CDK4-6/pRb pathway and telomerase activity were analyzed in 123 adult B-cell precursor (BCP) ALL cases included in the GRAALL/GRAAPH trials. We found a significantly increased expression of p16(INK4A) in BCP-ALLs with MLL rearrangement. Telomerase activity was significantly lower in Philadelphia chromosome-negative/IKAROS-deleted (BCR-ABL1(-)/IKAROS(del)) cases compared to Philadelphia chromosome-positive (BCR-ABL1+) BCP-ALLs. In BCR-ABL1+ ALLs, high CDK4 expression, phosphorylated pRb (p-pRb) and telomerase activity were significantly associated with a shorter disease-free survival (DFS) and event-free survival (EFS). Enhanced p16(INK4A) expression was only related to a significantly shorter DFS. In vitro analyses of normal stimulated lymphocytes after short- and long-term cultures demonstrated that the observed protein variations of poor prognosis in BCR-ABL1+ ALLs may be related to cell activation but not to cell aging. For these patients, our findings argue for the development of therapeutic strategies including the addition of new lymphocyte activation inhibitors to current treatments. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. The Use of Recombinant Pseudotype Virus-Like Particles Harbouring Inserted Target Antigen to Generate Antibodies against Cellular Marker p16INK4A

    PubMed Central

    Lasickienė, Rita; Gedvilaite, Alma; Norkiene, Milda; Simanaviciene, Vaida; Sezaite, Indre; Dekaminaviciute, Dovile; Shikova, Evelina; Zvirbliene, Aurelija

    2012-01-01

    Protein engineering provides an opportunity to generate new immunogens with desired features. Previously, we have demonstrated that hamster polyomavirus major capsid protein VP1-derived virus-like particles (VLPs) are highly immunogenic and can be employed for the insertion of foreign epitopes at certain surface-exposed positions. In the current study, we have designed pseudotype VLPs consisting of an intact VP1 protein and VP2 protein fused with the target antigen—cellular marker p16INK4A—at its N terminus. Both proteins coexpressed in yeast were self-assembled to pseudotype VLPs harbouring the inserted antigen on the surface. The pseudotype VLPs were used for generation of antibodies against p16INK4A that represents a potential biomarker for cells transformed by high-risk human papillomavirus (HPV). The pseudotype VLPs induced in immunized mice a strong immune response against the target antigen. The antisera raised against pseudotype VLPs showed specific immunostaining of p16INK4A protein in malignant cervical tissue. Spleen cells of the immunized mice were used to generate monoclonal antibodies against p16INK4A protein. The specificity of antibodies was proven by the immunostaining of HPV-transformed cells. In conclusion, the current study demonstrates the potential of pseudotype VLPs with inserted target antigen as a new type of immunogens to generate antibodies of high diagnostic value. PMID:22629125

  14. β-Catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development

    PubMed Central

    Delmas, Véronique; Beermann, Friedrich; Martinozzi, Silvia; Carreira, Suzanne; Ackermann, Julien; Kumasaka, Mayuko; Denat, Laurence; Goodall, Jane; Luciani, Flavie; Viros, Amaya; Demirkan, Nese; Bastian, Boris C.; Goding, Colin R.; Larue, Lionel

    2007-01-01

    Tumor progression is a multistep process in which proproliferation mutations must be accompanied by suppression of senescence. In melanoma, proproliferative signals are provided by activating mutations in NRAS and BRAF, whereas senescence is bypassed by inactivation of the p16Ink4a gene. Melanomas also frequently exhibit constitutive activation of the Wnt/β-catenin pathway that is presumed to induce proliferation, as it does in carcinomas. We show here that, contrary to expectations, stabilized β-catenin reduces the number of melanoblasts in vivo and immortalizes primary skin melanocytes by silencing the p16Ink4a promoter. Significantly, in a novel mouse model for melanoma, stabilized β-catenin bypasses the requirement for p16Ink4a mutations and, together with an activated N-Ras oncogene, leads to melanoma with high penetrance and short latency. The results reveal that synergy between the Wnt and mitogen-activated protein (MAP) kinase pathways may represent an important mechanism underpinning the genesis of melanoma, a highly aggressive and increasingly common disease. PMID:18006687

  15. 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.

  16. Alterations of pRb1-cyclin D1-cdk4/6-p16(INK4A) pathway in endometrial carcinogenesis.

    PubMed

    Semczuk, Andrzej; Jakowicki, Jerzy Andrzej

    2004-01-08

    The retinoblastoma protein pathway (pRb1-cyclin D1-cdk4/6-p16(INK4A)) participates in the regulation of the cellular processes at the transition of G1/S phases of the cell-cycle. Derailments of this pathway, caused either by lack of pRb1 or p16(INK4A) expression or overexpression of cyclin D1 and/or cdk4/6, are implicated in the deregulation of the cell-cycle machinery, resulting in uncontrolled cell proliferation, tumor heterogeneity, invasion and metastasis. Several studies conducted so far have assessed the deregulation of the pRb1-pathway components in various human tumors and cell-lines, provided these pathway alterations play an obligatory role in tumorigenesis. This review briefly summarizes the current information on the pRb1-cyclin D1-cdk4/6-p16(INK4A) alterations in sporadic uterine cancer, placing emphasis on the influence on the dualistic model of endometrial carcinogenesis.

  17. Id-1 stimulates serum independent prostate cancer cell proliferation through inactivation of p16(INK4a)/pRB pathway.

    PubMed

    Ouyang, Xue Song; Wang, Xianghong; Ling, Ming-Tat; Wong, Hing Lok; Tsao, Sai Wah; Wong, Y C

    2002-05-01

    It has been suggested that the helix-loop-helix protein Id-1 plays an important role in tumourigenesis in certain types of human cancer. Previously, we reported that Id-1 was up-regulated during sex hormone-induced prostate carcinogenesis in a Noble rat model (Ouyang et al. (2001) Carcinogenesis, 22, 965-973). In the present study, we investigated the direct effect of Id-1 expression on human prostate cancer cell proliferation by transfecting an Id-1 expression vector into a prostate cancer cell line LNCaP. Ten stable transfectant clones were isolated and the ectopic Id-1 expression resulted in both increased DNA synthesis rate and the percentage of S phase cells. To study the possible mechanisms involved in the Id-1 induced prostate cancer cell growth, we examined the expression of several factors responsible for G(1) to S phase progression. We found that Id-1 expression induced phosphorylation of RB and down-regulation of p16(INK4a) but not p21(Waf1)or p27(Kip1). Our results indicate that the Id-1 induced inactivation of p16(INK4a)/pRB pathway may be responsible for the increased cell proliferation in prostate cancer cells. Given the fact that both Id-1 over-expression and inactivation of p16(INK4a)/pRB are common events in prostate cancer, our results provide a possible mechanism on the molecular basis of prostate carcinogenesis.

  18. Interpretation of p16(INK4a) /Ki-67 dual immunostaining for the triage of human papillomavirus-positive women by experts and nonexperts in cervical cytology.

    PubMed

    Allia, Elena; Ronco, Guglielmo; Coccia, Anna; Luparia, Patrizia; Macrì, Luigia; Fiorito, Corinna; Maletta, Francesca; Deambrogio, Cristina; Tunesi, Sara; De Marco, Laura; Gillio-Tos, Anna; Sapino, Anna; Ghiringhello, Bruno

    2015-04-01

    The triage of human papillomavirus (HPV)-positive women is needed to avoid overreferral to colposcopy. p16(INK4a) immunostaining is an efficient triage method. p16(INK4a) /Ki-67 dual staining was introduced mainly to increase reproducibility and specificity compared with stand-alone p16(INK4a) staining. Within a pilot project, HPV-positive women were referred to colposcopy if cytology was abnormal or unsatisfactory or HPV testing was still positive after 1 year. For 500 consecutive women, a slide obtained during colposcopy was immunostained for p16(INK4a) /Ki-67 and independently interpreted by 7 readers without previous experience with dual staining. Four of these readers were experts in cervical pathology and 3 were not. Kappa values for multiple raters, sensitivity, and specificity for cervical intraepithelial neoplasia type 2-positive histology were computed. Because women with normal cytology were underrepresented, estimates for all HPV-positive women were obtained as weighted means of cytology-specific estimates. The overall kappa for HPV-positive women was 0.70 (95% confidence interval [95% CI], 0.60-0.77). Kappa values were not found to be significantly different between expert and nonexpert readers with regard to cervical cytology but were significantly increased (P =. 0066) after consensus discussion. The overall specificity estimate for HPV-positive women was 64.0% (95% CI, 57.4%-70.2%): 66.7% (95% CI, 59.8%-73.0%) for experts and 60.5% (95% CI, 59.8%-73.0%) for nonexperts. Among women with abnormal cytology, the sensitivity was 85.5% (95% CI, 77.9%-90.8%): 85.8% (95% CI, 77.9%-91.2%) for experts and 85.1% (95% CI, 76.6%-90.9%) for nonexperts. p16(INK4a) /Ki-67 immunostaining demonstrated good reproducibility and specificity when triaging HPV-positive women. Dual-staining interpretation can be performed, after short training, even by staff who are not experts in cervical cytology. This allows HPV-based screening with triage to be performed in settings in

  19. Evaluation of Telomerase (hTert), Ki67 and p16ink4a expressions in low and high-grade cervical intraepithelial lesions.

    PubMed

    Goulart, Ana Paula Szezepaniak; Gonçalves, Manoel Afonso Guimarães; DA-Silva, Vinicius Duval

    2017-01-01

    to study the association between the histological grading of cervical intraepithelial neoplasia (CIN I, CIN II and CIN III) and the immunohistochemical expression for p16ink4a, hTert and Ki67, as well as to evaluate the relationship of these markers with the risk of recurrence after surgical treatment. we studied a historical cohort of 94 women with intraepithelial lesions CIN I (low grade), CIN II and CIN III (high grades) submitted to conization or electrosurgical excision of the transformation zone. We evaluated all surgical specimens for immunohistochemical expression of p16ink4a, hTert and Ki67. the mean age was 38.2 years; p16ink4a was absent in most CIN I cases. In patients with CIN II or I/II (association of low and high-grade lesions), we observed p16ink4a ≤10%. In patients with CIN III, we found a higher expression frequency of p16ink4a >50%. In CIN I, the majority had Ki67≤10% and low frequency of Ki67>50%. In the CIN III category, there were fewer patients with Ki67≤10%, and Ki67 was absent in most patients of CIN II and III groups. There was no association between hTert expression and histologic grade. There were no statistically significant differences between the expression of the markers in patients with and without recurrence. there was a statistically significant association of p16ink4a and Ki67 with histological grade. The markers' expression, as for disease recurrence, was not statistically significant in the period evaluated. estudar a associação entre a graduação histológica das neoplasias intraepiteliais cervicais (NIC I, NIC II e NIC III) e a expressão imuno-histoquímica para p16ink4a, hTert e Ki67, assim como, avaliar a relação destes marcadores com o risco de recorrência após tratamento cirúrgico. estudo de coorte histórica de 94 mulheres portadoras de lesões intraepiteliais NIC I (baixo grau), NIC II e NIC III (altos graus), submetidas à conização ou à excisão eletrocirúrgica da zona de transformação. Todas as

  20. TMEM45A, SERPINB5 and p16INK4A transcript levels are predictive for development of high-grade cervical lesions

    PubMed Central

    Manawapat-Klopfer, Anna; Thomsen, Louise T; Martus, Peter; Munk, Christian; Russ, Rainer; Gmuender, Hans; Frederiksen, Kirsten; Haedicke-Jarboui, Juliane; Stubenrauch, Frank; Kjaer, Susanne K; Iftner, Thomas

    2016-01-01

    Women persistently infected with human papillomavirus (HPV) type 16 are at high risk for development of cervical intraepithelial neoplasia grade 3 or cervical cancer (CIN3+). We aimed to identify biomarkers for progression to CIN3+ in women with persistent HPV16 infection. In this prospective study, 11,088 women aged 20-29 years were enrolled during 1991-1993, and re-invited for a second visit two years later. Cervical cytology samples obtained at both visits were tested for HPV DNA by Hybrid Capture 2 (HC2), and HC2-positive samples were genotyped by INNO-LiPA. The cohort was followed for up to 19 years via a national pathology register. To identify markers for progression to CIN3+, we performed microarray analysis on RNA extracted from cervical swabs of 30 women with persistent HPV16-infection and 11 HPV-negative women. Six genes were selected and validated by quantitative PCR. Three genes were subsequently validated within a different and large group of women from the same cohort. Secondly, Kaplan-Meier and Cox-regression analyses were used to investigate whether expression levels of those three genes predict progression to CIN3+. We found that high transcript levels of TMEM45A, SERPINB5 and p16INK4a at baseline were associated with increased risk of CIN3+ during follow-up. The hazard ratios of CIN3+ per 10-fold increase in baseline expression level were 1.6 (95% CI: 1.1-2.3) for TMEM45A, 1.6 (95% CI: 1.1-2.5) for p16INK4a, and 1.8 (95% CI: 1.2-2.7) for SERPINB5. In conclusion, high mRNA expression levels of TMEM45A, SERPINB5 and p16INK4a were associated with increased risk of CIN3+ in persistently HPV16-infected women. PMID:27508094

  1. Localized juvenile spongiotic gingival hyperplasia featuring unusual p16INK4A labeling and negative human papillomavirus status by polymerase chain reaction.

    PubMed

    Argyris, Prokopios P; Nelson, Andrew C; Papanakou, Styliani; Merkourea, Stavroula; Tosios, Konstantinos I; Koutlas, Ioannis G

    2015-01-01

    Localized juvenile spongiotic gingival hyperplasia (LJSGH) is a distinct type of gingival hyperplastic lesion with specific clinicopathologic features. Evaluation of the morphological characteristics of LJSGH indicates the potential role of human papillomavirus (HPV) infection as an underlying etiopathogenetic mechanism. All cases of LJSGH from 2008 to present were retrieved. Clinical and demographic data were collected. HPV status was investigated by p16INK4A immunohistochemistry and HPV-Polymerase chain reaction (PCR). Twenty-one cases of LJSGH were identified, 14 (66.7%) affecting males and seven (33.3%) females (M:F = 2:1, age range: 8-36, mean: 13 years). All lesions were well-demarcated, exophytic, erythematous, and hemorrhagic with granular or slightly papillary surface. Preponderance for the maxillary gingiva (19, 90.5%) was observed. Two (9.5%) patients presented with recurrence 20 and 21 months after excision (mean follow-up: 18.7 months). Histopathologically, all LJSGH lesions featured epithelial hyperplasia with intense neutrophilic exocytosis and spongiosis. All cases demonstrated positivity for p16INK4A with the majority of specimens (47.6%) intensely decorated in >50% of the overlying epithelium with focal immunostaining observed in 47.6% and diffuse in 52.4%. Thirteen cases (61.9%) were negative for HPV DNA by PCR, while two (9.5%) were suspicious for the presence of low levels of HPV DNA but definitive genotyping was not possible. One case (4.8%) displayed positivity for HPV-31. The remaining five cases failed the PCR reaction. Human papillomavirus does not participate in the pathogenesis of LJSGH. P16INK4A expression in the absence of detectable HPV DNA can likely be attributed to the intense inflammation associated with LJSGH. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. 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.

  3. Post-transcriptional induction of p21cip1 protein in condylomata and dysplasias is inversely related to human papillomavirus activities.

    PubMed Central

    Schmidt-Grimminger, D. C.; Wu, X.; Jian, Y.; Broker, T. R.; Chow, L. T.

    1998-01-01

    Infections of the genital and oral epithelia by human papillomaviruses cause condylomata, papillomas, and squamous intraepithelial neoplasms, some of which can progress to invasive cancers. We describe an induction of p21cip1/WAF1/sdi1 protein in a fraction of the spinous cells in benign lesions and in cervical intraepithelial neoplasia grades I and II. The induction appears to be post-transcriptional and independent of p53. p21cip1 antigen-positive cells were sporadic in cervical intraepithelial neoplasia III and rare and focal in carcinomas. In contrast, p21cip1 protein was below or at the threshold of detection in the differentiated cells of normal squamous epithelia from different body sites despite an up-regulation of p21cip1 RNA. In cervical intraepithelial neoplasias from patients who were also positive for the human immunodeficiency virus, there was an additional increase in p21cip1 RNA in the upper spinous cells without concomitant p21cip1 protein induction. A consistent inverse relationship was observed between the p21cip1 protein induction and abundant human papillomavirus DNA and RNAs. We propose that p21cip1 protein induction is a novel host response that inhibits viral DNA replication and thus prevents elevated viral transcription. This hypothesis can partly account for the heterogeneity and the differentiation-dependent viral activities commonly observed in benign human papillomavirus lesions. Images Figure 1 Figure 2 Figure 3 PMID:9546362

  4. Molecular features of primary mediastinal B-cell lymphoma: involvement of p16INK4A, p53 and c-myc.

    PubMed

    Scarpa, A; Moore, P S; Rigaud, G; Inghirami, G; Montresor, M; Menegazzi, M; Todeschini, G; Menestrina, F

    1999-10-01

    Primary mediastinal B-cell lymphoma (PMBL) shows chromosome 9p anomalies in 50% of cases. Based on reports that p16INK4A gene, located on this chromosomal arm, is frequently altered in aggressive lymphomas, we analysed for alterations of this gene in 27 cases of PMBL, which were part of a series of 32 PMBL cases that have been characterized for alterations in c-myc, p53, N-ras, bcl-1, bcl-2, bcl-6 and for Epstein-Barr virus (EBV) infection. Four cases showed p16INK4A gene anomalies, including three with promoter methylation and one homozygous deletion. Eight PMBLs showed c-myc rearrangements. Three additional cases showed sequence variations in the c-myc P2 promoter, two of which consisted of the same germline variation involving a novel polymorphic XhoI site. Four tumours contained p53 gene mutations and three had clonal EBV infection. One case had a bcl-6 rearrangement. In conclusion, our study shows that p16INK4, c-myc and p53 alterations occur in 15%, 25% and 13% of PMBLs, respectively. EBV monoclonality was found in 9% of cases, whereas no abnormality was detected in bcl-1, bcl-2 and N-ras. Thus, none of the common genetic aberrations seen in other types of non-Hodgkin's lymphomas appears to be stringently involved in the pathogenesis of this unique lymphoma type.

  5. p16INK4A and p14ARF Gene Promoter Hypermethylation as Prognostic Biomarker in Oral and Oropharyngeal Squamous Cell Carcinoma: A Review

    PubMed Central

    Al-Kaabi, A.; van Bockel, L. W.; Pothen, A. J.; Willems, S. M.

    2014-01-01

    Head and neck squamous cell carcinoma is a heterogeneous group of tumors with each subtype having a distinct histopathological and molecular profile. Most tumors share, to some extent, the same multistep carcinogenic pathways, which include a wide variety of genetic and epigenetic changes. Epigenetic alterations represent all changes in gene expression patterns that do not alter the actual DNA sequence. Recently, it has become clear that silencing of cancer related genes is not exclusively a result of genetic changes such as mutations or deletions, but it can also be regulated on epigenetic level, mostly by means of gene promoter hypermethylation. Results from recent studies have demonstrated that DNA methylation patterns contain tumor-type-specific signatures, which could serve as biomarkers for clinical outcome in the near future. The topic of this review discusses gene promoter hypermethylation in oral and oropharyngeal squamous cell carcinoma (OSCC). The main objective is to analyse the available data on gene promoter hypermethylation of the cell cycle regulatory proteins p16INK4A and p14ARF and to investigate their clinical significance as novel biomarkers in OSCC. Hypermethylation of both genes seems to possess predictive properties for several clinicopathological outcomes. We conclude that the methylation status of p16INK4A is definitely a promising candidate biomarker for predicting clinical outcome of OSCC, especially for recurrence-free survival. PMID:24803719

  6. A simple stochastic model for the feedback circuit between p16INK4a and p53 mediated by p38MAPK: implications for senescence and apoptosis.

    PubMed

    de Oliveira, L R; Mombach, J C M; Castellani, G

    2015-11-01

    The mechanisms leading to the cell fate decision between apoptosis and senescence upon DNA damage are still unclear and have stochastic features. Cellular oxidative stress can generate DNA damage and activate the important mitogen-activated protein kinase 14 (p38MAPK) that is involved in pathologies like Alzheimer's disease. Based on experimental evidence we propose a simple network that might operate at the core of the cell control machinery for the choice between apoptosis and senescence involving the cross-talk between p38MAPK, the tumor suppressor protein p53 and the cyclin-dependent kinase inhibitor (p16INK4a). We have performed two types of analyses, deterministic and stochastic, exploring the system's parameter space, in the first, we calculated the fixed points of the deterministic model and, in the second, we numerically integrated the master equation for the stochastic version. The model shows a variety of behaviors dependent on the parameters including states of high expression levels of p53 or p16INK4a that can be associated with an apoptotic or senescent phenotype, respectively, in agreement with experimental data. In addition, we observe both monostable and bistable behavior (where bistability is a phenomenon in which two stable steady states coexist for a fixed set of control parameter values) which here we suggest to be involved in the cell fate decision problem.

  7. Role of p16(INK4a) and BMI-1 in oxidative stress-induced premature senescence in human dental pulp stem cells.

    PubMed

    Mas-Bargues, Cristina; Viña-Almunia, José; Inglés, Marta; Sanz-Ros, Jorge; Gambini, Juan; Ibáñez-Cabellos, José Santiago; García-Giménez, José Luis; Viña, José; Borrás, Consuelo

    2017-08-01

    Human dental pulp stem cells (hDPSCs) are a source for cell therapy. Before implantation, an in vitro expansion step is necessary, with the inconvenience that hDPSCs undergo senescence following a certain number of passages, loosing their stemness properties. Long-term in vitro culture of hDPSCs at 21% (ambient oxygen tension) compared with 3-6% oxygen tension (physiological oxygen tension) caused an oxidative stress-related premature senescence, as evidenced by increased β-galactosidase activity and increased lysil oxidase expression, which is mediated by p16(INK4a) pathway. Furthermore, hDPSCs cultured at 21% oxygen tension underwent a downregulation of OCT4, SOX2, KLF4 and c-MYC factors, which was recued by BMI-1 silencing. Thus, p16(INK4a) and BMI-1 might play a role in the oxidative stress-associated premature senescence. We show that it is important for clinical applications to culture cells at physiological pO2 to retain their stemness characteristics and to delay senescence. Copyright © 2017. Published by Elsevier B.V.

  8. Drm/Gremlin transcriptionally activates p21(Cip1) via a novel mechanism and inhibits neoplastic transformation.

    PubMed

    Chen, Bo; Athanasiou, Meropi; Gu, Qiuping; Blair, Donald G

    2002-08-02

    Drm/Gremlin, a member of the Dan family of BMP antagonists, is known to function in early embryonic development, but is also expressed in a tissue-specific fashion in adults and is significantly downregulated in transformed cells. In this report, we demonstrate that overexpression of Drm in the tumor-derived cell lines Daoy (primitive neuroectodermal) and Saos-2 (osteoblastic), either under ecdysone-inducible or constitutive promoters, significantly inhibits tumorigenesis. Furthermore, Drm overexpression in these cells increases the level of p21(Cip1) protein and reduces the level of phosphorylated p42/44 MAP kinase. Finally, our data indicate that Drm can induce p21(Cip1) transcriptionally via a novel pathway that is independent of p53 and the p38 and p42/44 MAP kinases. These results provide evidence that Drm can function as a novel transformation suppressor and suggest that this may occur through its affect on the levels of p21(Cip1) and phosphorylated p42/44 MAPK.

  9. Investigating Diagnostic Problems of CIN 1 and 2 Associated with High-Risk HPV by Combining the Novel Molecular Biomarker PanHPV E4 with P16ink4a

    PubMed Central

    Wu, Zhonglin; Soneji, Yasmina Jay; van de Sandt, Miekel; Arora, Rupali; van der Marel, Jacolien; ter Harmsel, Bram; Jach, Robert; Okon, Krzysztof; Huras, Hubert; Jenkins, David; Quint, Wim; Doorbar, John

    2015-01-01

    Grading cervical intraepithelial neoplasia (CIN) determines clinical management of women after abnormal cytology with potential for over-diagnosis and overtreatment. We studied a novel biomarker of HPV life-cycle completion (panHPVE4), in combination with the MCM cell-cycle marker and the p16INK4a transformation marker to improve CIN diagnosis and categorization. Scoring these biomarkers alongside CIN grading by three pathologists was performed on 114 cervical specimens with high-risk (HR-) HPV. Inter-observer agreement for histopathology was moderate (kappa (κ): 0.43 for CIN1/negative, 0.54 for CIN2/≤CIN1, and 0.36 for CIN3). Agreement was good or excellent for biomarker scoring (E4: κ=0.896; 95%CI: 0.763-0.969, p16INK4a: κ=0.798; 95%CI: 0.712-0.884, MCM: κ=0.894; 95%CI: n.c.). Biomarker expression was studied by immunofluorescence and immunohistochemistry and correlated with 104 final CIN diagnoses following histological review. All 25 histologically negative specimens were p16INK4a and panHPVE4 negative although 9 were MCM positive. There were variable extents of p16INK4a positivity in 11/11 CIN1, and extensive panHPVE4 staining in 9/11. Ten CIN2 lesions expressed panHPVE4 and p16INK4a and 13 CIN2 expressed only p16INK4a. CIN3 showed extensive p16INK4a positivity with no/minimal panHPVE4 staining. PanHPVE4, unlike MCM, distinguished CIN1 from negative. PanHPVE4 with p16INK4a separated CIN2/3 showing only expression of p16INK4a indicating transforming HR-HPV E7 expression, from CIN1/2 showing completion of HR-HPV life-cycle by E4 expression and variable p16INK4a expression. PanHPVE4 and p16INK4a staining are complementary markers that could provide simple, reliable support for diagnosing CIN. Their value in distinguishing CIN1/2 that supports HR-HPV life cycle completion (and which might ultimately regress), from purely transforming CIN2/3 needing treatment warrants further research. PMID:26379150

  10. Regulation of P21cip1 Expression by Growth Factors and the Extracellular Matrix Reveals a Role for Transient ERK Activity in G1 Phase

    PubMed Central

    Bottazzi, Maria Elena; Zhu, Xiaoyun; Böhmer, Ralph M.; Assoian, Richard K.

    1999-01-01

    We have examined the regulation of p21cip1 by soluble mitogens and cell anchorage as well as the relationship between the expression of p21cip1 and activation of the ERK subfamily of MAP kinases. We find that p21cip1 expression in G1 phase can be divided into two discrete phases: an initial induction that requires growth factors and the activation of ERK, and then a subsequent decline that is enhanced by cell anchorage in an ERK-independent manner. In contrast to the induction of cyclin D1, the induction of p21cip1 is mediated by transient ERK activity. Comparative studies with wild-type and p21cip1-null fibroblasts indicate that adhesion-dependent regulation of p21cip1 is important for proper control of cyclin E–cdk2 activity. These data lead to a model in which mitogens and anchorage act in a parallel fashion to regulate G1 phase expression of p21cip1. They also show that (a) growth factors and growth factor/extracellular matrix cooperation can have different roles in regulating G1 phase ERK activity and (b) both transient and sustained ERK signals have functionally significant roles in controlling cell cycle progression through G1 phase. PMID:10491389

  11. Oncogenic STRAP functions as a novel negative regulator of E-cadherin and p21(Cip1) by modulating the transcription factor Sp1.

    PubMed

    Jin, Lin; Datta, Pran K

    2014-01-01

    We have previously reported the identification of a novel WD-domain protein, STRAP that plays a role in maintenance of mesenchymal morphology by regulating E-cadherin and that enhances tumorigenicity partly by downregulating CDK inhibitor p21(Cip1). However, the functional mechanism of regulation of E-cadherin and p21(Cip1) by STRAP is unknown. Here, we have employed STRAP knock out and knockdown cell models (mouse embryonic fibroblast, human cancer cell lines) to show how STRAP downregulates E-cadherin and p21(Cip1) by abrogating the binding of Sp1 to its consensus binding sites. Moreover, ChIP assays suggest that STRAP recruits HDAC1 to Sp1 binding sites in p21(Cip1) promoter. Interestingly, loss of STRAP can stabilize Sp1 by repressing its ubiquitination in G1 phase, resulting in an enhanced expression of p21(Cip1) by >4.5-fold and cell cycle arrest. Using Bioinformatics and Microarray analyses, we have observed that 87% mouse genes downregulated by STRAP have conserved Sp1 binding sites. In NSCLC, the expression levels of STRAP inversely correlated with that of Sp1 (60%). These results suggest a novel mechanism of regulation of E-cadherin and p21(Cip1) by STRAP by modulating Sp1-dependent transcription, and higher expression of STRAP in lung cancer may contribute to downregulation of E-cadherin and p21(Cip1) and to tumor progression.

  12. p53 missense but not truncation mutations are associated with low levels of p21CIP1/WAF1 mRNA expression in primary human sarcomas

    PubMed Central

    Mousses, S; Gokgoz, N; Wunder, J S; Ozcelik, H; Bull, S; Bell, R S; Andrulis, I L

    2001-01-01

    Many growth-suppressing signals converge to control the levels of the CDK inhibitor p21CIP1/WAF1. Some human cancers exhibit low levels of expression of p21CIP1/WAF1and mutations in p53 have been implicated in this down-regulation. To evaluate whether the presence of p53 mutations was related to the in vivo expression of p21CIP1/WAF1 mRNA in sarcomas we measured the p21CIP1/WAF1 mRNA levels for a group of 71 primary bone and soft tissue tumours with known p53 status. As expected, most tumours with p53 mutations expressed low levels of p21CIP1/WAF1 mRNA. However, we identified a group of tumours with p53 gene mutations that exhibited normal or higher levels of p21CIP1/WAF1 mRNA. The p53 mutations in the latter group were not the common missense mutations in exons 4–9, but were predominantly nonsense mutations predicted to result in truncation of the p53 protein. The results of this study suggest that different types of p53 mutations can have different effects on the expression of downstream genes such as p21CIP1/WAF1 in human sarcomas. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11401317

  13. Abnormalities of the RB1 pathway in ovarian serous papillary carcinoma as determined by overexpression of the p16(INK4A) protein.

    PubMed

    Armes, Jane E; Lourie, Rohan; de Silva, Melanie; Stamaratis, Georgia; Boyd, Alison; Kumar, Beena; Price, Gareth; Hyde, Simon; Allen, David; Grant, Peter; Venter, Deon J

    2005-10-01

    Dysfunction of proteins involved in the G1 to S transition of the cell cycle, such as p16(INK4A) and RB1, is common in many cancer types. A screen of p16 protein expression was performed in benign, borderline, and invasive ovarian tumors, together with endometrial cancers, aligned on a tissue microarray. We observed frequent p16 overexpression in serous papillary carcinomas of ovarian and endometrial origin. An extended cohort of ovarian serous papillary carcinomas was examined to further evaluate the frequency of p16 overexpression. Strong, uniform staining in the majority of cancer cells occurred commonly in invasive serous papillary ovarian cancers, particularly in grade 3 carcinomas. RB1 protein expression abnormalities were rare. Our data indicate that abnormalities in the retinoblastoma pathway, as determined by p16 overexpression, are common in serous papillary carcinomas and are probably an early event.

  14. TLR9 re-expression in cancer cells extends the S-phase and stabilizes p16INK4a protein expression

    PubMed Central

    Parroche, P; Roblot, G; Le Calvez-Kelm, F; Tout, I; Marotel, M; Malfroy, M; Durand, G; McKay, J; Ainouze, M; Carreira, C; Allatif, O; Traverse-Glehen, A; Mendiola, M; Pozo-Kreilinger, J J; Caux, C; Tommasino, M; Goutagny, N; Hasan, U A

    2016-01-01

    Toll-like receptor 9 (TLR9) recognizes bacterial, viral or cell damage-associated DNA, which initiates innate immune responses. We have previously shown that TLR9 expression is downregulated in several viral induced cancers including HPV16-induced cervical neoplasia. Findings supported that downregulation of TLR9 expression is involved in loss of anti-viral innate immunity allowing an efficient viral replication. Here we investigated the role of TLR9 in altering the growth of transformed epithelial cells. Re-introducing TLR9 under the control of an exogenous promoter in cervical or head and neck cancer patient-derived cells reduced cell proliferation, colony formation and prevented independent growth of cells under soft agar. Neither TLR3, 7, nor the TLR adapter protein MyD88 expression had any effect on cell proliferation, indicating that TLR9 has a unique role in controlling cell growth. The reduction of cell growth was not due to apoptosis or necrosis, yet we observed that cells expressing TLR9 were slower in entering the S-phase of the cell cycle. Microarray-based gene expression profiling analysis highlighted a strong interferon (IFN) signature in TLR9-expressing head and neck cancer cells, with an increase in IFN-type I and IL-29 expression (IFN-type III), yet neither IFN-type I nor IL-29 production was responsible for the block in cell growth. We observed that the protein half-life of p16INK4a was increased in TLR9-expressing cells. Taken together, these data show for the first time that TLR9 affects the cell cycle by regulating p16INK4a post-translational modifications and highlights the role of TLR9 in the events that lead to carcinogenesis. PMID:27454079

  15. 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.

  16. 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

  17. Detection of MGMT, RASSF1A, p15INK4B, and p14ARF promoter methylation in circulating tumor-derived DNA of central nervous system cancer patients.

    PubMed

    Majchrzak-Celińska, Aleksandra; Paluszczak, Jarosław; Kleszcz, Robert; Magiera, Marta; Barciszewska, Anna-Maria; Nowak, Stanisław; Baer-Dubowska, Wanda

    2013-08-01

    Despite the growing understanding of the mechanisms of carcinogenesis, cancers of the central nervous system are usually associated with unfavorable prognosis. The use of an appropriate molecular marker may improve the treatment outcome by allowing early diagnosis and treatment susceptibility monitoring. Since methylation of tumor-derived DNA can be detected in the serum of cancer patients, this makes DNA methylation-based biomarkers one of the most promising diagnostic strategies. In this study, the methylation profiles of MGMT, RASSF1A, p15INK4B, and p14ARF genes were evaluated in serum free-circulating DNA and the corresponding tumor tissue in a group of 33 primary or metastatic central nervous system cancer patients. Gene promoter methylation was assessed using methylation-specific polymerase chain reaction (PCR). All the tested genes were found to be methylated to a different extent in both serum and tumor samples. In comparison to metastatic brain tumor patients, the patients with glial tumors were characterized by a higher frequency of gene hypermethylation. The hypermethylation of RASSF1A differentiated primary from metastatic brain cancers. Moreover, the gene methylation profiles observed in serum, in most cases, matched the methylation profiles detected in paired tumor samples.

  18. Diabetes-Induced Oxidative DNA Damage Alters p53-p21CIP1/Waf1 Signaling in the Rat Testis

    PubMed Central

    Al-Bader, Maie M.

    2015-01-01

    Diabetes is increasingly becoming a major cause of large-scale morbidity and mortality. Diabetes-induced oxidative stress alters numerous intracellular signaling pathways. Although testicular dysfunction is a major concern in diabetic men, the mechanistic alterations in the testes that lead to hypogonadism are not yet clear. Oxidative mitochondrial DNA damage, as indicated by 7,8-dihydro-8-oxo-2′-deoxyguanosine, and phosphorylation of p53 at ser315 residue (p-p53ser315) increased in a stage- and cell-specific manner in the testes of rats that were diabetic for 1 month (DM1). Prolongation of diabetes for 3 months (DM3) led to an increase in nuclear oxidative DNA damage in conjunction with a decrease in the expression of p-p53ser315. The nuclei of pachytene and preleptotene spermatocytes, steps 1, 11, and 12 spermatids, secondary spermatocytes and the Sertoli cells, and the meiotic figures showed an increase in the expression of p-p53ser315. An increase in the expression of a downstream target of p53 and protein 21cyclin-dependent kinase interacting protein 1/wild-type p53-activated factor 1 (p21CIP1/Waf1) in both diabetic groups did not show any time-dependent effects but occurred concurrent with an upregulation of p-p53ser315 in DM1 and a downregulation of the protein in DM3. In diabetic groups, the expression of p21CIP1/Waf1 was mainly cytoplasmic but also perinuclear in pachytene spermatocytes and round spermatids. The cytoplasmic localization of p21CIP1/Waf1 may be suggestive of an antiapoptotic role for the protein. The perinuclear localization is probably related to the cell cycle arrest meant for DNA damage repair. Diabetes upregulates p21CIP1/Waf1 signaling in testicular germ cells in association with alteration in p-p53ser315 expression, probably to counteract DNA damage-induced cell death. PMID:24828139

  19. Transcriptional Regulation of p21/CIP1 Cell Cycle Inhibitor by PDEF Controls Cell Proliferation and Mammary Tumor Progression*

    PubMed Central

    Schaefer, Jeremy S.; Sabherwal, Yamini; Shi, Heidi Y.; Sriraman, Venkataraman; Richards, JoAnne; Minella, Alex; Turner, David P.; Watson, Dennis K.; Zhang, Ming

    2010-01-01

    The Ets family of transcription factors control a myriad of cellular processes and contribute to the underlying genetic loss of cellular homeostasis resulting in cancer. PDEF (prostate-derived Ets factor) has been under investigation for its role in tumor development and progression. However, the role of PDEF in cancer development has been controversial. Some reports link PDEF to tumor promoter, and others show tumor-suppressing functions in various systems under different conditions. So far, there has been no conclusive evidence from in vivo experiments to prove the role of PDEF. We have used both in vitro and in vivo systems to provide a conclusive role of PDEF in the progression process. PDEF-expressing cells block the cell growth rate, and this retardation was reversible when PDEF expression was silenced with PDEF-specific small interfering RNA. When these PDEF-expressing cells were orthotopically implanted into the mouse mammary gland, tumor incidence and growth rate were significantly retarded. Cell cycle analysis revealed that PDEF expression partially blocked cell cycle progression at G1/S without an effect on apoptosis. PDEF overexpression resulted in an increase in p21/CIP1 at both the mRNA and protein levels, resulting in decreased Cdk2 activity. Promoter deletion analysis, electrophoresis mobility shift assays, and chromatin immunoprecipitation studies identified the functional Ets DNA binding site at −2118 bp of the p21/CIP1 gene promoter. This site is capable of binding and responding to PDEF. Furthermore, we silenced p21/CIP1 expression in PDEF-overexpressing cells by small interfering RNA. p21-silenced PDEF cells exhibited significantly increased cell growth in vitro and in vivo, demonstrating the p21 regulation by PDEF as a key player. These experiments identified PDEF as a new transcription factor that directly regulates p21/CIP1 expression under non-stressed conditions. This study conclusively proves that PDEF is a breast tumor suppressor for

  20. No evidence of oncogenic KRAS mutations in squamous cell carcinomas of the anogenital tract and head and neck region independent of human papillomavirus and p16(INK4a) status.

    PubMed

    Prigge, Elena-Sophie; Urban, Katharina; Stiegler, Sandrine; Müller, Meike; Kloor, Matthias; Mai, Sabine; Ottstadt, Martine; Lohr, Frank; Wenz, Frederik; Wagner, Steffen; Wittekindt, Claus; Klussmann, Jens Peter; Hampl, Monika; von Knebel Doeberitz, Magnus; Reuschenbach, Miriam

    2014-11-01

    Carcinogenesis of squamous cell carcinomas (SCCs) in the anogenital tract and head and neck region is heterogeneous. A substantial proportion of SCC in the vulva, anus, and head and neck follows a human papillomavirus (HPV)-induced carcinogenic pathway. However, the molecular pathways of carcinogenesis in the HPV-independent lesions are not completely understood. We hypothesized that oncogenic Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations might represent a carcinogenic mechanism in a proportion of those HPV-negative cancers. Considering the repeated observation of KRAS-associated p16(INK4a) overexpression in human tumors, it was assumed that KRAS mutations might be particularly present in the group of HPV-negative, p16(INK4a)-positive cancers. To test this hypothesis, we analyzed 66 anal, vulvar, and head and neck SCC with known immunohistochemical p16(INK4a) and HPV DNA status for KRAS mutations in exon 2 (codons 12, 13, and 15). We enriched the tumor collection with HPV DNA-negative, p16(INK4a)-positive cancers. A subset of 37 cancers was also analyzed for mutations in the B-Raf proto-oncogene, serine/threonine kinase (BRAF) gene. None of the 66 tumors harbored mutations in KRAS exon 2, thus excluding KRAS mutations as a common event in SCC of the anogenital and head and neck region and as a cause of p16(INK4a) expression in these tumors. In addition, no BRAF mutations were detected in the 37 analyzed tumors. Further studies are required to determine the molecular events underlying HPV-negative anal, vulvar, and head and neck carcinogenesis. Considering HPV-independent p16(INK4a) overexpression in some of these tumors, particular focus should be placed on alternative upstream activators and potential downstream disruption of the p16(INK4a) pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Concentrations of Pro-Inflammatory Cytokines Are Not Associated with Senescence Marker p16INK4a or Predictive of Intracellular Emtricitabine/Tenofovir Metabolite and Endogenous Nucleotide Exposures in Adults with HIV Infection

    PubMed Central

    Maas, Brian M.; Francis, Owen; Mollan, Katie R.; Lee, Cynthia; Cottrell, Mackenzie L.; Prince, Heather M. A.; Sykes, Craig; Trezza, Christine; Torrice, Chad; White, Nicole; Malone, Stephanie; Hudgens, Michael G.; Sharpless, Norman E.; Dumond, Julie B.

    2016-01-01

    Objectives As the HIV-infected population ages, the role of cellular senescence and inflammation on co-morbid conditions and pharmacotherapy is increasingly of interest. p16INK4a expression, a marker for aging and senescence in T-cells, is associated with lower intracellular concentrations of endogenous nucleotides (EN) and nucleos(t)ide reverse transcriptase inhibitors (NRTIs). This study expands on these findings by determining whether inflammation is contributing to the association of p16INK4a expression with intracellular metabolite (IM) exposure and endogenous nucleotide concentrations. Methods Samples from 73 HIV-infected adults receiving daily tenofovir/emtricitabine (TFV/FTC) with either efavirenz (EFV) or atazanavir/ritonavir (ATV/r) were tested for p16INK4a expression, and plasma cytokine and intracellular drug concentrations. Associations between p16INK4a expression and cytokine concentrations were assessed using maximum likelihood methods, and elastic net regression was applied to assess whether cytokines were predictive of intracellular metabolite/endogenous nucleotide exposures. Results Enrolled participants had a median age of 48 years (range 23–73). There were no significant associations between p16INK4a expression and cytokines. Results of the elastic net regression showed weak relationships between IL-1Ra and FTC-triphosphate and deoxyadenosine triphosphate exposures, and MIP-1β, age and TFV-diphosphate exposures. Conclusions In this clinical evaluation, we found no relationships between p16INK4a expression and cytokines, or cytokines and intracellular nucleotide concentrations. While inflammation is known to play a role in this population, it is not a major contributor to the p16INK4a association with decreased IM/EN exposures in these HIV-infected participants. PMID:28036343

  2. Up-regulation of survivin during immortalization of human myofibroblasts is linked to repression of tumor suppressor p16(INK4a) protein and confers resistance to oxidative stress.

    PubMed

    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

    2013-04-26

    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 p16(INK4a). Survivin and p16(INK4a) were functionally linked by assays that showed that either the up-regulation of survivin or repression of p16(INK4a) 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/p16(INK4a) 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 p16(INK4a) during telomerase-mediated immortalization. E2F1 binding to the BIRC5 was also enhanced in telomerase-transduced cells subjected to shRNA-mediated repression of p16(INK4a). Together, these data show that repression of p16(INK4a) 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.

  3. Skin epidermis lacking the c-myc gene is resistant to Ras-driven tumorigenesis but can reacquire sensitivity upon additional lossof the p21Cip1 gene

    PubMed Central

    Oskarsson, Thordur; Essers, Marieke Alida Gertruda; Dubois, Nicole; Offner, Sandra; Dubey, Christelle; Roger, Catherine; Metzger, Daniel; Chambon, Pierre; Hummler, Edith; Beard, Peter; Trumpp, Andreas

    2006-01-01

    The target gene(s) required for Myc-mediated tumorigenesis are still elusive. Here we show that while endogenous c-Myc is surprisingly dispensable for skin homeostasis and TPA-induced hyperplasia, c-Myc-deficient epidermis is resistant to Ras-mediated DMBA/TPAinduced tumorigenesis. This is mechanistically linked to p21Cip1, which is induced in tumors by the activated Ras–ERK pathway but repressed by c-Myc. Acute elimination of c-Myc in established tumors leads to the up-regulation of p21Cip1, and epidermis lacking both p21Cip1 and c-Myc reacquires normal sensitivity to DMBA/TPA-induced tumorigenesis. This identifies c-Myc-mediated repression of p21Cip1 as a key step for Ras-driven epidermal tumorigenesis. PMID:16882980

  4. AU-1 from Agavaceae plants causes transient increase in p21/Cip1 expression in renal adenocarcinoma ACHN cells in an miR-34-dependent manner.

    PubMed

    Fujino, Tomofumi; Yokosuka, Akihito; Higurashi, Hideaki; Yokokawa, Rina; Sakurai, Ryo; Harashima, Wataru; Miki, Yuichi; Fujiwara, Yasuyuki; Mimaki, Yoshihiro; Hayakawa, Makio

    2017-01-01

    Here, we show that AU-1, spirostanol saponin isolated from Agavaceae plants, causes a transient increase in cyclin-dependent kinase inhibitor (CDKI) p21/Cip1 through the upregulation of miRNAs, miR-34 and miR-21. AU-1 stimulated p21/Cip1 expression without exerting cytotoxicity against different types of carcinoma cell lines. In renal adenocarcinoma ACHN cells, AU-1 transiently elevated the expression level of p21/Cip1 protein without marked increases in p21/Cip1 mRNA levels. Rapid and transient increases in miR-34 and miR-21, both of which are known to upregulate p21/Cip1, were observed in AU-1-treated cells. Inhibitor for miR-34 and for miR-21 significantly blocked the AU-1-caused increase in p21/Cip1, indicating that elevation of p21/Cip1 protein by AU-1 is dependent on these microRNAs. We further clarified that NAD-dependent deacetylase SIRT1, a direct target of miR-34, is decreased by the treatment with AU-1. Furthermore, we found that SIRT1-knockdown increases p21/Cip1 protein levels in an miR-21-dependent manner. On the other hand, ectopic expression of p21/Cip1 resulted in the lowered expression of miR-34 and miR-21, suggesting that reciprocal regulation exists between p21/Cip1 and these miRNAs. We propose that the following feedback network composed of miR-34/SIRT1/miR-21/p21 is triggered by the treatment with AU-1: in cells treated with AU-1, transient elevation of miR-34 leads to the downregulation of SIRT1, thereby miR-21 is freed from SIRT1-dependent suppression. Then, elevated miR-21 upregulates p21/Cip1 protein, followed by the suppression of miR-34 expression.

  5. A role for transcriptional repression of p21CIP1 by c-Myc in overcoming transforming growth factor β-induced cell-cycle arrest

    PubMed Central

    Claassen, Gisela F.; Hann, Stephen R.

    2000-01-01

    c-Myc plays a vital role in cell-cycle progression. Deregulated expression of c-Myc can overcome cell-cycle arrest in order to promote cellular proliferation. Transforming growth factor β (TGFβ) treatment of immortalized human keratinocyte cells inhibits cell-cycle progression and is characterized by down-regulation of c-Myc followed by up-regulation of p21CIP1. A direct role of c-Myc in this pathway was demonstrated by the observation that ectopic expression of c-Myc overcame the cell-cycle block induced by TGFβ treatment. The induction of p21CIP1 transcription by TGFβ was blocked in human keratinocyte cells stably expressing c-Myc. Furthermore, overexpression of c-Myc in NIH 3T3 cells repressed the basal levels of p21CIP1 mRNA. Repression of p21CIP1 transcription by c-Myc occurred at the promoter level in a region near the start site of transcriptional initiation and was independent of histone deacetylase activity. These data suggest that the down-regulation of c-Myc after TGFβ signaling is important for subsequent regulation of p21CIP1 and cell-cycle inhibition. Thus, repression of the cell-cycle inhibitory gene p21CIP1 plays a role in c-Myc-dependent cell-cycle progression. PMID:10920185

  6. Clinical role of p16INK4a expression in liquid-based cervical cytology: correlation with HPV testing and histologic diagnosis.

    PubMed

    Benevolo, Maria; Vocaturo, Amina; Mottolese, Marcella; Mariani, Luciano; Vocaturo, Giuseppe; Marandino, Ferdinando; Sperduti, Isabella; Rollo, Francesca; Antoniani, Barbara; Donnorso, Raffaele Perrone

    2008-04-01

    p16INK4a is overexpressed in high-risk human papillomavirus (HR-HPV)-infected preneoplastic and neoplastic lesions of the uterine cervix. Our aim was to verify whether p16 is a diagnostic marker also in cervical liquid-based cytology. We performed p16 immunocytochemical analysis and the Hybrid Capture 2 (HC2) test (Digene, Gaithersburg, MD) for HR-HPV infection in 471 ThinPrep-processed (Cytyc, Boxborough, MA) cervicovaginal samples and correlated the results with histologic findings. A total of 32.3% of the specimens showed p16 immunoreactivity, whereas the HC2 test was positive in 41.2% of the cases (65.2% concordance rate). Correlating the cytologic, p16, and HPV results with histologic findings revealed HC2 as the most sensitive test for a diagnosis of cervical intraepithelial neoplasia 2 or worse, whereas cytologic examination was the most specific. The positive predictive value was significantly higher for cytologic examination than for p16 and HR-HPV testing. These data suggest that p16 evaluation in ThinPrep samples does not have better clinical effectiveness for identifying high-grade lesions than conventional morphologic examination and HPV testing.

  7. Effect of phosphorylation and methylation on the function of the p16INK4a protein in non-small cell lung cancer A549 cells

    PubMed Central

    MA, WEN-LONG; WANG, LIN; LIU, LING-XIA; WANG, XIU-LI

    2015-01-01

    The p16INK4a protein (p16) has been reported to be a tumor suppressor gene that suppresses the proliferation of cells through the direct inhibition of cell cycle progression. Accordingly, p16 is a potential target for cancer gene therapy. In the present study, the arginine 22, 131 and 138 residues of p16 were found to be methylation sites, as the mutation of these arginine residues to lysine resulted in the hypomethylation of p16. Furthermore, the protein arginine methyltransferases (PRMTs), such as PRMT1, PRMT4 and PRMT6, were determined to be involved in the methylation of the p16 arginine residues. PRMT6 effectively reduced the intensity of the association between p16 and CDK4, and also weakened the function of p16 in preventing cell proliferation. In addition, the p16 protein was found to be phosphorylated in various cell lines, and mutations in the serine residues weakened the cell cycle arrest and induction of apoptosis mediated by p16. Preliminarily, the crosstalk between the phosphorylation and arginine methylation modification of p16 was examined. These findings predict a role for serine phosphorylation against arginine methylation of p16. PMID:26622834

  8. Mutational analysis of the N-ras, p53, p16INK4a, CDK4, and MC1R genes in human congenital melanocytic naevi.

    PubMed

    Papp, T; Pemsel, H; Zimmermann, R; Bastrop, R; Weiss, D G; Schiffmann, D

    1999-08-01

    Eighteen human congenital melanocytic naevi (CMN) from 17 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 and for sequence variants in the MC1R gene by combined RFLP-PCR/SSCP analysis. In addition, all lesions were screened for deletions and point mutations in the tumour suppressor genes p53 and p16INK4a (CDKN2A) by combined multiplex PCR/SSCP analysis. Positive screening data were specified by sequencing of the corresponding PCR product. Activating point mutations in the N-ras gene (nine CAA (Gln) to AAA (Lys) transversions and one CAA (Gln) to CGA (Arg) transition at codon 61) were detected at high frequency (56%). Furthermore, three missense mutations (V92M) and two silent mutations (CGA (Arg) to CGG (Arg), codon 213, exon 6) were found in the MC1R and p53 genes, respectively. No mutations were found in p16 or CDK4. The activated N-ras oncogene, which is also found in human cutaneous melanomas, may constitute a potential risk factor for melanoma formation within CMN.

  9. Correlation of p16INK4a immunoexpression and human papillomavirus (HPV) detected by in-situ hybridization in cervical squamous neoplasia.

    PubMed

    Cheah, P L; Koh, C C; Nazarina, A R; Teoh, K H; Looi, L M

    2016-04-01

    Persistence and eventual integration of high-risk HPV (hrHPV) into the cervical cell is crucial to the progression of cervical neoplasia and it would be beneficial to morphologically identify this transformation in routine surgical pathology practice. Increased p16(INK4a) (p16) expression is a downstream event following HPV E7 binding to pRB. A study was conducted to assess the correlation between hrHPV detection using a commercial in-situ hybridization assay (Ventana INFORM HPV ISH) and p16 immunoexpression (CINtec Histology Kit) in cervical squamous intraepithelial lesions and squamous carcinoma. 27 formalin-fixed, paraffin-embedded cervical low-grade squamous intraepithelial lesions (LSIL), 21 high-grade squamous intraepithelial lesions (HSIL) and 51 squamous carcinoma (SCC) were interrogated. hrHPV was significantly more frequent in HSIL (76.2%) and SCC (88.2%) compared to LSIL(37.0%). p16 expression was similarly more frequent in HSIL (95.2%) and SCC (90.2%) compared to LSIL(3.7%). That the rates of hrHPV when compared with p16 expression were almost equivalent in HSIL and SCC while p16 was expressed in only 1 of the 10 LSIL with hrHPV, are expected considering the likelihood that transformation has occurred in HSIL and SCC but does not occur in majority of LSIL.

  10. Coincident inactivation of 14-3-3sigma and p16INK4a is an early event in vulval squamous neoplasia.

    PubMed

    Gasco, Milena; Sullivan, Alex; Repellin, Claire; Brooks, Louise; Farrell, Paul J; Tidy, John A; Dunne, Barbara; Gusterson, Barry; Evans, David J; Crook, Tim

    2002-03-14

    The structure and expression of 14-3-3 sigma(sigma) was analysed in squamous carcinomas (SCC) of the vulva and in the vulval pre-malignant lesion vulval intraepithelial neoplasia (VIN). Sequence analysis of the sigma coding region did not detect mutations in any case of SCC or VIN III and loss of heterozygosity (LOH) occurred in only 2 out of 27 informative cases. In contrast to the absence of genetic change, methylation-specific PCR (MSP) analysis revealed dense CpG methylation within the sigma gene in approximately 60% of cases of vulval SCC, but methylation was not detected in matched, normal epithelial tissue. Methylation was associated in all cases with reduced or absent expression of sigma mRNA. There was no correlation between sigma methylation and HPV or p53 status. Analysis of pre-malignant vulval intraepithelial neoplasia (VIN) revealed that sigma methylation was detectable early in neoplastic development. Co-incident methylation, accompanied by loss of expression, of sigma and p16INK4a was commonly detected in both SCC and VIN III, suggesting that epigenetic silencing of these two genes is an early and important event in vulval neoplasia.

  11. The homeoprotein SIX1 controls cellular senescence through the regulation of p16INK4A and differentiation-related genes.

    PubMed

    Adrados, I; Larrasa-Alonso, J; Galarreta, A; López-Antona, I; Menéndez, C; Abad, M; Gil, J; Moreno-Bueno, G; Palmero, I

    2016-07-07

    Cellular senescence is an antiproliferative response with essential functions in tumor suppression and tissue homeostasis. Here we show that SIX1, a member of the SIX family of homeobox transcriptional factors, is a novel repressor of senescence. Our data show that SIX1 is specifically downregulated in fibroblasts upon oncogenic stress and other pro-senescence stimuli, as well as in senescent skin premalignant lesions. Silencing of SIX1 in human fibroblasts suffices to trigger senescence, which is mediated by p16INK4A and lacks a canonical senescence-associated secretory phenotype. Interestingly, SIX1-associated senescence is further characterized by the expression of a set of development and differentiation-related genes that significantly overlap with genes associated with SIX1 in organogenesis or human tumors, and show coincident regulation in oncogene-induced senescence. Mechanistically, we show that gene regulation by SIX1 during senescence is mediated, at least in part, by cooperation with Polycomb repressive complexes. In summary, our results identify SIX1, a key development regulator altered in human tumors, as a critical repressor of cellular senescence, providing a novel connection between senescence, differentiation and tumorigenesis.

  12. NS1- and Minute Virus of Mice-Induced Cell Cycle Arrest: Involvement of p53 and p21cip1

    PubMed Central

    Op De Beeck, Anne; Sobczak-Thepot, Joelle; Sirma, Huseyin; Bourgain, Florence; Brechot, Christian; Caillet-Fauquet, Perrine

    2001-01-01

    The nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVMp) is cytolytic when expressed in transformed cells. Before causing extensive cell lysis, NS1 induces a multistep cell cycle arrest in G1, S, and G2, well reproducing the arrest in S and G2 observed upon MVMp infection. In this work we investigated the molecular mechanisms of growth inhibition mediated by NS1 and MVMp. We show that NS1-mediated cell cycle arrest correlates with the accumulation of the cyclin-dependent kinase (Cdk) inhibitor p21cip1 associated with both the cyclin A/Cdk and cyclin E/Cdk2 complexes but in the absence of accumulation of p53, a potent transcriptional activator of p21cip1. By comparison, MVMp infection induced the accumulation of both p53 and p21cip1. We demonstrate that p53 plays an essential role in the MVMp-induced cell cycle arrest in both S and G2 by using p53 wild-type (+/+) and null (−/−) cells. Furthermore, only the G2 arrest was abrogated in p21cip1 null (−/−) cells. Together these results show that the MVMp-induced cell cycle arrest in S is p53 dependent but p21cip1 independent, whereas the arrest in G2 depends on both p53 and its downstream effector p21cip1. They also suggest that induction of p21cip1 by the viral protein NS1 arrests cells in G2 through inhibition of cyclin A-dependent kinase activity. PMID:11602746

  13. Distinct susceptibility of induction of methylation of p16ink4a and p19arf CpG islands by X-radiation and chemical carcinogen in mice.

    PubMed

    Yang, Chen; Gu, Liankun; Deng, Dajun

    2014-07-01

    Inactivation of the tumor suppressor genes p16(ink4a) and p19(arf)/p14(arf) by hypermethylation of promoter CpG islands occurs frequently in various tumors. The aim of this study is to investigate the difference of susceptibility of methylation induced by carcinogens between p16(ink4a) and p19(arf). The methylation status of both genes was analyzed by denaturing high performance liquid chromatography (DHPLC) and bisulfite-sequencing, respectively. The expression level of P16 protein was analyzed by immunohistochemistry. Results showed that p16(ink4a) methylation was detected in the glandular stomach, small intestine and other organs of mice following X-radiation and subsequent bone marrow transplantation (BMT), but not in mock control mice. We found that the intestinal tract was the most sensitive organ for X-ray induced p16(ink4a) methylation. Loss of P16 protein expression was observed in the intestinal tissues of X-irradiated mice, but not in the mock control mice. Interestingly, p19(arf) methylation was not observed in the gastrointestinal tissues of the negative control mice following X-radiation/BMT. However, administration of N-nitrosomethylurea and/or Helicobacter felis infection promoted methylation of p19(arf) CpG islands in the gastrointestinal tracts, but did not promote p16(ink4a) methylation. In addition, p16(ink4a) methylation was detected not only in the X-irradiated GFP-negative tissue cells, but also in the GFP-positive bone marrow-derived cells that were transplanted into the BMT mice after X-radiation. In conclusion, the methylation susceptibility of p16(ink4a) and p19(arf) to carcinogen treatments was remarkably different: X-radiation indirectly induces systemic p16(ink4a) methylation, especially in the intestine; whereas N-nitrosomethylurea and/or H. felis infection induce p19(arf) methylation in their target organs. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Involvement of p21cip1/waf1 in the anti-proliferative effects of polyethylene glycol in colon carcinogenesis.

    PubMed

    Roy, Hemant K; Koetsier, Jennifer L; Tiwari, Ashish K; Joshi, Suhasini; Kunte, Dhananjay P; Ward, Tina P; Gandhi, Seema R; Wali, Ramesh K

    2011-02-01

    Polyethylene glycol (PEG) is a safe and effective chemopreventive agent against colorectal carcinogenesis in cell culture, animal models and human subjects. Although the precise molecular mechanism is unclear, we previously reported that PEG suppresses colonic epithelial proliferation. As cellular proliferation is driven by complex G1-S phase transition, we now characterize the role of PEG on cell cycle regulation. We focused our attention on the effect of PEG on the CDK inhibitor p21cip1/waf1, which is implicated in early colon carcinogenesis and is upregulated by non-steroidal anti-inflammatory drugs. These studies were done in the azoxymethane-treated (AOM) rat model as well as in HT-29 colon cancer cells. Immunohistochemical analysis revealed that while AOM decreased the p21 expression (75%, p<0.01) in the premalignant colonic mucosa, PEG induced p21 levels back to normal. These findings paralleled a decreased BrdUrd incorporation (78%, p<0.001) and hypophosphorylated retinoblastoma protein (Rb; by 47%) signifying PEG's antiproliferative activity. Furthermore, in HT-29 cells, PEG decreased proliferation as measured by PCNA (68% reduction), increased p21 expression (2.3-fold), induced cell cycle arrest during G0/G1 phase (45% reduction in S phase cells) and inhibited the phosphorylation of Rb (by 52% compared to untreated). PEG caused greater than a 2-fold induction of protein and mRNA level of p21cip1/waf1 in HT-29 cells. These results demonstrate for the first time that PEG is involved in p21 regulation concomitant with G1S phase cell cycle arrest and it is through these effects that it can exert its anti-proliferative and hence chemopreventive role.

  15. Persistent p21Cip1 induction mediates G(1) cell cycle arrest by methylseleninic acid in DU145 prostate cancer cells.

    PubMed

    Wang, Zhe; Lee, Hyo-Jeong; Chai, Yubo; Hu, Hongbo; Wang, Lei; Zhang, Yong; Jiang, Cheng; Lü, Junxuan

    2010-05-01

    The induction of G(1) cell cycle arrest and apoptosis by second-generation selenium compounds (e.g., methylselenol precursors such as methylseleninic acid, MSeA) may contribute to their anti-cancer activities. We have documented previously induction of G(1) arrest and apoptosis by MSeA in association with upregulation of cyclin-dependent kinase inhibitor (CDKI) proteins p21Cip1 and/or p27Kip1 in DU145 prostate cancer cells. However, whether these CDKIs play a critical mediator role in G(1) arrest and apoptosis by MSeA has not been addressed. In the present work, we show exposure of p53-mutant DU145 cells to sub-apoptotic concentrations of MSeA induced p21cip1 mRNA (3 h) and protein (6 h) much faster than p27kip1 mRNA (12 h) and protein (12 h). Knocking down of p21 by siRNA completely abolished G(1) arrest induction by MSeA in DU145 cells, yet si-p27 RNA had no attenuation effect on the G(1) arrest. Depletion of p21Cip1 alone or both p21Cip1 and p27Kip1 increased MSeA-induced caspase-mediated apoptosis. Immunoprecipitation detected increased binding of p21Cip1 to CDK2 and CDK6 in MSeA-exposed DU145 cells. In DU145 xenografts from mice acutely treated with MSeA p.o., the induction of p21Cip1 was observed at 72 h of daily exposure. In p53-wild type LNCaP PCa cells and p53-null PC-3 PCa cells, MSeA modestly and transiently upregulated p21Cip1 protein level, subsiding to basal level by 24 h, without affecting P27Kip1 abundance in the same duration. Si-p21 RNA knockdown in these cells have only a partial effect to reverse G(1) arrest induction by MSeA. Together, our data support persistent, p53-independent, p21Cip1 induction as a critical mediator of MSeA-induced G(1) arrest in DU145 PCa cells, however, p21Cip1 induction and G(1) arrest were not necessary for, and may antagonize, caspase-mediated apoptosis.

  16. MTHFR, MTR, and MTRR Polymorphisms in Relation to p16INK4A Hypermethylation in Mucosa of Patients with Colorectal Cancer

    PubMed Central

    Wettergren, Yvonne; Odin, Elisabeth; Carlsson, Göran; Gustavsson, Bengt

    2010-01-01

    We recently analyzed the hypermethylation status of the p16INK4a (p16) gene promoter in normal-appearing mucosa obtained from patients with colorectal cancer. Hypermethylation of p16 was associated with reduced survival of these patients. In the present study, germ line polymorphisms in the folate- and methyl-associated genes, methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and methionine synthase reductase (MTRR), were analyzed in the same patient cohort to find a possible link between these genetic variants and p16 hypermethylation. Genomic DNA was extracted from blood of patients (n = 181) and controls (n = 300). Genotype analyses were run on an ABI PRISM® 7900HT sequence-detection system (Applied Biosystems), using real-time polymerase chain reaction and TaqMan chemistry. The results showed that the genotype distributions of the patient and control groups were similar. No significant differences in cancer-specific or disease-free survival of stage I–III patients according to polymorphic variants were detected, nor were any differences in cancer-specific or disease-free survival detected when patients were subgrouped according to the MTHFR or MTR genotype groups and dichotomized by p16 hypermethylation status in mucosa. However, patients with the MTRR 66 AA/AG genotypes were found to have a significantly worse cancer-specific survival when the mucosa were positive, compared with negative, for p16 hypermethylation (hazard ratio 2.7; 95% confidence interval 1.2–6.4; P = 0.023). In contrast, there was no difference in survival among patients with the MTRR 66 GG genotype stratified by p16 hypermethylation status. These results indicate a relationship between genetic germ-line variants of the MTRR gene and p16 hypermethylation in mucosa, which may affect the clinical outcome of patients with colorectal cancer. PMID:20549016

  17. MTHFR, MTR, and MTRR polymorphisms in relation to p16INK4A hypermethylation in mucosa of patients with colorectal cancer.

    PubMed

    Wettergren, Yvonne; Odin, Elisabeth; Carlsson, Göran; Gustavsson, Bengt

    2010-01-01

    We recently analyzed the hypermethylation status of the p16INK4a (p16) gene promoter in normal-appearing mucosa obtained from patients with colorectal cancer. Hypermethylation of p16 was associated with reduced survival of these patients. In the present study, germ line polymorphisms in the folate- and methyl-associated genes, methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and methionine synthase reductase (MTRR), were analyzed in the same patient cohort to find a possible link between these genetic variants and p16 hypermethylation. Genomic DNA was extracted from blood of patients (n = 181) and controls (n = 300). Genotype analyses were run on an ABI PRISM(®) 7900HT sequence-detection system (Applied Biosystems), using real-time polymerase chain reaction and TaqMan chemistry. The results showed that the genotype distributions of the patient and control groups were similar. No significant differences in cancer-specific or disease-free survival of stage I-III patients according to polymorphic variants were detected, nor were any differences in cancer-specific or disease-free survival detected when patients were subgrouped according to the MTHFR or MTR genotype groups and dichotomized by p16 hypermethylation status in mucosa. However, patients with the MTRR 66 AA/AG genotypes were found to have a significantly worse cancer-specific survival when the mucosa were positive, compared with negative, for p16 hypermethylation (hazard ratio 2.7; 95% confidence interval 1.2-6.4; P = 0.023). In contrast, there was no difference in survival among patients with the MTRR 66 GG genotype stratified by p16 hypermethylation status. These results indicate a relationship between genetic germ-line variants of the MTRR gene and p16 hypermethylation in mucosa, which may affect the clinical outcome of patients with colorectal cancer.

  18. 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

  19. Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions.

    PubMed

    Benevolo, Maria; Mottolese, Marcella; Marandino, Ferdinando; Vocaturo, Giuseppe; Sindico, Roberto; Piperno, Giulia; Mariani, Luciano; Sperduti, Isabella; Canalini, Paola; Donnorso, Raffaele P; Vocaturo, Amina

    2006-03-01

    The p16(INK4a) is a cyclin-dependent kinase inhibitor that decelerates the cell cycle by inactivating the cyclin-dependent kinases involved in the phosphorylation of the retinoblastoma protein (RB). Expression of E6 and E7 oncogenes of high-risk (HR) human papillomavirus (HPV), affecting the RB-p16 pathway, leads to p16 upregulation. Although it is widely reported that p16 is overexpressed in a high percentage of preneoplastic lesions and in almost all carcinomas of the uterine cervix, protein upregulation and its correlation with HPV infection in low-grade lesions is still being debated. In this study, we investigated in parallel, p16 expression and HPV infection in 100 cervical biopsies (17 normal tissues, 54 CIN1, 10 CIN2, 11 CIN3, eight invasive squamous cancers). Results obtained demonstrated that none of the 17 normal cervical tissues, evaluated by immunohistochemistry, presented p16 positivity whereas, starting from CIN1 (31%) to CIN2 (90%), CIN3 (100%) and carcinomas (100%), a constant and significant increase of protein overexpression (P<0.0001) was observed. In addition, p16 overexpression consistently showed elevated sensitivity (84%) and specificity (98%) in detecting HR-HPV infection with a high positive predictive value (97%) and negative predictive value (86%). Of interest, 93% of the p16-positive CIN1 were also HR-HPV infected. Our findings confirmed that p16 overexpression is associated to high-grade precancerous lesions and cervical carcinomas, and further demonstrated that immunohistochemical evaluation of p16 may be a useful biomarker in identifying HR-HPV-infected low-grade lesions.

  20. Induction of p21WAF1/CIP1 and Inhibition of Cdk2 Mediated by the Tumor Suppressor p16INK4a

    PubMed Central

    Mitra, Jayashree; Dai, Charlotte Y.; Somasundaram, Kumaravel; El-Deiry, Wafik S.; Satyamoorthy, Kapaettu; Herlyn, Meenhard; Enders, Greg H.

    1999-01-01

    The tumor suppressor p16INK4a inhibits cyclin-dependent kinases 4 and 6. This activates the retinoblastoma protein (pRB) and, through incompletely understood events, arrests the cell division cycle. To permit biochemical analysis of the arrest, we generated U2-OS osteogenic sarcoma cell clones in which p16 transcription could be induced. In these clones, binding of p16 to cdk4 and cdk6 abrogated binding of cyclin D1, p27KIP1, and p21WAF1/CIP1. Concomitantly, the total cellular level of p21 increased severalfold via a posttranscriptional mechanism. Most cyclin E-cdk2 complexes associated with p21 and became inactive, expression of cyclin A was curtailed, and DNA synthesis was strongly inhibited. Induction of p21 alone, in a sibling clone, to the level observed during p16 induction substantially reproduced these effects. Overexpression of either cyclin E or A prevented p16 from mediating arrest. We then extended these studies to HCT 116 colorectal carcinoma cells and a p21-null clone derived by homologous recombination. In the parental cells, p16 expression also augmented total cellular and cdk2-bound p21. Moreover, p16 strongly inhibited DNA synthesis in the parental cells but not in the p21-null derivative. These findings indicate that p21-mediated inhibition of cdk2 contributes to the cell cycle arrest imposed by p16 and is a potential point of cooperation between the p16/pRB and p14ARF/p53 tumor suppressor pathways. PMID:10207115

  1. Human papillomavirus DNA and p16(INK4a) expression in hypopharyngeal cancer and in relation to clinical outcome, in Stockholm, Sweden.

    PubMed

    Dalianis, Tina; Grün, Nathalie; Koch, Jana; Vlastos, Andrea; Tertipis, Nikolaos; Nordfors, Cecilia; Näsman, Anders; Wendt, Malin; Romanitan, Mircea; Bersani, Cinzia; Munck-Wikland, Eva; Ramqvist, Torbjörn

    2015-09-01

    Hypopharyngeal cancer is a subset of head neck squamous cell carcinoma (HNSCC) with particularly poor prognosis. Human papillomavirus (HPV) is a risk factor for some HNSCC, and its presence is of prognostic value for certain subsites. However, its influence on survival in hypopharyngeal cancer has not been thoroughly investigated. Here we examine HPV DNA and p16(INK4a) (p16) overexpression in relation to clinical outcome. Hypopharyngeal tumour biopsies from 82 patients diagnosed 2008-2013 were examined for presence of HPV DNA by a bead-based multiplex assay and for p16 expression by immunohistochemistry, and the obtained data compared to that acquired previously from 109 patients diagnosed 2000-2007 at the same clinic. A survival analysis was then performed on 142 patients (from both studies) treated with curative intent and a 3-year follow-up time. Of the tumour biopsies 3/82 (3.7%) were HPV16 DNA and p16 positive, while 12/82 (14.6%) were p16 positive, equivalent to that in the previous study. Overall 3-year survival was significantly more favourable for patients with HPV16 DNA and p16 positive tumours as compared to survival of the other patients (86% vs. 31%, p=0.0185). A similar but not statistically significant trend was found for disease specific survival. HPV DNA and p16 positive hypopharyngeal cancer was rare and had not increased, but had a better clinical outcome as compared to other HPV-unrelated hypopharyngeal cancer. In addition, p16 overexpression was not a suitable surrogate marker for presence of HPV or for prediction of survival in this type of cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Ultrasensitive electrochemical immunosensor based on dual signal amplification process for p16(INK4a) cervical cancer detection in clinical samples.

    PubMed

    Duangkaew, Pattasuda; Tapaneeyakorn, Satita; Apiwat, Chayachon; Dharakul, Tararaj; Laiwejpithaya, Somsak; Kanatharana, Proespichaya; Laocharoensuk, Rawiwan

    2015-12-15

    The p16(INK4a) (p16) is a cyclin-dependent kinase inhibitor, which has been evaluated in several studies as a diagnostic marker of cervical cancer. Immunostaining using p16 specific antibody has confirmed an over-expression of p16 protein in cervical cancer cells and its association with disease progression. This article reports an ultrasensitive electrochemical immunosensor for specific detection of p16 and demonstrates its performance for detection of solubilized p16 protein in cell lysates obtained from patients. Sandwich-based immunoreaction couple with double signal amplification strategy based on catalytic enlargement of particle tag was used for high sensitivity and specificity. The conditions were optimized to create an immunoassay protocol. Disposable screen-printed electrode modified with capture antibodies (Ab1) was selected for further implementation towards point-of-care diagnostics. Small gold nanoparticles (15 nm diameter) conjugated with detection antibodies (Ab2) were found to better serve as a detection label due to limited interference with antigen-antibody interaction. Double signal enhancement was performed by sequential depositions of gold and silver layers. This gave the sensitivity of 1.78 μA mL(ng GST-p16)(-1) cm(-2) and detection limit of 1.3 ng mL(-1) for GST-p16 protein which is equivalent to 0.49 ng mL(-1) for p16 protein and 28 cells for HeLa cervical cancer cells. In addition to purified protein, the proposed immunosensor effectively detected elevated p16 level in cervical swab samples obtained from 10 patients with positive result from standard Pap smear test, indicating that an electrochemical immunosensors hold an excellent promise for detection of cervical cancer in clinical setting. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Tumor suppressor p16INK4A/Cdkn2a alterations in 7, 12-dimethylbenz(a)anthracene (DMBA)-induced hamster cheek pouch tumors

    PubMed Central

    Li, Junan; Warner, Blake; Casto, Bruce C.; Knobloch, Thomas J.; Weghorst, Christopher M.

    2014-01-01

    The prevalence of p16INK4A/CDKN2A genetic alterations in human oral cancers indicates that the p16 gene could be a potent and appropriate target for novel intervention. Chemically induced hamster cheek pouch (HCP) tumors are highly similar to human oral cancers in both histology and genetics and may be a good choice as a surrogate model for human oral cancers. Nevertheless, little is known about the genetic events in the p16 gene in the HCP tumor model. The purpose of this study was to evaluate chemically induced HCP tumor specimens for potential inactivating p16 alterations. HCP tumors were induced with 7, 12-dimethylbenz(a)anthracene (DMBA), and DNA extracted from 34 such specimens were analyzed for homozygous/hemizygous deletions, aberrant methylation of 5’ CpG islands, and point mutations using real-time multiplex PCR, methylation-specific PCR, and direct sequencing/cold single strand conformation polymorphism (SSCP), respectively. Homozygous deletions, hemizygous deletions, aberrant methylation of 5’-CpG islands, and point mutation were identified in 11, 4, 9, and 1 of 34 specimens, respectively. While the overall incidence of p16 alterations was 70.6% (24 of 34 specimens), the majority of inactivating events (67.6%) stemmed from deletion or methylation, which is consistent with the observations that in human oral SCC, the significant p16-inactivating events have been reported to be deletion and methylation. Our results strongly support the use of DMBA-induced HCP tumor model in evaluating novel p16-targeted therapy and prevention of human oral SCC. PMID:18247379

  4. Increased expression of senescence markers p14(ARF) and p16(INK4a) in breast cancer is associated with an increased risk of disease recurrence and poor survival outcome.

    PubMed

    Pare, Rahmawati; Shin, Joo-Shik; Lee, Cheok Soon

    2016-09-01

    Breast cancer is a hormonally driven disease. Cellular senescence is an age-related irreversible cell cycle arrest at the G1 phase upon induction. The aim of this study was to characterize the expression patterns of the senescence markers p14(ARF) , p16(INK4a) and p21(WAF1/Cip1) during breast cancer progression in a large patient cohort. We conducted a retrospective study of 1080 patients with invasive ductal carcinoma, no special type, over an 11-year period. We performed immunohistochemical staining on tissue microarrays that included normal, benign hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma tissue from each patient. Invasive ductal carcinomas showed higher expression of p14(ARF) and p16(INK4a) but lower expression of p21(WAF1/Cip1) than non-malignant tissues. There were significant correlations of normal, benign, preinvasive and malignant tissues with p14(ARF) , p16(INK4a) and p21(WAF1/Cip1) expression (P < 0.05). Univariate comparison showed a correlation between high p16(INK4a) expression and poor survival (P = 0.000) and an increased risk of relapse (P = 0.000), whereas high p14(ARF) expression correlated only with an increased risk of relapse (P = 0.038). Multivariate analysis showed p16(INK4a) to be an important prognostic factor for overall survival (P = 0.011) and disease-free survival (P = 0.004), with p14(ARF) also being a significant prognostic factor for disease-free survival (P = 0.043). Moreover, patients showing both high p16(INK4a) expression and and high p14(ARF) expression had an adjusted three-fold increased risk of disease recurrence (P < 0.05) and a two-fold increased risk of all-cause-related death (P < 0.05). These finding suggest p16(INK4a) expression and p14(ARF) expression may play an important role in the progression of proliferative breast tissue to invasive cancer, and may be useful as prognostic factors. © 2016 John Wiley & Sons Ltd.

  5. ROCK mediates phorbol ester-induced apoptosis in prostate cancer cells via p21Cip1 up-regulation and JNK.

    PubMed

    Xiao, Liqing; Eto, Masumi; Kazanietz, Marcelo G

    2009-10-23

    It is established that androgen-dependent prostate cancer cells undergo apoptosis upon treatment with phorbol esters and related analogs, an effect primarily mediated by PKCdelta. Treatment of LNCaP prostate cancer cells with phorbol 12-myristate 13-acetate (PMA) causes a strong and sustained activation of RhoA and its downstream effector ROCK (Rho kinase) as well as the formation of stress fibers. These effects are impaired in cells subjected to PKCdelta RNA interference depletion. Functional studies revealed that expression of a dominant negative RhoA mutant or treatment with the ROCK inhibitor Y-27632 inhibits the apoptotic effect of PMA in LNCaP cells. Remarkably, the cytoskeleton inhibitors cytochalasin B and blebbistatin blocked not only PMA-induced apoptosis but also the activation of JNK, a mediator of the cell death effect by the phorbol ester. In addition, we found that up-regulation of the cell cycle inhibitor p21(Cip1) is required for PMA-induced apoptosis and that inhibitors of ROCK or the cytoskeleton organization prevent p21(Cip1) induction. Real time PCR analysis and reporter gene assay revealed that PMA induces p21(Cip1) transcriptionally in a ROCK- and cytoskeleton-dependent manner. p21(Cip1) promoter analysis revealed that PMA induction is dependent on Sp1 elements in the p21(Cip1) promoter but independent of p53. Taken together, our studies implicate ROCK-mediated up-regulation of p21(Cip1) and the cytoskeleton in PKCdelta-dependent apoptosis in prostate cancer cells.

  6. Induction of anergy in Th1 cells associated with increased levels of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1.

    PubMed

    Jackson, S K; DeLoose, A; Gilbert, K M

    2001-01-15

    Th1 cells exposed to Ag and the G(1) blocker n-butyrate in primary cultures lose their ability to proliferate in Ag-stimulated secondary cultures. The ability of n-butyrate to induce anergy in Ag-stimulated, but not resting, Th1 cells was shown here to be blocked by cycloheximide. Subsequent experiments to delineate the nature of the protein apparently required for n-butyrate-induced Th1 cell anergy focused on the role of cyclin-dependent kinase (cdk) inhibitors p21(Cip1) and p27(Kip1). Normally, entry into S phase by Th1 cells occurs around 24 h after Ag stimulation and corresponds with relatively low levels of both p21(Cip1) and p27(Kip1). However, unlike control Th1 cells, anergic Th1 cells contained high levels of both p21(Cip1) and p27(Kip1) when examined 24 h after Ag stimulation. The increase in p21(Cip1) observed in Ag-stimulated anergic Th1 cells appeared to be initiated in primary cultures. In contrast, the increase in p27(Kip1) observed in these anergic Th1 cells appears to represent a re-expression of the protein much earlier than control cells following Ag stimulation in secondary cultures. The anergic Th1 cells contained functionally active cdk inhibitors capable of inhibiting the activity of both endogenous and exogenous cdks. Consequently, it appears that n-butyrate-induced anergy in Th1 cells correlated with the up-regulation of p21(Cip1) and perhaps the downstream failure to maintain low levels of p27(Kip1). Increased levels of both p21(Cip1) and p27(Kip1) at the end of G(1) could prevent cdk-mediated entry into S phase, and thus help maintain the proliferative unresponsiveness found in the anergic Th1 cells.

  7. Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition

    PubMed Central

    1995-01-01

    Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells. PMID:7559780

  8. 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

  9. Low p16(INK4a) 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

  10. 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.

  11. [The natural history of cervical intraepithelial neoplasia I and the clinical significance of p16(INK4a) protein as a marker of progression in cervical intraepithelial neoplasia I].

    PubMed

    Wang, Rongmin; Li, Xuejie; Qian, Min; Niu, Jianghua; You, Zhixue

    2015-03-01

    To describe the natural history of cervical intraepithelial neoplasia (CIN) I and the biologic factors associated with the progression of CIN I and to analyze the predictive values of p16(INK4a) protein for the progression of CIN I. From August 2010 to July 2013, 104 patients referred for abnormal cytology [≤ low-grade squamous intraepithelial lesion (LSIL); including negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells of undetermined significance (ASCUS), LSIL] and high-risk (HR) HPV positive, and were diagnosed CIN I by colposcopy-assisted biopsy and followed at 1-year intervals in the First Affiliated Hospital of Nanjing Medical University. In order to analyze the relationship between the progression of CIN I with clinical biologic factors, including patient age, cervical cytology before colposcopy, loads of HR HPV, HPV L1 capsid protein, p16(INK4a) protein, χ(2) tests was used to compare the different frequencies of factors in groups of progressed and persisted/regressed CIN I, then five factors with progressed CIN I were processed into binary logistic regression analysis. (1) In the first year of follow-up, among 104 patients (including 15 cases NILM, 78 cases ASCUS, 11 cases LSIL), 52 cases of them were NILM and HR HPV negative, 30 cases were negative for intraepithelial lesion, 10 cases were CIN I, 5 cases were CIN II and 7 cases were CIN III. In total, 82 cases (78.8%, 82/104) cases had regressed, 10 cases (9.6%, 10/104) persisted, 12 cases (11.5%, 12/104) progressed [including 5 cases (4.8% , 5/104) progressed to CIN II, 7 cases (6.7% , 7/104) progressed to CIN III, none progressed to invasive cancer]. (2) All patients, 88 cases of them accepted immunohistochemical detection the expression of p16(INK4a) protein. The result shown that 30 cases (34%, 30/88) were positive and 58 cases (66%, 58/88) were negative. And 94 cases accepted immunocytochemical detection the expression of HPV L1 capsid protein, 49 cases (52% , 49

  12. [Prognostic and predictive value of koilocytosis, expression of e6 hpv types 16/18, p16ink4a, p53 in locally advanced squamous cell carcinomas of oral cavity and oropharynx, associated with human papillomavirus].

    PubMed

    Riaboshapka, A N

    2014-11-01

    To determine the predictive and prognostic value of koilocytosis, expression of E6 HPV types 16/18, p16INK4a, p53 in patients with locally advanced HPV-associated squamous cell carcinoma of oral cavity and oropharynx. In biopsy specimens of squamous cell carcinomas of oral cavity and oropharynx from 60 patients performed koylocytes count, immunohistochemical detection of HPV 16/18 types E6 protein, proteins p16INK4a and p53. Koilocytosis was detected in 50 patients (83.3%); in all 60 patients (100%) were simultaneous expression of p16INK4a and E6 HPV types 16/18; p53 expression was found in 37 patients (61.7%). After combined treatment (induction chemotherapy followed by radiotherapy) stable disease (SD) was detected in 11 patients (18.3%), partial response (PR) - in 25 patients (41.7%), complete response (CR) - in 24 patients (40.0%). There were no cases of disease progression. Treatment effect correlated with expression of p16INK4a (ρ = 0.3, p = 0.024) and expression of p53 (ρ = - 0.3, p = 0.019). Patients with a low expression of p16INK4a (2 points) and high expression of p53 (4 "+") had a high level of SD and had no CR. For all patients, the median of overall survival (OS) was 17 months, 1-year cumulative survival rate was 66.7%, 2-year cumulative survival rate - 35.0%. Median of overall survival was correlated with koilocytosis (ρ=0.5, p<0,001) and expression of E6 HPV types 16/18 (ρ=0.9, p<0.001), p16INK4a (ρ=0.9, p=0.037), p53 (ρ=-0.9; p<0.001). Patients with low expression of p53 (0 and 1 "+") had cumulative 1-year survival rates 87% and 90%, respectively (p<0.001), 2-year survival rates - 52% and 80%, respectively (p=0.015). In the Cox proportional hazards model the significant prognostic factors were prevalence of primary tumor (OR 2.2, 95% CI 1.3 - 3.5, p=0.003) and p53 expression (OR 1.3, 95% CI 1.1=1.7, p=0.016). High expression of p16INK4a associated with a high effect of combined treatment, high expression of a p53 - with low effect of

  13. Cigarette smoke triggers code red: p21CIP1/WAF1/SDI1 switches on danger responses in the lung.

    PubMed

    Tuder, Rubin M; Yun, Jeong H; Graham, Brian B

    2008-07-01

    The article by Yao and coworkers in this issue (Am. J. Respir. Cell Mol. Biol. 2008;39:7-18) reveals that the cyclin-dependent kinase inhibitor p21CIP1/WAF1/SDI1 (designated hereafter as p21), which has been linked to cell cycle growth arrest due to stress or danger cell responses, may modulate alveolar inflammation and alveolar destruction, and thus enlightens our present understanding of how the lung senses injury due to cigarette smoke and integrates these responses with those that activate inflammatory pathways potentially harmful to the lung. Furthermore, the interplay of p21 and cellular processes involving cell senescence and the imbalance of cell proliferation/apoptosis may provide us with a more logical explanation of how p21, acting as a sensor of cellular stress, might have such potent and wide roles in lung responses triggered by cigarette smoke. Molecular switches, ontologically designed for the protection of the host, are now hijacked by injurious stresses (such as cigarette smoke), leading to organ damage.

  14. BRG1 controls the activity of the retinoblastoma protein via regulation of p21CIP1/WAF1/SDI.

    PubMed

    Kang, Hyeog; Cui, Kairong; Zhao, Keji

    2004-02-01

    The ubiquitous mammalian chromatin-remodeling SWI/SNF-like BAF complexes play critical roles in tumorigenesis. It was suggested that the direct interaction of BRG1 with the retinoblastoma protein pRB is required for regulation of cell cycle progression by pRB. We present evidence that the BRG1-containing complexes regulate the expression of the cdk inhibitor p21(CIP1/WAF1/SDI). Furthermore, we show that the physical interaction between BRG1 and pRB is not required for induction of cell growth arrest and transcriptional repression of E2F target genes by pRB. Instead, BRG1 activates pRB by inducing its hypophosphorylation through up-regulation of the cdk inhibitor p21. The hypophosphorylation of pRB is reinforced by down-regulation of critical components, including cdk2, cyclin E, and cyclin D, in the pRB regulatory network. We demonstrate that up-regulation of p21 by BRG1 is necessary to induce formation of flat cells, growth arrest, and finally, cell senescence. Our results suggest that the BRG1-containing complexes control cellular proliferation and senescence by modulating the pRB pathway via multiple mechanisms.

  15. Loss of p21Cip1/CDKN1A renders cancer cells susceptible to Polo-like kinase 1 inhibition.

    PubMed

    Kreis, Nina-Naomi; Louwen, Frank; Zimmer, Brigitte; Yuan, Juping

    2015-03-30

    The deregulation of Polo-like kinase 1 is inversely linked to the prognosis of patients with diverse human tumors. Targeting Polo-like kinase 1 has been widely considered as one of the most promising strategies for molecular anticancer therapy. While the preclinical results are encouraging, the clinical outcomes are rather less inspiring by showing limited anticancer activity. It is thus of importance to identify molecules and mechanisms responsible for the sensitivity of Polo-like kinase 1 inhibition. We have recently shown that p21Cip1/CDKN1A is involved in the regulation of mitosis and its loss prolongs the mitotic duration accompanied by defects in chromosome segregation and cytokinesis in various tumor cells. In the present study, we demonstrate that p21 affects the efficacy of Polo-like kinase 1 inhibitors, especially Poloxin, a specific inhibitor of the unique Polo-box domain. Intriguingly, upon treatment with Polo-like kinase 1 inhibitors, p21 is increased in the cytoplasm, associated with anti-apoptosis, DNA repair and cell survival. By contrast, deficiency of p21 renders tumor cells more susceptible to Polo-like kinase 1 inhibition by showing a pronounced mitotic arrest, DNA damage and apoptosis. Furthermore, long-term treatment with Plk1 inhibitors induced fiercely the senescent state of tumor cells with functional p21. We suggest that the p21 status may be a useful biomarker for predicting the efficacy of Plk1 inhibition.

  16. Mitotic p21Cip1/CDKN1A is regulated by cyclin-dependent kinase 1 phosphorylation

    PubMed Central

    Kreis, Nina-Naomi; Friemel, Alexandra; Zimmer, Brigitte; Roth, Susanne; Rieger, Michael A.; Rolle, Udo; Louwen, Frank; Yuan, Juping

    2016-01-01

    The multifunctional protein p21Cip1/CDKN1A (p21) is an important and universal Cdk-interacting protein. Recently, we have reported that p21 is involved in the regulation of the mitotic kinase Cdk1/cyclin B1 and critical for successful mitosis and cytokinesis. In the present work we show that S130 of p21 is phosphorylated by Cdk1/cyclin B1 during mitosis, which reduces p21′s stability and binding affinity to Cdk1/cyclin B1. Interfering with this phosphorylation results in extended mitotic duration and defective chromosome segregation, indicating that this regulation ensures proper mitotic progression. Given that p53, the major transcriptional activator of p21, is the most frequently mutated gene in human cancer and that deregulated Cdk1 associates with the development of different types of cancer, this work provides new insight into the understanding of how deregulated p21 contributes to chromosomal instability and oncogenesis. PMID:27384476

  17. 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

  18. A phase 1/2a study to test the safety and immunogenicity of a p16(INK4a) peptide vaccine in patients with advanced human papillomavirus-associated cancers.

    PubMed

    Reuschenbach, Miriam; Pauligk, Claudia; Karbach, Julia; Rafiyan, Mohammad-Reza; Kloor, Matthias; Prigge, Elena-Sophie; Sauer, Madeleine; Al-Batran, Salah-Eddin; Kaufmann, Andreas M; Schneider, Achim; Jäger, Elke; von Knebel Doeberitz, Magnus

    2016-05-01

    The cyclin-dependent kinase inhibitor p16(INK4a) is strongly and consistently overexpressed in all human papillomavirus (HPV)-associated cancers. Therefore, the authors hypothesized that p16(INK4a) may be a vaccine target antigen for HPV-associated cancers. To test this hypothesis, the authors performed a phase 1/2a first-in-human trial to evaluate the safety and immunogenicity of a p16(INK4a) -based peptide vaccine. A total of 26 patients with different, advanced, p16(INK4a) -overexpressing, HPV DNA-positive cancers were included after the completion of standard treatment. According to protocol, 12 subcutaneous injections of a p16(INK4) peptide (P16_37-63) mixed in a water-in-oil emulsion with immunoadjuvant activity (Montanide ISA-51 VG) were administered over a 6-month period. A total of 20 patients received at least 4 injections and were evaluable for immune responses against P16_37-63. Clusters of differentiation (CD) 4 T cells were detected in 14 of 20 patients (3 of whom had preexisting CD4 T cells before vaccination), CD8 T cells were detected in 5 of 20 patients, and antibodies were detected in 14 of 20 patients (1 of whom had preexisting antibodies). No suspected unexpected serious adverse reaction or serious adverse drug reaction was documented. All reported serious adverse events were expected and not considered to be related to study therapy. None of the patients discontinued trial participation due to unacceptable toxicities and no dose-limiting toxicities occurred. Tumor response could be assessed in 14 patients. Of these, 9 patients (64%) had stable disease as their best overall response and 5 patients (36%) developed progressive disease. Vaccination with the p16(INK4a) -derived peptide P16_37-63 appears to induce cellular and humoral immune responses and does not cause severe toxicities. The results of the current study pave the way for the further clinical development of p16(INK4a) -based cancer immunotherapeutics. Cancer 2016

  19. Degradation of p21Cip1 through anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20) ubiquitin ligase complex-mediated ubiquitylation is inhibited by cyclin-dependent kinase 2 in cardiomyocytes.

    PubMed

    Yamada, Kazuhiko; Tamamori-Adachi, Mimi; Goto, Ikuko; Iizuka, Masayoshi; Yasukawa, Takashi; Aso, Teijiro; Okazaki, Tomoki; Kitajima, Shigetaka

    2011-12-23

    Cyclin-dependent kinase inhibitor p21Cip1 plays a crucial role in regulating cell cycle arrest and differentiation. It is known that p21Cip1 increases during terminal differentiation of cardiomyocytes, but its expression control and biological roles are not fully understood. Here, we show that the p21Cip1 protein is stabilized in cardiomyocytes after mitogenic stimulation, due to its increased CDK2 binding and inhibition of ubiquitylation. The APC/CCdc20 complex is shown to be an E3 ligase mediating ubiquitylation of p21Cip1 at the N terminus. CDK2, but not CDC2, suppressed the interaction of p21Cip1 with Cdc20, thereby leading to inhibition of anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20)-mediated p21Cip1 ubiquitylation. It was further demonstrated that p21Cip1 accumulation caused G2 arrest of cardiomyocytes that were forced to re-enter the cell cycle. Taken together, these data show that the stability of the p21Cip1 protein is actively regulated in terminally differentiated cardiomyocytes and plays a role in inhibiting their uncontrolled cell cycle progression. Our study provides a novel insight on the control of p21Cip1 by ubiquitin-mediated degradation and its implication in cell cycle arrest in terminal differentiation.

  20. Quantitative analysis of CDKN2A methylation, mRNA, and p16(INK4a) protein expression in children and adolescents with Burkitt lymphoma: biological and clinical implications.

    PubMed

    Robaina, Marcela Cristina S; Faccion, Roberta Soares; Arruda, Viviane Oliveira; de Rezende, Lidia Maria Magalhães; Vasconcelos, Gisele Moledo; Apa, Alexandre Gustavo; Bacchi, Carlos E; Klumb, Claudete Esteves

    2015-02-01

    CDKN2A is a tumor suppressor gene critical in the cell cycle regulation. Little is known regarding the role of CDKN2A methylation in the pathogenesis of Burkitt lymphoma (BL). CDKN2A methylation was investigated using pyrosequencing in 51 tumor samples. p16(INK4a) mRNA and protein levels were measured using real-time PCR and immunohistochemistry, respectively. CDKN2A methylation was detectable in 72% cases. Nuclear expression of p16(INK4a) was not detected in 41% cases. There was an association between methylation and absence of CDKN2A mRNA (P=0.003). In conclusion, CDKN2A methylation occurs at a high frequency suggesting a role in BL pathogenesis and potential therapeutic implications.

  1. Prediction of clinical outcome using p16INK4a immunocytochemical expression in low-grade squamous intraepithelial lesions and high-risk HPV-positive atypical squamous cells of undetermined significance in patients with and without colposcopic evident cervical disease

    PubMed Central

    LUKIC, ANKICA; SBENAGLIA, GIORGIO; CARICO, ELISABETTA; DI PROPERZIO, MATILDE; GIARNIERI, ENRICO; FREGA, ANTONIO; NOBILI, FLAVIA; MOSCARINI, MASSIMO; GIOVAGNOLI, MARIA ROSARIA

    2011-01-01

    p16INK4a as a diagnostic marker of a cervical intraepithelial neoplasia of grade 2+ (CIN2+) in atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LSIL) cytological samples has been analyzed, but has not yet been included in clinical routine practice. One hundred and ninety-one patients with an abnormal Pap test (84 ASC-US and 107 LSILs) who underwent colposcopy were selected for this study. At enrollment, 96 patients (Group 1) had a positive colposcopy and therefore underwent a cervical biopsy, while 95 (Group 2) had a negative colposcopy and were followed up for up to 1 year. Both groups were tested for p16INK4a using immunocytochemical methods, and the p16INK4a results were correlated with histology or follow-up outcome. In Group 1 ASC-US cases, 82% of lesions less than CIN2 were p16INK4a-negative and all CIN2 cases were p16INK4a-positive (p=0.00044). In Group 1 LSIL cases, 71% of lesions less than CIN2 were p16INK4a-negative and 87% of CIN2/3 were p16INK4a-positive (p=0.00033). Seventy-seven percent of Group 2 ASC-US patients with a negative 1-year follow-up (NF-U) were p16INK4a-negative at enrollment, while all patients with positive follow-up (PF-U) were p16INK4a-positive (p=0.00113). In Group 2 LSIL cases, 83% of patients with NF-U were p16INK4a-negative, while 65% of patients with PF-U were p16INK4a-positive at enrollment (p=0.0014). In fact, 39% of the positive p16INK4a LSIL patients had CIN2+ histological lesions. The positive predictive value of p16INK4a for CIN2+ was 50% in ASC-US and 52% in LSIL cases; the negative predictive value was 100 and 94%, respectively. In conclusion, in our patients, a negative p16INK4a appears to be a marker of the absence of CIN3, while a positive p16INK4a can be correlated with the presence of histological CIN2+ found at enrollment or during the subsequent follow-up. Thus, its clinical predictive value is independent from the colposcopic aspect at enrollment. PMID

  2. Expression of a novel form of p21Cip1/Waf1 in UV-irradiated and transformed cells.

    PubMed

    Poon, R Y; Hunter, T

    1998-03-12

    The tumor suppressor p53 and its target the CDK inhibitor p21 (Cip1/Waf1) are key components of the cellular response to DNA damage. Insight into how p21 is regulated in normal cells, and how it may be deregulated in tumor cells is important for the understanding of tumorigenesis. p21 was induced in normal human diploid fibroblasts after UV irradiation-induced DNA damage, but, at a high dose of UV irradiation, a faster mobility form of p21 on SDS-PAGE (designated p21delta) was expressed. Surprisingly, in a variety of growing transformed cell lines, the level of p21 was low but p21delta was prominent. We found that p21delta appeared to be derived through a loss of around 10 amino acids from the C-terminus of p21, which theoretically would remove the PCNA binding domain, a second cyclin binding domain and the nuclear localization signal sequence. Several characteristics distinguish p21 from p21delta. Both the full length p21 and p21delta could be stabilized by a proteasome inhibitor, but only the full length p21 was associated with Cdk2 and PCNA. Consistent with this, gel filtration chromatography revealed that all the full length p21 in the cell was complexed to other proteins, whereas a significant portion of p21delta was in monomeric form. Moreover, p21 was mainly localized to the nucleus, but p21delta was mainly localized to the cytoplasm. We propose that the decrease in p21 and increase in p21delta could contribute to the deregulation of the cell cycle, and could be a mechanism involved in cellular transformation.

  3. Primary Squamous Cell Carcinoma of the Upper Genital Tract: Utility of p16INK4a Expression and HPV DNA Status in its Differential Diagnosis from Extended Cervical Squamous Cell Carcinoma

    PubMed Central

    Yoo, Su Hyun; Son, Eun-Mi; Sung, Chang Okh

    2013-01-01

    Background Primary squamous cell carcinoma (SCC) of the upper genital tract, including the endometrium, fallopian tubes, and ovaries, is extremely rare. It must be distinguished from the mucosal extension of primary cervical SCC because determination of the primary tumor site is important for tumor staging. However, patients with SCC of the fallopian tubes or ovarian surface have often undergone prior hysterectomy with inadequate examination of the cervix, making it difficult to determine the primary site. Methods We compared histologic findings, p16INK4a expression, and human papillomavirus (HPV) DNA status in four patients with primary SCC of the upper genital tract and five patients with primary cervical SCC extending to the mucosa of the upper genital tract. Results All five SCCs of cervical origin showed strong expression of p16INK4a, whereas all four SCCs of the upper genital tract were negative, although one showed weak focal staining. Three of the five cervical SCCs were positive for HPV16 DNA, whereas all four primary SCCs of the upper genital tract were negative for HPV DNA. Conclusions Although a thorough histological examination is important, immunonegativity for p16INK4a and negative for HPV DNA may be useful adjuncts in determining primary SCCs of the upper genital tract. PMID:24421848

  4. Clinicopathological Comparison of Adenocarcinoma of Cervix and Endometrium Using Cell Cycle Markers: P16ink4a, P21waf1, and p27Kip1 on 132 Cancers

    PubMed Central

    Marican Abu Backer, Farveen; Nik Mustapha, Nik Raihan; Othman, Nor Hayati

    2011-01-01

    Objective. We studied the clinicopathological parameters of adenocarcinoma arising from endocervix (ECA) and from endometrium (EMA) based on the expression of P16ink4a, P21waf1, and p27Kip1 proteins. Study Design. Immunohistochemistry was done on sections of confirmed ECA and EMA from hysterectomy specimens which have had no prior chemotherapy/radiotherapy. Results. There were 40 ECAs and 92 EMAs. The mean age of ECA was 49.82 (SD 10.29); the youngest was 30 years old and the oldest 75 years old. The mean age of EMA was 54.45 (SD 10.92); the youngest was 30 years old and the oldest was 82 years old. For ECA, the size of the tumour is significantly associated with age and with depth of infiltration. FIGO stage is associated with histological grade. p21WAF1 expression is significantly associated with infiltration of the corpus and lymph node metastasis. p27Kip1 expression is significantly associated with lymph node invasion. The presence of lymph node metastasis is strongly associated when p16INK4a and p27Kip1 expressions are analyzed in combination. For EMA, p16INK4a expression is associated with histologic grade. Conclusion. Our study shows that we could use these cell cycle markers as predictors for more aggressive subsets of adenocarcinoma of the cervix and endometrium. PMID:22114462

  5. [Gastric carcinoma and chronic gastritis: epigenetic regulation of CDH1 (E-Cadherin), CDKN2A (p16INK4A), PTGS2 (COX-2) and EGFR genes through methylation].

    PubMed

    Silva, Maria; Azenha, Diana; Pereira, César; Almeida, Anabela; Balseiro, Sandra; Sampaio, Ana Maria; Santos, Paulo; Carvalho, Lina

    2010-01-01

    The genetic and epigenetic alterations are being studied as one of the causes of gastric cancer (GC) progression and development. DNA methylation is an epigenetic alteration which leads to suppressor gene silencing and proto-oncogene activation, playing an important role in carcinogenesis. The histological types of gastric carcinoma have different genetic paths and the knowledge of the molecular bases of tumoral progression leads to diagnostic accuracy and attempted therapy. CDH1 (E-cadherin) and CDKN2A (p16(INK4A)) genes are thought to be tumoral suppressor genes and PTGS2 (COX-2) and genes are involved in tumour regulation and growth. In one hand, gene silencing as an epigenetic phenomenon, and in the other hand, gene expression enhancement due to possible demethylation, simultaneously, can facilitate carcinogénesis and tumoral progression. Our aim was to relate CDH1, p16(INK4A), COX-2 and EGFR genes DNA methylation with the several histological types of gastric carcinoma and chronic gastritis. We studied 55 formalin fixed paraffin embedded gastric biopsies: 35 were GC specimens (12 diffuse type, 15 intestinal type and 8 indeterminate type, according to Laurén's classification) and 20 samples had chronic gastritis (CG). The DNA was treated with sodium bisulfite after extraction and then performed Methylation Specific PCR (MSP). Statistical analysis was based on chi-square test and Exact Fisher's test. CpG island methylation was detected in 94% of the GC samples for CDH1, 91% for COX-2, 80% for p16(INK4A) and no methylation was detected in EGFR gene (0%). In CG, CpG island methylation was found in 100% for CDH1 and COX-2 genes, 90% for p16(INK4A) and 20% for EGFR. These results reveal significant differences in EGFR gene methylation distinguishing GC from CG (p < 0, 01), suggesting that gene demethylation leads to malignant transformation and favours the use of tyrosine-kinase inhibitors in its treatment. Genes COX2 e p16INK4A lower methylation in intestinal and

  6. p16(INK4a) as a complementary marker of high-grade intraepithelial lesions of the uterine cervix. I: Experience with squamous lesions in 189 consecutive cervical biopsies.

    PubMed

    Dray, Michael; Russell, Peter; Dalrymple, Chris; Wallman, Neil; Angus, George; Leong, Adelyn; Carter, Jonathan; Cheerala, Bharathi

    2005-04-01

    To test the usefulness of p16(INK4a) immunostaining for improving the diagnostic accuracy of cervical punch biopsies referred to a routine laboratory setting during the investigation of women with abnormal Papanicolaou smears. A total of 188 consecutive and unselected colposcopically directed cervical biopsies and a single contemporaneous cervical polyp were accessioned prospectively over a 3-month period, step-serially sectioned and examined by H&E and immunostained for p16(INK4a). The clinical context, results of concurrent Papanicolaou smears/ThinPrep slides and Digene hybrid capture tests for high-risk human papillomavirus (HPV) subtypes, as well as follow-up cervical smears/ThinPrep, biopsies and loop excisions of transformation zones or cone biopsies were all correlated with the morphological and immunohistochemical findings. Seventy-seven biopsies (40.7%) displayed a high-grade squamous intraepithelial lesion (HGSIL; cervical intraepithelial neoplasia [CIN] 2-3), 27 (14.3%) showed a low grade squamous intraepithelial lesion (HPV +/- CIN1) and 85 (45%) showed a range of non-dysplastic (inflammatory or reactive) changes. Diffuse strong parabasal immunostaining for p16(INK4a), suggestive of integrated high-risk HPV DNA into the host genome, was observed in 81 biopsies (42.9%, including the cervical polyp) and correlated (>90%) with HGSIL in the H&E sections. Only one case revealed irreconcilable discordance between the histological features and this strong parabasal immunostaining pattern. Focal and weaker midzonal or superficial p16(INK4a) immunostaining, suggestive of episomal HPV infection, was noted in 19 biopsies (10%) and these biopsies exhibited a range of histological changes but predominantly low grade squamous intraepithelial lesion (LGSIL). No staining of the squamous epithelium was seen in 89 biopsies (47.1%). Again, only one case revealed irreconcilable discordance between the histological features and this negative immunostaining pattern. On

  7. Characterization of p21Cip1/Waf1 peptide domains required for cyclin E/Cdk2 and PCNA interaction.

    PubMed

    Chen, I T; Akamatsu, M; Smith, M L; Lung, F D; Duba, D; Roller, P P; Fornace, A J; O'Connor, P M

    1996-02-01

    The cyclin-dependent kinase inhibitor p21Cip1/Waf1 is responsible for the p53-dependent growth arrest of cells in G1 phase following DNA damage. In the present study we investigated regions of p21 involved in inhibition of the G1/S phase cyclin-dependent kinase, cyclin E/Cdk2, as well as regions of p21 important for binding to this kinase and recombinant PCNA. To perform these studies we synthesized a series of overlapping peptides spanning the entire p21 sequence and used them in in vitro assays with cyclin E/Cdk2-immune complexes and with recombinant p21 and PCNA proteins. One amino-terminal p21 peptide spanning amino acids 15-40, antagonized p21 binding and inhibition of cyclin E/Cdk2 kinase. Antagonism of p21 binding was, however, lost in a similar peptide lacking amino acids 15-20, or in a peptide in which cysteine-18 was substituted for a serine. These results suggest that this peptide region is important for p21 interaction with cyclin E/Cdk2. A second peptide (amino acids 58-77) also antagonized p21-activity, but this peptide did not affect the ability of p21 to interact with cyclin E/Cdk2. A region of p21 larger than 26 amino acids is presumably required for Cdk-inhibition because none of the peptides we tested inhibited cyclin E/Cdk2. We also found that a peptide spanning amino acids 21-45 bound recombinant p21 in ELISA assays, and additional studies revealed a requirement for amino acids 26 through 45 for this interaction. A p21 peptide spanning amino acids 139-164 was found to bind PCNA in a filter binding assay and this peptide suppressed recombinant p21-PCNA interaction. Conformational analysis revealed that peptides spanning amino acids 21-45 and 139-164 tended towards an alpha-helical conformation in trifluoroethanol buffer, indicating that these regions are probably in a coiled conformation in the native protein. Taken together, our results provide an insight into domains of p21 that are involved in cyclin E/Cdk2 and PCNA interaction. Our results

  8. Cholesterol Retards Senescence in Bone Marrow Mesenchymal Stem Cells by Modulating Autophagy and ROS/p53/p21(Cip1/Waf1) Pathway.

    PubMed

    Zhang, Mingyu; Du, Yue; Lu, Renzhong; Shu, You; Zhao, Wei; Li, Zhuoyun; Zhang, Yu; Liu, Ruixue; Yang, Ti; Luo, Shenjian; Gao, Ming; Zhang, Yue; Zhang, Guiye; Liu, Jiaqi; Lu, Yanjie

    2016-01-01

    In the present study, we demonstrated that bone marrow mesenchymal stem cells (BMSCs) of the 3rd passage displayed the senescence-associated phenotypes characterized with increased activity of SA-β-gal, altered autophagy, and increased G1 cell cycle arrest, ROS production, and expression of p53 and p21(Cip1/Waf1) compared with BMSCs of the 1st passage. Cholesterol (CH) reduced the number of SA-β-gal positive cells in a dose-dependent manner in aging BMSCs induced by H2O2 and the 3rd passage BMSCs. Moreover, CH inhibited the production of ROS and expression of p53 and p21(Cip1/Waf1) in both cellular senescence models and decreased the percentage of BMSCs in G1 cell cycle in the 3rd passage BMSCs. CH prevented the increase in SA-β-gal positive cells induced by RITA (reactivation of p53 and induction of tumor cell apoptosis, a p53 activator) or 3-MA (3-methyladenine, an autophagy inhibitor). Our results indicate that CH not only is a structural component of cell membrane but also functionally contributes to regulating cellular senescence by modulating cell cycle, autophagy, and the ROS/p53/p21(Cip1/Waf1) signaling pathway.

  9. IL-12-dependent innate immunity arrests endothelial cells in G0-G1 phase by a p21(Cip1/Waf1)-mediated mechanism.

    PubMed

    Napione, Lucia; Strasly, Marina; Meda, Claudia; Mitola, Stefania; Alvaro, Maria; Doronzo, Gabriella; Marchiò, Serena; Giraudo, Enrico; Primo, Luca; Arese, Marco; Bussolino, Federico

    2012-12-01

    Innate immunity may activate paracrine circuits able to entail vascular system in the onset and progression of several chronic degenerative diseases. In particular, interleukin (IL)-12 triggers a genetic program in lymphomononuclear cells characterized by the production of interferon-γ and specific chemokines resulting in an angiostatic activity. The aim of this study is to identify molecules involved in the regulation of cell cycle in endothelial cells co-cultured with IL-12-stimulated lymphomonuclear cells. By using a transwell mediated co-culture system we demonstrated that IL-12-stimulated lymphomonuclear cells induce an arrest of endothelial cells cycle in G1, which is mainly mediated by the up-regulation of p21(Cip1/Waf1), an inhibitor of cyclin kinases. This effect requires the activation of STAT1, PKCδ and p38 MAPK, while p53 is ineffective. In accordance, siRNA-dependent silencing of these molecules in endothelial cells inhibited the increase of p21(Cip1/Waf1) and the modification in cell cycle promoted by IL-12-stimulated lymphomonuclear cells. These results indicate that the angiostatic action of IL-12-stimulated lymphomononuclear cells may lie in the capability to arrest endothelial cells in G1 phase through a mechanisms mainly based on the specific up-regulation of p21(Cip1/Waf1) induced by the combined activity of STAT1, PKCδ and p38 MAPK.

  10. Long-term arsenic exposure induces histone H3 Lys9 dimethylation without altering DNA methylation in the promoter region of p16(INK4a) and down-regulates its expression in the liver of mice.

    PubMed

    Suzuki, Takehiro; Nohara, Keiko

    2013-09-01

    Long-term exposure of humans to high concentrations of arsenic is associated with an increased risk of cancer. Previous studies have suggested that arsenic exposure promotes tumorigenesis by inducing changes in the expression of tumor-related genes by dysregulating DNA methylation at tumor-related gene loci. However, the causal relationships between epigenetic changes and both arsenic exposure and tumorigenesis are still unclear. In the present study, we investigated whether arsenic can change the expression of tumor-related genes by inducing epigenetic modifications before tumorigenesis. We did so by investigating the effects of long-term arsenic exposure on representative epigenetic modifications, DNA methylation and histone modifications, in the tumor-free normal liver of C57Bl/6 mice. We focused on the tumor-related genes, p16(INK4a) , RASSF1A, Ha-ras and ER-α as target genes, because their expression and promoter methylation status in mice have been reported to be affected by long-term arsenic exposure. The results showed that long-term arsenic exposure induced a significant decrease in expression of p16(INK4a) associated with an increase in level of dimethylated histone H3 lysine 9 (H3K9), a transcription-suppressive histone modification, in the promoter region, but that DNA methylation of the promoter region was unaffected. The results also showed a significant increase in recruitment of H3K9 histone methyltransferase G9a to the promoter after arsenic exposure. These findings suggest that long-term arsenic exposure may induce down-regulation of p16(INK4a) by targeting recruitment of G9a and H3K9 dimethylation without altering DNA methylation before tumorigenesis in the liver. Copyright © 2012 John Wiley & Sons, Ltd.

  11. Individuals with presumably hereditary uveal melanoma do not harbour germline mutations in the coding regions of either the P16INK4A, P14ARF or cdk4 genes

    PubMed Central

    Soufir, N; Bressac-de Paillerets, B; Desjardins, L; Lévy, C; Bombled, J; Gorin, I; Schlienger, P; Stoppa-Lyonnet, D

    2000-01-01

    In familial cutaneous malignant melanoma (CMM), disruption of the retinoblastoma (pRB) pathway frequently occurs through inactivating mutations in the p16 (p16INK4A/CDKN2A/MTS1) gene or activating mutations in the G1-specific cyclin dependent kinase 4 gene (CDK4). Uveal malignant melanoma (UMM) also occurs in a familial setting, or sometimes in association with familial or sporadic CMM. Molecular studies of sporadic UMM have revealed somatic deletions covering the INK4A-ARF locus (encoding P16INK4Aand P14ARF) in a large proportion of tumours. We hypothesized that germline mutations in the p16INK4A, p14ARFor CDK4 genes might contribute to some cases of familial UMM, or to some cases of UMM associated with another melanoma. Out of 155 patients treated at the Institut Curie for UMM between 1994 and 1997, and interviewed about their personal and familial history of melanoma, we identified seven patients with a relative affected with UMM (n = 6) or CMM (n = 1), and two patients who have had, in addition to UMM, a personal history of second melanoma, UMM (n = 1), or CMM (n = 1). We screened by polymerase chain reaction single-strand conformation polymorphism the entire coding sequence of the INK4A-ARF locus (exon 1α from p16INK4A, exon 1β from p14ARF, and exons 2 and 3, common to both genes), as well as the exons 2, 5 and 8 of the CDK4 gene, coding for the functional domains involved in p16 and/or cyclin D1 binding. A previously reported polymorphism in exon 3 of the INK4A-ARF locus was found in one patient affected with bilateral UMM, but no germline mutations were detected, either in the p16INK4A, p14ARFor CDK4 genes. Our data support the involvement of other genes in predisposition to uveal melanoma. © 2000 Cancer Research Campaign PMID:10732752

  12. Limoniastrum guyonianum aqueous gall extract induces apoptosis in human cervical cancer cells involving p16 INK4A re-expression related to UHRF1 and DNMT1 down-regulation.

    PubMed

    Krifa, Mounira; Alhosin, Mahmoud; Muller, Christian D; Gies, Jean-Pierre; Chekir-Ghedira, Leila; Ghedira, Kamel; Mély, Yves; Bronner, Christian; Mousli, Marc

    2013-05-20

    Several reports have described the potential effects of natural compounds as anti-cancer agents in vitro as well as in vivo. The aim of this study was to evaluate the anti-cancer effect of Limoniastrum guyonianum aqueous gall extract (G extract) and luteolin in the human cervical cancer HeLa cell line, and, if so, to clarify the underlying mechanism. Our results show that G extract and luteolin inhibited cell proliferation and induced G2/M cell cycle arrest in a concentration and time-dependent manner. Both natural products induced programmed cell death as confirmed by the presence of hypodiploid G0/G1 cells. These effects are associated with an up-regulation of the expression of the tumor suppressor gene p16INK4A and a down-regulation of the expression of the anti-apoptotic actor UHRF1 and its main partner DNMT1. Moreover, G extract- and luteolin-induced UHRF1 and DNMT1 down-regulation is accompanied with a global DNA hypomethylation in HeLa cell line. Altogether our results show that G extract mediates its growth inhibitory effects on human cervical cancer HeLa cell line likely via the activation of a p16INK4A-dependent cell cycle checkpoint signalling pathway orchestrated by UHRF1 and DNMT1 down-regulation.

  13. RXR antagonism induces G0 /G1 cell cycle arrest and ameliorates obesity by up-regulating the p53-p21(Cip1) pathway in adipocytes.

    PubMed

    Nakatsuka, Atsuko; Wada, Jun; Hida, Kazuyuki; Hida, Aya; Eguchi, Jun; Teshigawara, Sanae; Murakami, Kazutoshi; Kanzaki, Motoko; Inoue, Kentaro; Terami, Takahiro; Katayama, Akihiro; Ogawa, Daisuke; Kagechika, Hiroyuki; Makino, Hirofumi

    2012-04-01

    The peroxisome proliferator activated receptor-γ (PPARγ) agonist, pioglitazone (PIO), exerts anti-diabetic properties associated with increased fat mass, whereas the retinoid X receptor (RXR) antagonist HX531 demonstrates anti-obesity and anti-diabetic effects with reduced body weight and fat pad mass. The cell cycle abnormality in adipocytes has not been well-investigated in obesity or during treatment with modulators of nuclear receptors. We therefore investigated cell size and cell cycle distributions of adipocytes in vivo and examined the expression of cell cycle regulators in cultured human visceral preadipocytes. The cell size distribution and cell cycle analyses of in vivo adipocytes derived from OLETF rats demonstrated that HX531 brought about G0/G1 cell cycle arrest associated with the inhibition of cellular hypertrophy, which resulted in the reduction of fat pad mass. In contrast, PIO promoted proliferation activities associated with the increase in M + late M:G0 + G1 ratio and the appearance of both small and hypertrophied adipocytes. In cultured human visceral preadipocytes HX531 up-regulated cell cycle regulators, p53, p21(Cip1), cyclin D1, Fbxw7 and Skp2, which are known contributors towards G0 /G1 cell cycle arrest. The knockdown of p53 with a shRNA lentivirus reversed the HX531-induced up-regulation of p21(Cip1), which is one of the major p53-effector molecules. We conclude that HX531 exerts anti-obesity and anti-diabetes properties by up-regulating the p53-p21(Cip1) pathway, resulting in G0/G1 cell cycle arrest and the inhibition of cellular hypertrophy of adipocytes. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  14. FoxF1 and FoxF2 transcription factors synergistically promote Rhabdomyosarcoma carcinogenesis by repressing transcription of p21Cip1 CDK inhibitor

    PubMed Central

    Cai, Yuqi; Le, Tien; Turpin, Brian; Kalinichenko, Vladimir V.; Kalin, Tanya V.

    2016-01-01

    The role of Forkhead Box F1 (FoxF1) transcription factor in carcinogenesis is not well characterized. Depending on tissue and histological type of cancer, FoxF1 was shown to be either oncogene or tumor suppressor. Alveolar rhabdomyosarcoma (RMS) is the most aggressive pediatric soft tissue sarcoma. While FoxF1 is highly expressed in alveolar RMS, the functional role of FoxF1 in RMS is unknown. The present study demonstrates that expression of FoxF1 and its closely related transcription factor FoxF2 are essential for rhabdomyosarcoma tumor growth. Depletion of FoxF1 or FoxF2 in rhabdomyosarcoma cells decreased tumor growth in orthotopic mouse models of RMS. The decreased tumorigenesis was associated with the reduced tumor cell proliferation. Cell cycle regulatory proteins Cdk2, Cdk4/6, Cyclin D1 and Cyclin E2 were decreased in FoxF1- and FoxF2-deficient RMS tumors. Depletion of either FoxF1 or FoxF2 delayed G1-S cell cycle progression, decreased levels of phosphorylated Rb and increased protein levels of the CDK inhibitors, p21Cip1 and p27Kip1. Depletion of both FoxF1 and FoxF2 in tumor cells completely abrogated RMS tumor growth in mice. Overexpression of either FoxF1 or FoxF2 in tumor cells was sufficient to increase carcinogenesis in orthotopic RMS mouse model. FoxF1 and FoxF2 directly bound to and repressed transcriptional activity of p21Cip1 promoter through −556/−545 bp region, but did not affect p27Kip1 transcription. Knockdown of p21Cip1 restored cell cycle progression in the FoxF1- or FoxF2-deficient tumor cells. Altogether, FoxF1 and FoxF2 promoted RMS tumorigenesis by inducing tumor cell proliferation via transcriptional repression of p21Cip1 gene promoter. Due to robust oncogenic activity in RMS tumors, FoxF1 and FoxF2 may represent promising targets for anti-tumor therapy. PMID:27425595

  15. Tumour suppressor p16(INK4a) - anoikis-favouring decrease in N/O-glycan/cell surface sialylation by down-regulation of enzymes in sialic acid biosynthesis in tandem in a pancreatic carcinoma model.

    PubMed

    Amano, Maho; Eriksson, Hanna; Manning, Joachim C; Detjen, Katharina M; André, Sabine; Nishimura, Shin-Ichiro; Lehtiö, Janne; Gabius, Hans-Joachim

    2012-11-01

    Tumour suppressor p16(INK4a) is known to exert cell-cycle control via cyclin-dependent kinases. An emerging aspect of its functionality is the orchestrated modulation of N/O-glycosylation and galectin expression to induce anoikis in human Capan-1 pancreatic carcinoma cells. Using chemoselective N/O-glycan enrichment technology (glycoblotting) and product characterization, we first verified a substantial decrease in sialylation. Tests combining genetic (i.e. transfection with α2,6-sialyltransferase-specific cDNA) or metabolic (i.e. medium supplementation with N-acetylmannosamine to track down a bottleneck in sialic acid biosynthesis) engineering with cytofluorometric analysis of lectin binding indicated a role of limited substrate availability, especially for α2,6-sialylation, which switches off reactivity for anoikis-triggering homodimeric galectin-1. Quantitative MS analysis of protein level changes confirmed an enhanced galectin-1 presence along with an influence on glycosyltransferases (β1,4-galactosyltransferase-IV, α2,3-sialyltransferase-I) and detected p16(INK4a) -dependent down-regulation of two enzymes in the biosynthesis pathway for sialic acid [i.e. the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) and N-acetylneuraminic acid 9-phosphate synthase] (P < 0.001). By contrast, quantitative assessment for the presence of nuclear CMP-N-acetylneuraminic acid synthase (which is responsible for providing the donor for enzymatic sialylation that also acts as feedback inhibitor of the epimerase activity of GNE) revealed a trend for an increase. Partial restoration of sialylation in GNE-transfected cells supports the implied role of sialic acid availability for the glycophenotype. Fittingly, the extent of anoikis was reduced in double-transfected (p16(INK4a) /GNE) cells. Thus, a second means of modulating cell reactivity to the growth effector galectin-1 is established in addition to the common route of altering α2

  16. Effects of adenovirus-mediated expression of p27Kip1, p21Waf1 and p16INK4A in cell lines derived from t(2;5) anaplastic large cell lymphoma and Hodgkin's disease.

    PubMed

    Turturro, Franceso; Arnold, Marilyn D; Frist, Audrey Y; Seth, Prem

    2002-06-01

    We investigated the response of SUDHL-1 and L428 cells, derived from t(2;5)-anaplastic large cell lymphoma (ALCL) and Hodgkin's disease (HD), respectively, to recombinant adenoviruses expressing cyclin-dependent kinase inhibitors (CDKIs) p27Kip1 (Adp27), p21Waf1 (Adp21) and p16INK4A (Adp16). Cell cycle analysis of SUDHL-1 cells after 24 h of infection with 200 multiplicity of infection (MOI) of Adp27, Adp21, and Adp16, showed very high levels of cell debris in the subG1 area. The magnitude of cell debris-events was Adp27/Adp21 > Adp16. Cell cycle analysis of L428 cells revealed absence of cell debris and increased G2 phase in all the groups of cells tested as compared to the controls (mock and AdNull). A minimal increase in G1 phase was also evident in cells infected with Adp27 (52%) compared to uninfected cells (43%), AdNull (45%) and to cells infected with Adp21 (37%) and Adp16 (31%). The presence of significant levels of Coxsackie-adenovirus receptor (CAR) on the cell surface of L428 cells excluded the cell membrane-barrier as responsible for the differences in cell observed in response to the recombinant adenovirus-mediated CDKIs expression as compared to SUDHL-1. We also showed that the recombinant adenovirus-mediated cytotoxicity measured as apoptosis was MOI- and vector-dependent in SUDHL-1 cells at lower MOI (100). In conclusion, the therapeutic effect induced by recombinant adenoviruses expressing p27Kip1, p21Waf1 and p16INK4A is cell-dependent in cells derived from selected lymphoid malignancies. Biochemical cellular differences more than cell surface barriers seem to be responsible for differences in response to recombinant adenovirus-mediated expression of cytotoxic genes. Moreover, the cytotoxicity of recombinant adenoviruses expressing p27Kip1, p21Waf1 and p16INK4A may be further explored as a tool for gene therapy of t(2;5)-derived ALCL.

  17. Nucleolar GTP-binding Protein-1 (NGP-1) Promotes G1 to S Phase Transition by Activating Cyclin-dependent Kinase Inhibitor p21Cip1/Waf1*

    PubMed Central

    Datta, Debduti; Anbarasu, Kumaraswamy; Rajabather, Suryaraja; Priya, Rangasamy Sneha; Desai, Pavitra; Mahalingam, Sundarasamy

    2015-01-01

    Nucleolar GTP-binding protein (NGP-1) is overexpressed in various cancers and proliferating cells, but the functional significance remains unknown. In this study, we show that NGP-1 promotes G1 to S phase transition of cells by enhancing CDK inhibitor p21Cip-1/Waf1 expression through p53. In addition, our results suggest that activation of the cyclin D1-CDK4 complex by NGP-1 via maintaining the stoichiometry between cyclin D1-CDK4 complex and p21 resulted in hyperphosphorylation of retinoblastoma protein at serine 780 (p-RBSer-780) followed by the up-regulation of E2F1 target genes required to promote G1 to S phase transition. Furthermore, our data suggest that ribosomal protein RPL23A interacts with NGP-1 and abolishes NGP-1-induced p53 activity by enhancing Mdm2-mediated p53 polyubiquitination. Finally, reduction of p-RBSer-780 levels and E2F1 target gene expression upon ectopic expression of RPL23a resulted in arrest at the G1 phase of the cell cycle. Collectively, this investigation provides evidence that NGP-1 promotes cell cycle progression through the activation of the p53/p21Cip-1/Waf1 pathway. PMID:26203195

  18. The effects of short-term hypoxia on human mesenchymal stem cell proliferation, viability and p16INK4A mRNA expression: Investigation using a simple hypoxic culture system with a deoxidizing agent

    PubMed Central

    Ito, Akira; Aoyama, Tomoki; Yoshizawa, Makoto; Nagai, Momoko; Tajino, Junichi; Yamaguchi, Shoki; Iijima, Hirotaka; Zhang, Xiangkai; Kuroki, Hiroshi

    2015-01-01

    A hypoxic environment is thought to be important for the maintenance of stemness and suppressing cell senescence, in stem cells. Therefore, a hypoxic condition is induced during cell expansion and/or induction of intended differentiation. However, the induction of these conditions requires a specially equipped hypoxia chamber and expensive gas mixtures, which are expensive and space-consuming. Owing to these restrictions, appropriate hypoxic conditions cannot be provided during cell transportation, which is increasingly required for regenerative medicine. Hence, a simple and economical culture system is required. The purpose of this study was to investigate the effects of short-term hypoxic conditions on human mesenchymal stem cell (MSC) proliferation, viability, and senescence, utilizing the CulturePal system (CulturePal-Zero and CulturePal-Five), a novel and simple hypoxic culture system with a built-in deoxidizing agent. The O2 concentration in the CulturePal-Zero was observed to reduce to <0.1% within 1 h, and to 5% within 24h in the CulturePal-Five system. Cell proliferation under these hypoxic conditions showed a sharp increase at 5% O2 concentration, and no noticeable cell death was observed even at severe hypoxic conditions (<0.1% O2) up to 72h. The p16INK4A (cell senescence marker) mRNA expression was retained under hypoxic conditions up to 72h, but it was up-regulated under normoxic conditions. Interestingly, the p16INK4A expression altered proportionately to the O2 concentration. These results indicated that the short-term hypoxic condition, at an approximate O2 concentration of 5%, would be suitable for promoting cell proliferation and repressing cell senescence, without aggravating the MSC viability. Therefore, the CulturePal systems may be suitable for providing an appropriate hypoxic condition in stem cell research and transportation. PMID:26195892

  19. Tumor escape in a Wnt1-dependent mouse breast cancer model is enabled by p19Arf/p53 pathway lesions but not p16 Ink4a loss.

    PubMed

    Debies, Michael T; Gestl, Shelley A; Mathers, Jessica L; Mikse, Oliver R; Leonard, Travis L; Moody, Susan E; Chodosh, Lewis A; Cardiff, Robert D; Gunther, Edward J

    2008-01-01

    Breast cancers frequently progress or relapse during targeted therapy, but the molecular mechanisms that enable escape remain poorly understood. We elucidated genetic determinants underlying tumor escape in a transgenic mouse model of Wnt pathway-driven breast cancer, wherein targeted therapy is simulated by abrogating doxycycline-dependent Wnt1 transgene expression within established tumors. In mice with intact tumor suppressor pathways, tumors typically circumvented doxycycline withdrawal by reactivating Wnt signaling, either via aberrant (doxycycline-independent) Wnt1 transgene expression or via acquired somatic mutations in the gene encoding beta-catenin. Germline introduction of mutant tumor suppressor alleles into the model altered the timing and mode of tumor escape. Relapses occurring in the context of null Ink4a/Arf alleles (disrupting both the p16 Ink4a and p19 Arf tumor suppressors) arose quickly and rarely reactivated the Wnt pathway. In addition, Ink4a/Arf-deficient relapses resembled p53-deficient relapses in that both displayed morphologic and molecular hallmarks of an epithelial-to-mesenchymal transition (EMT). Notably, Ink4a/Arf deficiency promoted relapse in the absence of gross genomic instability. Moreover, Ink4a/Arf-encoded proteins differed in their capacity to suppress oncogene independence. Isolated p19 Arf deficiency mirrored p53 deficiency in that both promoted rapid, EMT-associated mammary tumor escape, whereas isolated p16 Ink4a deficiency failed to accelerate relapse. Thus, p19 Arf/p53 pathway lesions may promote mammary cancer relapse even when inhibition of a targeted oncogenic signaling pathway remains in force.

  20. Implication of polycomb members Bmi-1, Mel-18, and Hpc-2 in the regulation of p16INK4a, p14ARF, h-TERT, and c-Myc expression in primary breast carcinomas.

    PubMed

    Silva, Javier; García, José M; Peña, Cristina; García, Vanesa; Domínguez, Gemma; Suárez, Dolores; Camacho, Francisca I; Espinosa, Ruth; Provencio, Mariano; España, Pilar; Bonilla, Félix

    2006-12-01

    Deregulation of mammalian Polycomb group (PcG) members may contribute to human carcinogenesis. p16INK4a and p14ARF tumor suppressors, human telomerase reverse transcriptase (h-TERT), and oncoprotein c-Myc have been implicated in the regulation of the cell cycle and proliferation mediated by PcG proteins, mainly Bmi-1, in mice and in cell culture experiments. Here, we examine whether these in vitro findings can be extrapolated to the in vivo situation. We measure the expression of PcG members Bmi-1, Mel-18, and Hpc-2 and their potential targets by reverse transcription-PCR, immunostaining, and Western blotting in a series of 134 breast carcinomas and correlate the data with several clinical-pathologic variables of the tumors. Expression of PcG genes was variably detected, but overexpression of Bmi-1 was the most frequent PcG alteration observed. In addition, statistical direct correlation in expression level of the three PcG members was detected. A correlation between c-Myc and Bmi-1 expression levels was observed; however, there was no correlation between expression of Bmi-1 and p16INK4a, p14ARF, or h-TERT. However, expression of the other PcG members Mel-18 and Hpc-2 correlated with the cell cycle regulators. Moreover, PcG mRNA-altered expression correlated significantly with certain clinical-pathologic variables associated with poor prognosis. Our data suggest that the oncogenic role of Bmi-1 in human primary breast carcinomas is not determined by its capacity to inhibit INK4a/ARF proteins or to induce telomerase activity.

  1. BMP-4 Induction of Arrest and Differentiation of Osteoblast-Like Cells via p21CIP1 and p27KIP1 Regulation

    PubMed Central

    Chang, Shun-Fu; Chang, Ting-Kuo; Peng, Hsin-Hsin; Yeh, Yi-Ting; Lee, Ding-Yu; Yeh, Chiuan-Ren; Zhou, Jing; Cheng, Cheng-Kung; Chang, Cheng Allen; Chiu, Jeng-Jiann

    2009-01-01

    Cell cycle regulation by differentiation signals is critical for eukaryote development. We investigated the roles of bone morphogenetic protein (BMP)-4, an important stimulator of osteoblast differentiation and bone formation, in regulating cell cycle distribution in four osteoblast-like cell lines and mouse primary osteoblasts, and the underlying mechanisms. In all cells used, BMP-4 induced G0/G1 arrest. The molecular basis of the BMP-4 effect was analyzed, and the presentation on molecular mechanism is focused on human MG63 cells. BMP-4 induced p21CIP1 and p27KIP1 expressions and hence cell differentiation but had no effects on the expressions of cyclins A, B1, D1, and E, cyclin-dependent protein kinase-2, -4, and -6. Using specific small interfering RNA (siRNA), we found that BMP-4-induced G0/G1 arrest, and p21CIP1 and p27KIP1 expressions were mediated by BMP receptor type IA (BMPRIA)-specific Sma- and Mad-related protein (Smad)1/5. BMP-4 induced transient phosphorylations of ERK; transfection of MG63 cells with ERK2, but not ERK1, -specific siRNA inhibited the BMP-4-induced responses in MG63 cells. Pretreatment of MG63 cells with Arg-Gly-Asp-Ser, which blocks the cell-extracellular matrix interaction, or transfection with β3 integrin-specific siRNA inhibited BMP-4-induced ERK and Smad1/5 phosphorylations. BMP-4 induced transient increases in associations of β3-integrin with focal adhesion kinase and Shc, the dominant-negative mutants of which inhibited BMP-4-induced ERK and Smad1/5 phosphorylations. Our results indicate that BMP-4 induces G0/G1 arrest and hence differentiation in osteoblast-like cells through increased expressions of p21CIP1 and p27KIP1, which are mediated by BMPRIA-specific Smad1/5. The extracellular matrix/β3 integrin/ focal adhesion kinase/Shc/ERK2 signaling pathway is involved in these BMP-4-induced responses in osteoblast-like cells. PMID:19819988

  2. Caffeic Acid Phenethyl Ester Causes p21Cip1 Induction, Akt Signaling Reduction, and Growth Inhibition in PC-3 Human Prostate Cancer Cells

    PubMed Central

    Lin, Hui-Ping; Jiang, Shih Sheng; Chuu, Chih-Pin

    2012-01-01

    Caffeic acid phenethyl ester (CAPE) treatment suppressed proliferation, colony formation, and cell cycle progression in PC-3 human prostate cancer cells. CAPE decreased protein expression of cyclin D1, cyclin E, SKP2, c-Myc, Akt1, Akt2, Akt3, total Akt, mTOR, Bcl-2, Rb, as well as phosphorylation of Rb, ERK1/2, Akt, mTOR, GSK3α, GSK3β, PDK1; but increased protein expression of KLF6 and p21Cip1. Microarray analysis indicated that pathways involved in cellular movement, cell death, proliferation, and cell cycle were affected by CAPE. Co-treatment of CAPE with chemotherapeutic drugs vinblastine, paclitaxol, and estramustine indicated synergistic suppression effect. CAPE administration may serve as a potential adjuvant therapy for prostate cancer. PMID:22347457

  3. Apoptotic effect of ethyl-4-isothiocyanatobutanoate is associated with DNA damage, proteasomal activity and induction of p53 and p21cip1/waf1.

    PubMed

    Bodo, Juraj; Jakubikova, Jana; Chalupa, Ivan; Bartosova, Zdena; Horakova, Katarina; Floch, Lubomir; Sedlak, Jan

    2006-08-01

    The effect of synthetic isothiocyanate ethyl-4-isothiocyanatobutanoate (E-4IB) on survival of mismatch repair-proficient TK6 and -deficient MT1 cell lines as well as the influence of proteasomal inhibitor MG132, caspase inhibitor Z-VAD-fmk, and ATM inhibitor caffeine on E-4IB modulation of cell cycle and apoptosis was evaluated. Flow cytometric analyses of DNA double strand breaks (gamma-H2AX), mitotic fraction (phospho-histone H3), cell cycle modulation, apoptosis induction (sub-G(0) fraction and fluorescein diacetate staining), and dissipation of transmembrane mitochondrial potential (JC-1 staining) were performed. Western blotting was used for the evaluation of ERK activation, expression of p53, p21(cip1/waf1) and GADD45alpha proteins, as well as PARP fragmentation. Analysis of mitotic nuclei was performed for chromosomal aberrations assessment. MT1 cells were more resistant to E-4IB treatment then TK6 cells (IC(50) 8 muM vs. 4 muM). In both cell lines E-4IB treatment induced phosphorylation of H2AX, increase of p53 protein level, phospho-histone H3 staining, and G(2)/M arrest. The sub-G(0) fragmentation was accompanied by PARP degradation, decreased mitochondrial transmembrane potential, and diminished p21(cip1/waf1) protein expression in TK6 cells. Caspase inhibitor Z-VAD-fmk decreased E-4IB induced sub-G(0) fragmentation and extent of apoptosis in TK6 cells, while proteasome inhibitor MG132 increased number of apoptotic cells in both cell lines tested. A number of aberrant metaphases and clastogenic effect of high E-4IB concentration was observed. The synthetic isothiocyanate E-4IB induced DNA strand breaks, increased mitotic fraction and apoptosis potentiated by MG132 inhibitor in both mismatch repair-proficient and -deficient cell lines.

  4. Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21(cip1) in vascular remodelling during vein arterialization in the rat.

    PubMed

    Borin, Thaiz Ferraz; Miyakawa, Ayumi Aurea; Cardoso, Leandro; de Figueiredo Borges, Luciano; Gonçalves, Giovana Aparecida; Krieger, Jose Eduardo

    2009-06-01

    Neo-intima development and atherosclerosis limit long-term vein graft use for revascularization of ischaemic tissues. Using a rat model, which is technically less challenging than smaller rodents, we provide evidence that the temporal morphological, cellular, and key molecular events during vein arterialization resemble the human vein graft adaptation. Right jugular vein was surgically connected to carotid artery and observed up to 90 days. Morphometry demonstrated gradual thickening of the medial layer and important formation of neo-intima with deposition of smooth muscle cells (SMC) in the subendothelial layer from day 7 onwards. Transmission electron microscopy showed that SMCs switch from the contractile to synthetic phenotype on day 3 and new elastic lamellae formation occurs from day 7 onwards. Apoptosis markedly increased on day 1, while alpha-actin immunostaining for SMC almost disappeared by day 3. On day 7, cell proliferation reached the highest level and cellular density gradually increased until day 90. The relative magnitude of cellular changes was higher in the intima vs. the media layer (100 vs. 2 times respectively). Cyclin-dependent kinase inhibitors (CDKIs) p27(Kip1) and p16(INKA) remained unchanged, whereas p21(Cip1) was gradually downregulated, reaching the lowest levels by day 7 until day 90. Taken together, these data indicate for the first time that p21(Cip1) is the main CDKI protein modulated during the arterialization process the rat model of vein arterialization that may be useful to identify and validate new targets and interventions to improve the long-term patency of vein grafts.

  5. Altered expression of G1/S regulatory genes occurs early and frequently in lung carcinogenesis in transforming growth factor-beta1 heterozygous mice.

    PubMed

    Kang, Yang; Ozbun, Laurent L; Angdisen, Jerry; Moody, Terry W; Prentice, Margaret; Diwan, Bhalchandra A; Jakowlew, Sonia B

    2002-07-01

    We developed the AJBL6 transforming growth factor-beta 1 (TGF-beta1) heterozygous (HT) mouse by mating A/J mice with C57BL/6 TGF-beta1 HT mice that shows increased carcinogen-induced lung lesions with decreased latency to examine progressive events in lung tumorigenesis. Mouse cDNA macroarrays were used to identify cell cycle genes that are differentially regulated in ethyl carbamate-induced lung adenocarcinomas compared with normal lung tissue in AJBL6 TGF-beta1 HT mice using probes that were generated from tissues isolated using laser capture microdissection. While expression of the genes for cyclin D1, CDK4, and E2F1 increased in lung adenocarcinomas relative to normal lung, expression of p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), p57(Kip2), and pRb genes decreased in comparison. Competitive RT-PCR showed that the levels of cyclin D1 and CDK4 mRNAs were 2- and 3-fold higher, respectively, in lung adenocarcinomas than in normal lung, while the mRNAs for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb were 3- to 4-fold lower in adenocarcinomas than in normal lung, thus validating the macroarray findings. Competitive RT-PCR of microdissected lesions also showed that the levels of cyclin D1 and CDK4 mRNAs increased significantly, while the mRNAs for p15(Ink4b) and p27(Kip1) decreased significantly as lung tumorigenesis progressed. Immunohistochemical staining for cyclin D1 and CDK4 showed staining in >80% of nuclei in adenocarcinomas compared with fewer than 20% of nuclei staining positively in normal lung. In contrast, while >60% of normal lung cells showed immunostaining for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb, staining for these proteins decreased in hyperplasias, adenomas, and adenocarcinomas. These data show that multiple components of the cyclin D1/CDK4/p16(Ink4a)/pRb signaling pathway are frequently altered early in lung lesions of AJBL6 TGF-beta1 HT mice that are induced by ethyl carbamate as a function of progressive lung

  6. The Cyclin-dependent Kinase Inhibitor p16INK4a Physically Interacts with Transcription Factor Sp1 and Cyclin-dependent Kinase 4 to Transactivate MicroRNA-141 and MicroRNA-146b-5p Spontaneously and in Response to Ultraviolet Light-induced DNA Damage*

    PubMed Central

    Al-Khalaf, Huda H.; Mohideen, Peer; Nallar, Shreeram C.; Kalvakolanu, Dhananjaya V.; Aboussekhra, Abdelilah

    2013-01-01

    p16INK4a is a tumor suppressor protein involved in several stress-related cellular responses, including apoptosis. Recent lines of evidence indicate that p16INK4a is also a modulator of gene expression. However, the molecular mechanisms underlying this novel function are still obscure. Here, we present clear evidence that p16INK4a modulates the levels of various microRNAs, with marked positive effect on miR-141 and miR-146b-5p. This effect is mediated through the formation of the p16-CDK4-Sp1 heterocomplex, which binds to Sp1 consensus-binding motifs present in the promoters of miR-141 and miR-146b-5p, and it enables their transcription. In addition, we have shown that p16INK4a interacts with Sp1 through the fourth ankyrin repeat, which is crucial for Sp1 binding to the miR-141 and miR-146b-5p promoters and their transcriptional activation. The physiological importance of this association was revealed by the inability of cancer-related p16INK4a mutants to interact with Sp1. Moreover, we have shown p16-CDK4-Sp1-dependent up-regulation of miR-141 and miR-146b-5p following UV light-induced DNA damage and the role of these two microRNAs in mediating p16-related induction of apoptosis in response to this genotoxic stress. Together, these results indicate that p16INK4a associates with CDK4 not only to inhibit the cell cycle but also to enable the transcription of two important onco-microRNAs, which act as downstream effectors. PMID:24163379

  7. p15RS/RPRD1A (p15INK4b-related Sequence/Regulation of Nuclear Pre-mRNA Domain-containing Protein 1A) Interacts with HDAC2 in Inhibition of the Wnt/β-Catenin Signaling Pathway*

    PubMed Central

    Liu, Chunxiao; Zhang, Yanquan; Li, Jun; Wang, Yinyin; Ren, Fangli; Zhou, Yifan; Wu, Yinyuan; Feng, Yarui; Zhou, Yu; Su, Fuqin; Jia, Baoqing; Wang, Dong; Chang, Zhijie

    2015-01-01

    We previously reported that p15RS (p15INK4b-related sequence), a regulation of nuclear pre-mRNA domain containing protein, inhibited Wnt signaling by interrupting the formation of the β-catenin·TCF4 complex. However, how p15RS functions as an intrinsic repressor to repress transcription remains unclear. In this study, we show that p15RS, through a specific interaction with HDAC2 (histone deacetylase 2), a deacetylase that regulates gene transcription, maintains histone H3 in a deacetylated state in the promoter region of Wnt-targeted genes where β-catenin·TCF4 is bound. We observed that histone deacetylase inhibitors impair the ability of p15RS in inhibiting Wnt/β-catenin signaling. Depletion of HDAC2 markedly disabled p15RS inhibition of Wnt/β-catenin-mediated transcription. Interestingly, overexpression of p15RS decreases the level of acetylated histone H3 in the c-MYC promoter. Finally, we demonstrate that p15RS significantly enhances the association of HDAC2 and TCF4 and enhances the occupancy of HDAC2 to DNA, resulting in the deacetylation of histone H3 and the failure of β-catenin interaction. We propose that p15RS acts as an intrinsic transcriptional repressor for Wnt/β-catenin-mediated gene transcription at least partially through recruiting HDAC2 to occupy the promoter and maintaining deacetylated histone H3. PMID:25697359

  8. Translational Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress

    PubMed Central

    Lehman, Stacey L.; Cerniglia, George J.; Johannes, Gregg J.; Ye, Jiangbin; Ryeom, Sandra; Koumenis, Constantinos

    2015-01-01

    Multiple transcripts encode for the cell cycle inhibitor p21Cip1. These transcripts produce identical proteins but differ in their 5’ untranslated regions (UTRs). Although several stresses that induce p21 have been characterized, the mechanisms regulating the individual transcript variants and their functional significance are unknown. Here we demonstrate through 35S labeling, luciferase reporter assays, and polysome transcript profiling that activation of the Integrated Stress Response (ISR) kinase GCN2 selectively upregulates the translation of a p21 transcript variant containing 5’ upstream open reading frames (uORFs) through phosphorylation of the eukaryotic translation initiation factor eIF2α. Mutational analysis reveals that the uORFs suppress translation under basal conditions, but promote translation under stress. Functionally, ablation of p21 ameliorates G1/S arrest and reduces cell survival in response to GCN2 activation. These findings uncover a novel mechanism of p21 post-transcriptional regulation, offer functional significance for the existence of multiple p21 transcripts, and support a key role for GCN2 in regulating the cell cycle under stress. PMID:26102367

  9. Cooperative role between p21cip1/waf1 and p27kip1 in premature senescence in glandular proliferative lesions in mice.

    PubMed

    García-Fernández, R A; García-Palencia, P; Suarez, C; Sánchez, M A; Gil-Gómez, G; Sánchez, B; Rollán, E; Martín-Caballero, J; Flores, J M

    2014-03-01

    Cellular senescence has been considered a novel target for cancer therapy. It has also been pointed out that p21(cip1/waf1) and p27(kip1) cyclin-dependent kinase inhibitors (CKIs) play a role in cellular senescence in some tumor types. Therefore, in order to address the possibility of a cooperative role between p21 and p27 proteins in senescence in vivo we analyzed cellular senescence in spontaneous glandular proliferative lesions (adrenal, thyroid and pituitary glands) in a double-KO mice model, using γH2AX, p53, p16, PTEN and Ki67 as senescence markers. The results obtained showed that p21p27 double-null mice had the lowest number of γH2AX positive cells in glandular hyperplasias and benign tumors. Also, in this group, Ki67 proliferation index correlated with a lower immunohistochemical expression of γH2AX and p53. The expression of p16 and PTEN do not seem to cause synergism of senescence in the benign lesions analyzed in p21p27 double-KO mice. These observations suggest an intrinsic cooperation between p21 and p27 CKIs in the activation of stress-induced cellular senescence and tumor progression in vivo, which would be a physiological mechanism to prevent tumor cell proliferation.

  10. Cdkn2a, the cyclin-dependent kinase inhibitor encoding p16INK4a and p19ARF, is a candidate for the plasmacytoma susceptibility locus, Pctr1

    PubMed Central

    Zhang, Shuling; Ramsay, Edward S.; Mock, Beverly A.

    1998-01-01

    Plasma cell tumor induction in mice by pristane is under multigenic control. BALB/c mice are susceptible to tumor development; whereas DBA/2 mice are resistant. Restriction fragment length polymorphisms between BALB/c and DBA/2 for Cdkn2a(p16) and Cdkn2b(p15), and between BALB/c and Mus spretus for Cdkn2c(p18INK4c) were used to position these loci with respect to the Pctr1 locus. These cyclin-dependent kinase (CDK) inhibitors mapped to a 6 cM interval of chromosome 4 between Ifna and Tal1. C.D2-Chr 4 congenic strains harboring DBA/2 alleles associated with the Pctr1 locus contained DBA/2 “resistant” alleles of the CDK4/CDK6 inhibitors p16 and p15. On sequencing p16 and p18 cDNAs, two different allelic variants within ankyrin repeat regions of p16 were found between BALB/c and DBA/2 mice. By using an assay involving PCR amplification and restriction enzyme digestion, allelic variants were typed among several inbred strains of mice. One of the variants, G232A, was specific to two inbred strains, BALB/cAn and ABP/Le, of mice and occurred in a highly conserved amino acid in both human and rat p16. When tested with wild-type (DBA/2) p16, both A134C and G232A BALB/c-specific variants of p16 were inefficient in their ability to inhibit the activity of cyclin D2/CDK4 in kinase assays with retinoblastoma protein, suggesting this defective, inherited allele plays an important role in the genetic susceptibility of BALB/c mice for plasmacytoma induction and that p16INK4a is a strong candidate for the Pctr1 locus. PMID:9482902

  11. Cytotoxicity of 5-Aza-2'-deoxycytidine against gastric cancer involves DNA damage in an ATM-P53 dependent signaling pathway and demethylation of P16(INK4A).

    PubMed

    Liu, Juan; Xie, Yi-Shan; Wang, Fu-Liang; Zhang, Li-Jun; Zhang, Yan; Luo, He-Sheng

    2013-02-01

    The DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) has increasingly attracted worldwide attention for its antineoplastic potential. The cytotoxitic mechanisms, however, especially, the relative contribution of silenced genes reactivation by demethylation and enzyme-DNA adduct formation to the efficacy of 5-Aza-CdR is still a crucial unresolved question. In this investigation, we demonstrated that 5-Aza-CdR treatment resulted in growth suppression in a concentration and time-dependent manner and G2 phrase arrest - hallmarks of a DNA damage response in gastric cancer AGS cells. Formation of DNA double-strand breaks, as monitored by comet assay was examined in an ATM (ataxia-telangiectasia mutated)-dependent manner based on the fact that PI3K inhibitor Wortmannin abolished the action of cytotoxicity of 5-Aza-CdR. Upon treatment with 5-Aza-CdR, ATM activation was clearly associated with P53 phosphorylation at Ser(15), which was directly responsible for 5-Aza-CdR modified P21(Waf1/Cip1) expression. Further exploration revealed that demethylation of P16(INK4A) correlated with the strikingly down-regulated expressions of DNA methyltransferase 3A as well as 3B was, at least in part, attributed to the cytotoxicity of 5-Aza-CdR in AGS cells. Conclusively, these results greatly enhance our understanding of the mechanisms of cytotoxicity of 5-Aza-CdR and strongly provide the preclinical rationale for an assessment of 5-Aza-CdR to ameliorate patient outcome with gastric cancer. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  12. Co-Expression of p16INK4A and Laminin 5 γ2 by Microinvasive and Superficial Squamous Cell Carcinomas in Vivo and by Migrating Wound and Senescent Keratinocytes in Culture

    PubMed Central

    Natarajan, Easwar; Saeb, Marcela; Crum, Christopher P.; Woo, Sook B.; McKee, Phillip H.; Rheinwald, James G.

    2003-01-01

    The high frequency of mutation, deletion, and promoter silencing of the gene encoding p16INK4A (p16) in premalignant dysplasias and squamous cell carcinomas (SCC) of epidermis and oral epithelium classifies p16 as a tumor suppressor. However, the point during neoplastic progression at which this protein is expressed and presumably impedes formation of an SCC is unknown. Induction of p16 has been found to be responsible for the senescence arrest of normal human keratinocytes in culture, suggesting the possibility that excessive or spatially abnormal cell growth in vivo triggers p16 expression. We examined 73 skin and oral mucosal biopsy specimens immunohistochemically to test this hypothesis. p16 was not detectable in benign hyperplastic lesions, but instead was expressed heterogeneously in some dysplastic and carcinoma in situ lesions and consistently at areas of microinvasion and at superficial margins of advanced SCCs. p16-positive cells in these regions coexpressed the γ2 chain of laminin 5, identified previously as a marker of invasion in some carcinomas. Normal keratinocytes undergoing senescence arrest in culture proved to coordinately express p16 and γ2 and this was frequently associated with increased directional motility. Keratinocytes at the edges of wounds made in confluent early passage cultures also coexpressed p16 and γ2, accompanying migration to fill the wound. These results have identified the point during neoplastic progression in stratified squamous epithelial at which the tumor suppressor p16 is expressed and suggest that normal epithelia may use the same mechanism to generate non-dividing, motile cells for wound repair. PMID:12875969

  13. Epigenetic changes in the CDKN2A locus are associated with differential expression of P16INK4A and P14ARF in HPV-positive oropharyngeal squamous cell carcinoma

    PubMed Central

    Schlecht, Nicolas F; Ben-Dayan, Miriam; Anayannis, Nicole; Lleras, Roberto A; Thomas, Carlos; Wang, Yanhua; Smith, Richard V; Burk, Robert D; Harris, Thomas M; Childs, Geoffrey; Ow, Thomas J; Prystowsky, Michael B; Belbin, Thomas J

    2015-01-01

    Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is recognized as a distinct disease entity associated with improved survival. DNA hypermethylation profiles differ significantly by HPV status suggesting that a specific subset of methylated CpG loci could give mechanistic insight into HPV-driven OPSCC. We analyzed genome-wide DNA methylation of primary tumor samples and adjacent normal mucosa from 46 OPSCC patients undergoing treatment at Montefiore Medical Center, Bronx, NY using the Illumina HumanMethylation27 beadchip. For each matched tissue set, we measured differentially methylated CpG loci using a change in methylation level (M value). From these analyses, we identified a 22 CpG loci panel for HPV+ OPSCC that included four CDKN2A loci downstream of the p16(INK4A) and p14(ARF) transcription start sites. This panel was significantly associated with overall HPV detection (P < 0.05; ROC area under the curve = 0.96, 95% CI: 0.91–1.0) similar to the subset of four CDKN2A-specific CpG loci (0.90, 95% CI: 0.82–0.99) with equivalence to the full 22 CpG panel. DNA hypermethylation correlated with a significant increase in alternative open reading frame (ARF) expression in HPV+ OPSCC primary tumors, but not to the other transcript variant encoded by the CDKN2A locus. Overall, this study provides evidence of epigenetic changes to the downstream region of the CDKN2A locus in HPV+ oropharyngeal cancer that are associated with changes in expression of the coded protein products. PMID:25619363

  14. Epigenetic changes in the CDKN2A locus are associated with differential expression of P16INK4A and P14ARF in HPV-positive oropharyngeal squamous cell carcinoma.

    PubMed

    Schlecht, Nicolas F; Ben-Dayan, Miriam; Anayannis, Nicole; Lleras, Roberto A; Thomas, Carlos; Wang, Yanhua; Smith, Richard V; Burk, Robert D; Harris, Thomas M; Childs, Geoffrey; Ow, Thomas J; Prystowsky, Michael B; Belbin, Thomas J

    2015-03-01

    Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is recognized as a distinct disease entity associated with improved survival. DNA hypermethylation profiles differ significantly by HPV status suggesting that a specific subset of methylated CpG loci could give mechanistic insight into HPV-driven OPSCC. We analyzed genome-wide DNA methylation of primary tumor samples and adjacent normal mucosa from 46 OPSCC patients undergoing treatment at Montefiore Medical Center, Bronx, NY using the Illumina HumanMethylation27 beadchip. For each matched tissue set, we measured differentially methylated CpG loci using a change in methylation level (M value). From these analyses, we identified a 22 CpG loci panel for HPV+ OPSCC that included four CDKN2A loci downstream of the p16(INK4A) and p14(ARF) transcription start sites. This panel was significantly associated with overall HPV detection (P < 0.05; ROC area under the curve = 0.96, 95% CI: 0.91-1.0) similar to the subset of four CDKN2A-specific CpG loci (0.90, 95% CI: 0.82-0.99) with equivalence to the full 22 CpG panel. DNA hypermethylation correlated with a significant increase in alternative open reading frame (ARF) expression in HPV+ OPSCC primary tumors, but not to the other transcript variant encoded by the CDKN2A locus. Overall, this study provides evidence of epigenetic changes to the downstream region of the CDKN2A locus in HPV+ oropharyngeal cancer that are associated with changes in expression of the coded protein products. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  15. All-trans retinoic acid enhances the effect of 5-aza-2'-deoxycytidine on p16INK4a demethylation, and the two drugs synergistically activate retinoic acid receptor β gene expression in the human erythroleukemia K562 cell line.

    PubMed

    Xiang, Lili; Dong, Weimin; Wang, Rong; Wei, Jiang; Qiu, Guoqiang; Cen, Jiannong; Chen, Zixing; Zheng, Xiao; Hu, Shaoyan; Xie, Xiaobao; Cao, Xiangshan; Gu, Weiying

    2014-07-01

    The aim of the current study was to investigate the antineoplastic activities of 5-aza-2'-deoxycytidine (also known as decitabine; DAC) and all-trans retinoic acid (ATRA), administered alone or in combination, in K562 cells in vitro, as well as the effects on the expression of the tumor suppressor genes, p16INK4a (p16) and retinoic acid receptor β (RAR-β). Cell growth inhibition, differentiation and apoptosis in K562 cells treated with DAC and/or ATRA were detected. The methylation of the p16 and RAR-β genes in the K562 cells was detected using the methylation-specific polymerase chain reaction (PCR) method. Quantitative PCR was used for the detection of the mRNA expression of the p16 and RAR-β genes, and western blot analysis was used to detect protein expression. DAC and ATRA, alone or in combination, had no effect on the growth inhibition, differentiation and apoptosis of the K562 cells. DAC alone induced the demethylation of the p16 gene, and combination of DAC and ATRA demonstrated more evident demethylation of the p16 gene, however, ATRA alone had no effect on methylation. The RAR-β promoter region was not methylated in the K562 cells. DAC in combination with ATRA appeared to produce a greater activation of the RAR-β gene, which led to the upregulation of the RAR-β expression level. ATRA enhanced the effect of DAC on p16 demethylation, and the combination of the two drugs was found to activate RAR-β expression, which indicated that DAC used in combination with ATRA has clinical potential in the treatment of human erythroleukemia.

  16. Single-cell analysis of p16(INK4a) and p21(WAF1) expression suggests distinct mechanisms of senescence in normal human and Li-Fraumeni Syndrome fibroblasts.

    PubMed

    Mirzayans, Razmik; Andrais, Bonnie; Scott, April; Paterson, Malcolm C; Murray, David

    2010-04-01

    Herein we used single-cell observation methods to gain insight into the roles of p16(INK4A) and p21(WAF1) (hereafter p16 and p21) in replicative senescence and ionizing radiation-induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li-Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence-associated beta-galactosidase. In addition, proliferating early-passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53-proficient (normal and AT) fibroblasts. In p53-deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation-triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild-type p53 activity. The possibility that one of the tumor-suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results. J. Cell. Physiol. 223: 49-56, 2010. (c) 2009 Wiley-Liss, Inc.

  17. The Cell Cycle Inhibitors p21(Cip1) and p27(Kip1) Control Proliferation but Enhance DNA Damage Resistance of Glioma Stem Cells.

    PubMed

    Morris-Hanon, Olivia; Furmento, Verónica Alejandra; Rodríguez-Varela, María Soledad; Mucci, Sofía; Fernandez-Espinosa, Damián Darío; Romorini, Leonardo; Sevlever, Gustavo Emilio; Scassa, María Elida; Videla-Richardson, Guillermo Agustín

    2017-07-01

    High-grade gliomas are the most prevalent and lethal primary brain tumors. They display a hierarchical arrangement with a population of self-renewing and highly tumorigenic cells called cancer stem cells. These cells are thought to be responsible for tumor recurrence, which make them main candidates for targeted therapies. Unbridled cell cycle progression may explain the selective sensitivity of some cancer cells to treatments. The members of the Cip/Kip family p21(Cip1) and p27(Kip1) were initially considered as tumor suppressors based on their ability to block proliferation. However, they are currently looked at as proteins with dual roles in cancer: one as tumor suppressor and the other as oncogene. Therefore, the aim of this study was to determine the functions of these cell cycle inhibitors in five patient-derived glioma stem cell-enriched cell lines. We found that these proteins are functional in glioma stem cells. They negatively regulate cell cycle progression both in unstressed conditions and in response to genotoxic stress. In addition, p27(Kip1) is upregulated in nutrient-restricted and differentiating cells, suggesting that this Cip/Kip is a mediator of antimitogenic signals in glioma cells. Importantly, the lack of these proteins impairs cell cycle halt in response to genotoxic agents, rendering cells more vulnerable to DNA damage. For these reasons, these proteins may operate both as tumor suppressors, limiting cell proliferation, and as oncogenes, conferring cell resistance to DNA damage. Thus, deepening our knowledge on the biological functions of these Cip/Kips may shed light on how some cancer cells develop drug resistance. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Proto-oncogene FBI-1 represses transcription of p21CIP1 by inhibition of transcription activation by p53 and Sp1.

    PubMed

    Choi, Won-Il; Jeon, Bu-Nam; Yun, Chae-Ok; Kim, Pyung-Hwan; Kim, Sung-Eun; Choi, Kang-Yell; Kim, Se Hoon; Hur, Man-Wook

    2009-05-08

    Aberrant transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as the driving force for tumorigenesis. FBI-1 (formerly called Pokemon) is a member of the POK family of transcriptional repressors. Recently, FBI-1 was characterized as a critical oncogenic factor that specifically represses transcription of the tumor suppressor gene ARF, potentially leading indirectly to p53 inactivation. Our investigations on transcriptional repression of the p53 pathway revealed that FBI-1 represses transcription of ARF, Hdm2 (human analogue of mouse double minute oncogene), and p21CIP1 (hereafter indicated as p21) but not of p53. FBI-1 showed a more potent repressive effect on p21 than on p53. Our data suggested that FBI-1 is a master controller of the ARF-Hdm2-p53-p21 pathway, ultimately impinging on cell cycle arrest factor p21, by inhibiting upstream regulators at the transcriptional and protein levels. FBI-1 acted as a competitive transcriptional repressor of p53 and Sp1 and was shown to bind the proximal Sp1-3 GC-box and the distal p53-responsive elements of p21. Repression involved direct binding competition of FBI-1 with Sp1 and p53. FBI-1 also interacted with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylating Ac-H3 and Ac-H4 histones at the promoter. FBI-1 caused cellular transformation, promoted cell cycle proliferation, and significantly increased the number of cells in S phase. FBI-1 is aberrantly overexpressed in many human solid tumors, particularly in adenocarcinomas and squamous carcinomas. The role of FBI-1 as a master controller of the p53 pathway therefore makes it an attractive therapeutic target.

  19. Pim-1 kinase-dependent phosphorylation of p21Cip1/WAF1 regulates its stability and cellular localization in H1299 cells.

    PubMed

    Zhang, Yandong; Wang, Zeping; Magnuson, Nancy S

    2007-09-01

    Previous studies from our laboratory showed that p21Cip1/WAF1 can be phosphorylated by Pim-1 kinase in vitro, implying that part of the function of Pim-1 might involve influencing the cell cycle. In the present study, site-directed mutagenesis and phosphorylated-specific antibodies were used as tools to identify the sites phosphorylated by Pim-1 and the consequences of this phosphorylation. What we found was that Pim-1 can efficiently phosphorylate p21 on Thr145 in vitro using recombinant protein and in vivo in intact cells. Unexpectedly, we found that Ser146 is a second site that is phosphorylated in vivo, but this phosphorylation event seems to be an indirect result of Pim-1 expression. More importantly, the consequences of phosphorylation of either Thr145 or Ser146 are distinct. When p21 is phosphorylated on Thr145, it localizes to the nucleus and results in the disruption of the association between proliferating cell nuclear antigen and p21. Furthermore, phosphorylation of Thr145 promotes stabilization of p21. On the other hand, when p21 is phosphorylated on Ser146, it localizes primarily in the cytoplasm and the effect of phosphorylation on stability is minimal. Cotransfection of wild-type Pim-1 with p21 increases the rate of proliferation compared with cotransfection of p21 with kinase-dead Pim-1. Knocking down Pim-1 expression greatly decreases the rate of proliferation of H1299 cells and their ability to grow in soft agar. These data suggest that Pim-1 overexpression may contribute to tumorigenesis in part by influencing the cellular localization and stability of p21 and by promoting cell proliferation.

  20. TOK-1, a novel p21Cip1-binding protein that cooperatively enhances p21-dependent inhibitory activity toward CDK2 kinase.

    PubMed

    Ono, T; Kitaura, H; Ugai, H; Murata, T; Yokoyama, K K; Iguchi-Ariga, S M; Ariga, H

    2000-10-06

    A p21(Cip1/Waf1/Sdi1) is known to act as a negative cell-cycle regulator by inhibiting kinase activity of a variety of cyclin-dependent kinases. In addition to binding of the cyclin-dependent kinase to the N-terminal region of p21, p21 is also bound at its C-terminal region by proliferating cell nuclear antigen (PCNA), SET/TAF1, and calmodulin, indicating the versatile function of p21. In this study, we cloned cDNA encoding a novel protein named TOK-1 as a p21 C-terminal-binding protein by a two-hybrid system. Two splicing isoforms of TOK-1, TOK-1alpha and TOK-1beta, comprising 322 and 314 amino acids, respectively, were co-localized with p21 in nuclei and showed a similar expression profile to that of p21 in human tissues. TOK-1alpha, but not TOK-1beta, directly bound to the C-terminal proximal region of p21, and both were expressed at the G(1)/S boundary of the cell cycle. TOK-1alpha also preferentially bound to an active form of cyclin-dependent kinase 2 (CDK2) via p21, and these made a ternary complex in human cells. Furthermore, the results of three different types of experiments showed that TOK-1alpha enhanced the inhibitory activity of p21 toward histone H1 kinase activity of CDK2. TOK-1alpha is thus thought to be a new type of CDK2 modulator.

  1. ABT-263 enhances sorafenib-induced apoptosis associated with Akt activity and the expression of Bax and p21(CIP1/WAF1) in human cancer cells

    PubMed Central

    Li, Jingru; Chen, Yicheng; Wan, Jiali; Liu, Xin; Yu, Chunrong; Li, Wenhua

    2014-01-01

    BACKGROUND AND PURPOSE Sorafenib, a potent inhibitor that targets several kinases associated with tumourigenesis and cell survival, has been approved for clinical treatment as a single agent. However, combining sorafenib with other agents improves its anti-tumour efficacy in various preclinical tumour models. ABT-263, a second-generation BH3 mimic, binds to the anti-apoptotic family members Bcl-2, Bcl-xL and Bcl-w, and has been demonstrated to enhance TNFSF10 (TRAIL)-induced apoptosis in human hepatocarcinoma cells. Hence, we investigated the effects of ABT-263 treatment combined with sorafenib. EXPERIMENTAL APPROACH The effects of ABT-263 combined with sorafenib were investigated in vitro, on cell viability, clone formation and apoptosis, and the mechanism examined using western blot and flow cytometry. This combination was also evaluated in vivo, in a mouse xenograft model; tumour growth, volume and weights were measured and a TUNEL assay performed. KEY RESULTS ABT-263 enhanced sorafenib-induced apoptosis while sparing non-tumourigenic cells. Although ABT-263 plus sorafenib significantly stimulated intracellular reactive oxygen species production and subsequent mitochondrial depolarization, this was not sufficient to trigger cell apoptosis. ABT-263 plus sorafenib significantly decreased Akt activity, which was, at least partly, involved in its effect on apoptosis. Bax and p21 (CIP1/WAF1) were shown to play a critical role in ABT-263 plus sorafenib-induced apoptosis. Combining sorafenib with ABT-263 dramatically increased its efficacy in vivo. CONCLUSION AND IMPLICATIONS The anti-tumour activity of ABT-263 plus sorafenib may involve the induction of intrinsic cell apoptosis via inhibition of Akt, and reduced Bax and p21 expression. Our findings offer a novel effective therapeutic strategy for tumour treatment. PMID:24571452

  2. Human papilloma virus early proteins E6 (HPV16/18-E6) and the cell cycle marker P16 (INK4a) are useful prognostic markers in uterine cervical carcinomas in Qassim Region--Saudi Arabia.

    PubMed

    Omran, O M; AlSheeha, M

    2015-01-01

    Cervical cancer is a common and an important public health problem for adult women in developing countries. In contrast, cervical cancer incidence is low in Saudi Arabia. High-risk types of human papilloma viruses (HPV16 and HPV18) are the most significant risk factors for cervical cancer. HPV16/18-E6 oncoprotein is associated with HPV etiology, viral persistence and epithelial transformation. Cell cycle protein p16 INK4a (p16) plays an important role in the pathophysiology of cervical carcinomas. The aims of this study were to investigate the expression of HPV16/18-E6 and p16 in uterine cervical carcinomas in Qassim Region--Saudi Arabia, and to relate the results to the established clinicopathological prognostic parameters (age of the patient, educational level, birth control methods, number of pregnancy, smoking status, degree of histological differentiation, clinical stage, and lymph node metastasis) The study included 40 specimens of uterine cervical squamous cell carcinomas diagnosed and confirmed by biopsy. Histopathological classification of cervical tumors cases was performed according to the International Federation of Gynecology and Obstetrics (FIGO). Immunohistochemical analysis for HPV16/18-E6 and p16 were carried out on formalin-fixed paraffin-embedded sections of cervical tissues using avidin-biotin peroxidase method. There was a significant statistical correlation between HPV16/18-E6 expression in cervical carcinoma and nationality, smoking status and size of the tumor. HPV16/18-E6 oncoprotein expression in normal lymphocytes and endothelial cells in the tumor tissues and the adjacent normal cervical tissues suggest the possibility that HPV infection might spread to other organs through blood circulation. P16 expression has been correlated with high grade, stage of cervical SCC and HPV16/18-E6 expression. The current study supports the critical function of p16 and HPV16/18-E6 as specific markers for cervical carcinoma. However the potential for usage

  3. Role of p16(INK4a) cytology testing as an adjunct to enhance the diagnostic specificity and accuracy in human papillomavirus-positive women within an organized cervical cancer screening program.

    PubMed

    Gustinucci, Daniela; Passamonti, Basilio; Cesarini, Elena; Butera, Daniela; Palmieri, Emiliano Antonio; Bulletti, Simonetta; Carlani, Angela; Staiano, Maria; D'Amico, Maria Rosaria; D'Angelo, Valentina; Di Dato, Eugenio; Martinelli, Nadia; Malaspina, Morena; Spita, Nicoletta; Tintori, Beatrice; Fulciniti, Franco

    2012-01-01

    We evaluated the performance of cytologic p16(INK4a) (p16) immunostaining within a cervical cancer screening program for the categories of atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LS after triage with high-risk human papillomavirus (HR-HPV) testing and atypical squamous cells, cannot exclude high-grade intraepithelial squamous lesion (ASC-H) and high-grade squamous intraepithelial lesion (HSIL). We also verified whether the routine introduction of p16 staining might enhance the specificity and positive predictive value (PPV) for cervical intraepithelial neoplasia grade 2 or higher (CIN2+) lesions predicted by a cytological screening test. Performance of the p16 cytology test was estimated in 578 cytological samples, of which 213 were HR-HPV+ ASC-US, 186 were HR-HPV+ LSIL, 74 were ASC-H, 56 were HSIL-CIN2 and 49 were HSIL-CIN3. All samples had histological follow-up. In the ASC-US category, p16 sensitivity was 91% for CIN2+ and 100% for CIN3, while specificity was 64 and 58%, respectively, negative predictive value (NPV) was 96 and 100%, respectively, and PPV was 39%. In the LSIL category, sensitivity was 77 and 75%, respectively, for CIN2+ and CIN3, while specificity was 64 and 57%, NPV was 93 and 98% and PPV was 30%. Sensitivity for ASC-H and HSIL-CIN3 was 100% for CIN2+ and CIN3, while for HSIL-CIN2 it was 91 and 95%, respectively; NPV for ASC-H was 100%, and for HSIL-CIN2 it was 43 and 86%, respectively. Follow-up examinations of 8 cases diagnosed as p16+ ASC-H and HSIL-CIN3, but histologically negative or CIN1 on the first biopsy, showed 4 CIN2 and 4 CIN3 lesions. Sensitivity, specificity, PPV and NPV confirm the importance of the utilization of p16 in the categories ASC-US and LSIL after triage with an HR-HPV test. In the ASC-H and HSIL-CIN3 lesions, p16 was shown to be an excellent marker for picking up CIN2+ lesions, especially in cases with cytohistological discordance. Copyright © 2012 S

  4. Role of senescence marker p16 INK4a measured in peripheral blood T-lymphocytes in predicting length of hospital stay after coronary artery bypass surgery in older adults.

    PubMed

    Pustavoitau, Aliaksei; Barodka, Viachaslau; Sharpless, Norman E; Torrice, Chad; Nyhan, Daniel; Berkowitz, Dan E; Shah, Ashish S; Bandeen Roche, Karen J; Walston, Jeremy D

    2016-02-01

    Adults older than 65 years undergo more than 120,000 coronary artery bypass (CAB) procedures each year in the United States. Chronological age alone, though commonly used in prediction models of outcomes after CAB, does not alone reflect variability in aging process; thus, the risk of complications in older adults. We performed a prospective study to evaluate a relationship between senescence marker p16(INK4a) expression in peripheral blood T-lymphocytes (p16 levels in PBTLs) with aging and with perioperative outcomes in older CAB patients. We included 55 patients age 55 and older, who underwent CAB in Johns Hopkins Hospital between September 1st, 2010 and March 25th, 2013. Demographic, clinical and laboratory data following outline of the Society of Thoracic Surgeons data collection form was collected, and p16 mRNA levels in PBTLs were measured using TaqMan® qRT-PCR. Associations between p16 mRNA levels in PBTLs with length of hospital stay, frailty status, p16 protein levels in the aortic and left internal mammary artery tissue, cerebral oxygen saturation, and augmentation index as a measure of vascular stiffness were measured using regression analyses. Length of hospital stay was the primary outcome of interest, and major organ morbidity, mortality, and discharge to a skilled nursing facility were secondary outcomes. In secondary analysis, we evaluated associations between p16 mRNA levels in PBTLs and interleukin-6 levels using regression analyses. Median age of enrolled patients was 63.5 years (range 56-81 years), they were predominantly male (74.55%), of Caucasian descent (85.45%). Median log2(p16 levels in PBTLs) were 4.71 (range 1.10-6.82). P16 levels in PBTLs were significantly associated with chronological age (mean difference 0.06 for each year increase in age, 95% CI 0.01-0.11) and interleukin 6 levels (mean difference 0.09 for each pg/ml increase in IL-6 levels, 95% CI 0.01-0.18). There were no significant associations with frailty status, augmentation

  5. p21(Cip-1/SDI-1/WAF-1) expression via the mitogen-activated protein kinase signaling pathway in insulin-induced chondrogenic differentiation of ATDC5 cells.

    PubMed

    Nakajima, Masahiro; Negishi, Yoichi; Tanaka, Hiroyasu; Kawashima, Kohtaro

    2004-08-06

    The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21(Cip-1/SDI-1/WAF-1), is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory effects of U0126 or stimulatory effects of SP600125 on insulin-induced chondrogenic differentiation were observed when these inhibitors exist in the early phase of differentiation, suggesting that MEK/ERK and JNK act on early phase differentiation. SB202580, however, is necessary not only for early phase but also for late phase differentiation, indicating that p38 MAP kinase stimulates differentiation by acting during the entire period of cultivation. These results for the first time demonstrate that up-regulation of p21 expression by ERK1/2 and p38 MAP kinase is required for chondrogenesis, and that JNK acts as a suppressor of chondrogenesis by down-regulating p21 expression.

  6. p21/Cip1 and p27/Kip1 Are essential molecular targets of inositol hexaphosphate for its antitumor efficacy against prostate cancer.

    PubMed

    Roy, Srirupa; Gu, Mallikarjuna; Ramasamy, Kumaraguruparan; Singh, Rana P; Agarwal, Chapla; Siriwardana, Sunitha; Sclafani, Robert A; Agarwal, Rajesh

    2009-02-01

    Inositol hexaphosphate (IP6) causes G(1) arrest and increases cyclin-dependent kinase inhibitors p21/Cip1 and p27/Kip1 protein levels in human prostate cancer (PCa) DU145 cells lacking functional p53. However, whether cyclin-dependent kinase inhibitor I induction by IP6 plays any role in its antitumor efficacy is unknown. Herein, we observed that either p21 or p27 knockdown by small interfering RNA has no considerable effect on IP6-induced G(1) arrest, growth inhibition, and death in DU145 cells; however, the simultaneous knockdown of both p21 and p27 reversed the effects of IP6. To further confirm these findings both in vitro and in vivo, we generated DU145 cell variants with knockdown levels of p21 (DU-p21), p27 (DU-p27), or both (DU-p21+p27) via retroviral transduction of respective short hairpin RNAs. Knocking down p21 or p27 individually did not alter IP6-caused cell growth inhibition and G(1) arrest; however, their simultaneous ablation completely reversed the effects of IP6. In tumor xenograft studies, IP6 (2% w/v, in drinking water) caused a comparable reduction in tumor volume (40-46%) and tumor cell proliferation (26-28%) in DU-EV (control), DU-p21, and DU-p27 tumors but lost most of its effect in DU-p21+p27 tumors. IP6-caused apoptosis also occurred in a Cip/Kip-dependent manner because DU-p21+p27 cells were completely resistant to IP6-induced apoptosis both in cell culture and xenograft. Together, these results provide evidence, for the first time, of the critical role of p21 and p27 in mediating the anticancer efficacy of IP6, and suggest their redundant role in the antiproliferative and proapoptotic effects of IP6 in p53-lacking human PCa cells, both in vitro and in vivo.

  7. Pharmacodynamic study of the 7,8-dihydroxy-4-methylcoumarin-induced selective cytotoxicity toward U-937 leukemic cells versus mature monocytes: cytoplasmic p21(Cip1/WAF1) as resistance factor.

    PubMed

    Vázquez, Ramiro; Riveiro, María Eugenia; Mondillo, Carolina; Perazzo, Juan Carlos; Vermeulen, Mónica; Baldi, Alberto; Davio, Carlos; Shayo, Carina

    2013-07-15

    The development of tumor-selective drugs with low systemic toxicity has always been a major challenge in cancer treatment. Our group previously identified the 7,8-dihydroxy-4-methylcoumarin (DHMC) as a potential chemotherapeutic agent due to its potent, selective anti-proliferative and apoptosis-inducing effects on several cancer cell lines over peripheral blood mononuclear cells. However, there are still no published reports that can explain such selectivity of action. Herein, we addressed this question by using the U-937 promonocytic leukemia cell line, which can be forced to differentiate into a monocyte-like phenotype in vitro. U-937 cells differentiation is dependent on the nuclear expression of p21(Cip1/WAF1), a protein that is absent in immature U-937 cells but present in both the nucleus and the cytoplasm of normal DHMC-resistant monocytes. Considering that induction of differentiation rendered U-937 cells resistant to DHMC, we evaluated the possible causal role of cytoplasmic p21(Cip1/WAF1) in the onset of such resistance by employing U-937 cells stably transfected with a ZnCl2-inducible p21(Cip1/WAF1) variant lacking the nuclear localization signal (U-937/CB6-ΔNLS-p21 cells). Expression of cytoplasmic p21(Cip1/WAF1) did not induce differentiation of the cells but turned them resistant to DHMC through inhibition of JNK, a crucial mediator of DHMC-induced apoptosis in U-937 cells. Sub-acute toxicity evaluation of DHMC in Balb/c mice indicated that DHMC administered intraperitoneally at doses up to 100mg/kg induced no systemic damage. Collectively, our results explain for the first time the selective cytotoxicity of DHMC for tumor cells over normal monocytes, and encourage further in vivo studies on this compound as potential anti-leukemic agent.

  8. Risk of high-grade cervical intraepithelial neoplasia during follow-up in HPV-positive women according to baseline p16-INK4A results: a prospective analysis of a nested substudy of the NTCC randomised controlled trial.

    PubMed

    Carozzi, Francesca; Gillio-Tos, Anna; Confortini, Massimo; Del Mistro, Annarosa; Sani, Cristina; De Marco, Laura; Girlando, Salvatore; Rosso, Stefano; Naldoni, Carlo; Dalla Palma, Paolo; Zorzi, Manuel; Giorgi-Rossi, Paolo; Segnan, Nereo; Cuzick, Jack; Ronco, Guglielmo

    2013-02-01

    Immunostaining for p16-INK4A (henceforth p16) is a sensitive and specific method for detection of high-grade cervical intraepithelial neoplasia (CIN) in women infected with human papillomavirus (HPV), but longitudinal data have not been obtained. We investigated the relation between p16 status and risk of CIN during 3 years of follow-up. Women aged 25-60 years were enrolled between June 10, 2003, and Dec 31, 2004, in a multicentre randomised trial comparing HPV testing with cytology. HPV-positive women were referred for colposcopy and, in seven of nine centres, were tested for p16 overexpression by immunostaining. If no CIN was detected, these women were followed up at yearly intervals until clearance of HPV infection. The primary endpoint was histologically confirmed CIN of grade 2 or worse (CIN of grade 2 [CIN2], CIN of grade 3 [CIN3], or invasive cervical cancer) at recruitment or during follow-up. We calculated the absolute and relative risks by p16 status at recruitment. We also calculated the longitudinal sensitivity of p16 testing. Additionally, we assessed the relative sensitivity of an alternative strategy (referral to colposcopy and follow-up of only HPV-positive, p16-positive women) versus conventional cytology in two age groups. Percentages were weighted by the inverse of the tested fraction. The trial in which this study is nested is registered, number ISRCTN81678807. Of 1042 HPV-positive women who were tested for p16 with no CIN detected during the first round of screening, 944 (91%) had further HPV tests. 793 (84%) of these 944 were followed up until detection of CIN2 or worse, HPV infection clearance, or for at least 3 years. CIN2 or worse was detected during follow-up in more p16-positive women (31 of 365, 8·8% [95% CI 5·8-11·8]) than in p16-negative women (17 of 579, 3·7% [1·9-5·4]; relative risk [RR] 2·61 [95% CI 1·49-4·59]). RR was higher in women aged 35-60 years at recruitment (3·37 [1·39-8·15]) than in those aged 25-34 years (2

  9. Cytoplasmic p21(CIP1/WAF1), ERK1/2 activation, and cytoskeletal remodeling are associated with the senescence-like phenotype after airborne particulate matter (PM(10)) exposure in lung cells.

    PubMed

    Sánchez-Pérez, Yesennia; Chirino, Yolanda I; Osornio-Vargas, Álvaro Román; Herrera, Luis A; Morales-Bárcenas, Rocío; López-Saavedra, Alejandro; González-Ramírez, Imelda; Miranda, Javier; García-Cuellar, Claudia María

    2014-02-10

    The exposure to particulate matter with a mean aerodynamic diameter ≤10 μm (PM10) from urban zones is considered to be a risk factor in the development of cancer. The aim of this work was to determine if PM10 exposure induces factors related to the acquisition of a neoplastic phenotype, such as cytoskeletal remodeling, changes in the subcellular localization of p21(CIP1/WAF1), an increase in β-galactosidase activity and changes in cell cycle. To test our hypothesis, PM10 from an industrial zone (IZ) and a commercial zone (CZ) were collected, and human adenocarcinoma lung cell cultures (A549) were exposed to a sublethal PM10 concentration (10 μg/cm(2)) for 24 h and 48 h. The results showed that PM10 exposure induced an increase in F-actin stress fibers and caused the cytoplasmic stabilization of p21(CIP1/WAF1) via phosphorylation at Thr(145) and Ser(146) and the phosphorylation of ERK1/2 on Thr(202). Changes in the cell cycle or apoptosis were not observed, but an increase in β-galactosidase activity was detected. The PM10 from CZ caused more dramatic effects in lung cells. We conclude that PM10 exposure induced cytoplasmic p21(CIP1/WAF1) retention, ERK1/2 activation, cytoskeleton remodeling and the acquisition of a senescence-like phenotype in lung cells. These alterations could have mechanistic implications regarding the carcinogenic potential of PM10. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1.

    PubMed

    Lin, Hui-Ping; Lin, Ching-Yu; Huo, Chieh; Hsiao, Ping-Hsuan; Su, Liang-Cheng; Jiang, Shih Sheng; Chan, Tzu-Min; Chang, Chung-Ho; Chen, Li-Tzong; Kung, Hsing-Jien; Wang, Horng-Dar; Chuu, Chih-Pin

    2015-03-30

    Prostate cancer (PCa) patients receiving the androgen ablation therapy ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1-3 years. Treatment with caffeic acid phenethyl ester (CAPE) suppressed cell survival and proliferation via induction of G1 or G2/M cell cycle arrest in LNCaP 104-R1, DU-145, 22Rv1, and C4-2 CRPC cells. CAPE treatment also inhibited soft agar colony formation and retarded nude mice xenograft growth of LNCaP 104-R1 cells. We identified that CAPE treatment significantly reduced protein abundance of Skp2, Cdk2, Cdk4, Cdk7, Rb, phospho-Rb S807/811, cyclin A, cyclin D1, cyclin H, E2F1, c-Myc, SGK, phospho-p70S6kinase T421/S424, phospho-mTOR Ser2481, phospho-GSK3α Ser21, but induced p21Cip1, p27Kip1, ATF4, cyclin E, p53, TRIB3, phospho-p53 (Ser6, Ser33, Ser46, Ser392), phospho-p38 MAPK Thr180/Tyr182, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and phospho-p90RSK Ser380. CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group. Overexpression of Skp2, or siRNA knockdown of p21Cip1, p27Kip1, or p53 blocked suppressive effect of CAPE treatment. Co-treatment of CAPE with PI3K inhibitor LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects. Our finding suggested that CAPE treatment induced cell cycle arrest and growth inhibition in CRPC cells via regulation of Skp2, p53, p21Cip1, and p27Kip1.

  11. 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.

  12. Suppression of Transforming Growth Factor-β Signaling Delays Cellular Senescence and Preserves the Function of Endothelial Cells Derived from Human Pluripotent Stem Cells.

    PubMed

    Bai, Hao; Gao, Yongxing; Hoyle, Dixie L; Cheng, Tao; Wang, Zack Z

    2017-02-01

    Transplantation of vascular cells derived from human pluripotent stem cells (hPSCs) offers an attractive noninvasive method for repairing the ischemic tissues and for preventing the progression of vascular diseases. Here, we found that in a serum-free condition, the proliferation rate of hPSC-derived endothelial cells is quickly decreased, accompanied with an increased cellular senescence, resulting in impaired gene expression of endothelial nitric oxide synthase (eNOS) and impaired vessel forming capability in vitro and in vivo. To overcome the limited expansion of hPSC-derived endothelial cells, we screened small molecules for specific signaling pathways and found that inhibition of transforming growth factor-β (TGF-β) signaling significantly retarded cellular senescence and increased a proliferative index of hPSC-derived endothelial cells. Inhibition of TGF-β signaling extended the life span of hPSC-derived endothelial and improved endothelial functions, including vascular network formation on Matrigel, acetylated low-density lipoprotein uptake, and eNOS expression. Exogenous transforming growth factor-β1 increased the gene expression of cyclin-dependent kinase inhibitors, p15(Ink4b) , p16(Ink4a) , and p21(CIP1) , in endothelial cells. Conversely, inhibition of TGF-β reduced the gene expression of p15(Ink4b) , p16(Ink4a) , and p21(CIP1) . Our findings demonstrate that the senescence of newly generated endothelial cells from hPSCs is mediated by TGF-β signaling, and manipulation of TGF-β signaling offers a potential target to prevent vascular aging. Stem Cells Translational Medicine 2017;6:589-600.

  13. Suppression of Transforming Growth Factor-β Signaling Delays Cellular Senescence and Preserves the Function of Endothelial Cells Derived From Human Pluripotent Stem Cells.

    PubMed

    Bai, Hao; Gao, Yongxing; Hoyle, Dixie L; Cheng, Tao; Wang, Zack Z

    2016-09-20

    : Transplantation of vascular cells derived from human pluripotent stem cells (hPSCs) offers an attractive noninvasive method for repairing the ischemic tissues and for preventing the progression of vascular diseases. Here, we found that in a serum-free condition, the proliferation rate of hPSC-derived endothelial cells is quickly decreased, accompanied with an increased cellular senescence, resulting in impaired gene expression of endothelial nitric oxide synthase (eNOS) and impaired vessel forming capability in vitro and in vivo. To overcome the limited expansion of hPSC-derived endothelial cells, we screened small molecules for specific signaling pathways and found that inhibition of transforming growth factor-β (TGF-β) signaling significantly retarded cellular senescence and increased a proliferative index of hPSC-derived endothelial cells. Inhibition of TGF-β signaling extended the life span of hPSC-derived endothelial and improved endothelial functions, including vascular network formation on Matrigel, acetylated low-density lipoprotein uptake, and eNOS expression. Exogenous transforming growth factor-β1 increased the gene expression of cyclin-dependent kinase inhibitors, p15(Ink4b), p16(Ink4a), and p21(CIP1), in endothelial cells. Conversely, inhibition of TGF-β reduced the gene expression of p15(Ink4b), p16(Ink4a), and p21(CIP1). Our findings demonstrate that the senescence of newly generated endothelial cells from hPSCs is mediated by TGF-β signaling, and manipulation of TGF-β signaling offers a potential target to prevent vascular aging.

  14. Eukaryotic translation initiator protein 1A isoform, CCS-3, enhances the transcriptional repression of p21CIP1 by proto-oncogene FBI-1 (Pokemon/ZBTB7A).

    PubMed

    Choi, Won-Il; Kim, Youngsoo; Kim, Yuri; Yu, Mi-young; Park, Jungeun; Lee, Choong-Eun; Jeon, Bu-Nam; Koh, Dong-In; Hur, Man-Wook

    2009-01-01

    FBI-1, a member of the POK (POZ and Kruppel) family of transcription factors, plays a role in differentiation, oncogenesis, and adipogenesis. eEF1A is a eukaryotic translation elongation factor involved in several cellular processes including embryogenesis, oncogenic transformation, cell proliferation, and cytoskeletal organization. CCS-3, a potential cervical cancer suppressor, is an isoform of eEF1A. We found that eEF1A forms a complex with FBI-1 by co-immunoprecipitation, SDS-PAGE, and MALDI-TOF Mass analysis of the immunoprecipitate. GST fusion protein pull-downs showed that FBI-1 directly interacts with eEF1A and CCS-3 via the zinc finger and POZ-domain of FBI-1. FBI-1 co-localizes with either eEF1A or CCS-3 at the nuclear periplasm. CCS-3 enhances transcriptional repression of the p21CIP1 gene (hereafter referred to as p21) by FBI-1. The POZ-domain of FBI-1 interacts with the co-repressors, SMRT and BCoR. We found that CCS-3 also interacts with the co-repressors independently. The molecular interaction between the co-repressors and CCS-3 at the POZ-domain of FBI-1 appears to enhance FBI-1 mediated transcriptional repression. Our data suggest that CCS-3 may be important in cell differentiation, tumorigenesis, and oncogenesis by interacting with the proto-oncogene FBI-1 and transcriptional co-repressors.

  15. Correlation among 16 biological factors [p53, p21(waf1), MIB-1 (Ki-67), p16(INK4A), cyclin D1, E-cadherin, Bcl-2, TNF-α, NF-κB, TGF-β, MMP-7, COX-2, EGFR, HER2/neu, ER, and HIF-1α] and clinical outcomes following curative chemoradiation therapy in 10 patients with esophageal squamous cell carcinoma.

    PubMed

    Shibata-Kobayashi, Shino; Yamashita, Hideomi; Okuma, Kae; Shiraishi, Kenshiro; Igaki, Hiroshi; Ohtomo, Kuni; Nakagawa, Keiichi

    2013-03-01

    The expression levels of 16 proteins were analyzed to identify prognostic correlations in esophageal squamous cell carcinoma (ESCC) treated with concurrent chemoradiation therapy (CCRT). The immunohistochemical expression levels of p53, p21(waf1), molecular immunology borstel-1 (MIB-1, Ki-67), p16(INK4A), cyclin D1, E-cadherin, Bcl-2, tumor necrosis factor (TNF)-α, nuclear factor (NF)-κB, transforming growth factor (TGF)-β, matrix metalloproteinase (MMP)-7, cyclooxygenase (COX)-2, epidermal growth factor receptor (EGFR), human EGFR type 2 (HER2/neu), estrogen receptor (ER) and hypoxia-inducible factor (HIF)-1α were studied in 10 cases of ESCC treated with CCRT. The patients underwent CCRT between 2000 and 2010. The mean patient age was 68.1 years (range, 46-80 years). The numbers of patients in stages I, II, III and IV of the disease were 2, 2, 3 and 3, respectively. Of the tumors, 8 were positive for p53, 6 for p21(waf1, 7) for MIB-1 (Ki-67), 7 for p16(INK4A), 7 for cyclin D1, 8 for E-cadherin, 3 for Bcl-2, 0 for TNF-α, 5 for NF-κB, 7 for TGF-β, 9 for MMP-7, 7 for COX-2, 5 for EGFR, 1 for HER2/neu, 1 for ER and 7 for HIF-1α. The 2-year overall survival rate of patients expressing high levels of MIB-1 was 71% (±17%) compared with 0% (P=0.019) for those expressing low levels. For NF-κB, the rate was 0% for patients with high levels compared with 100% (P<0.018) for those with low levels. The 2-year local control rates of HER2/neu were 0% in patients expressing high levels and 88% (±12%) in patients expressing low levels (P=0.027). The 2-year disease-free survival rates of HER2/neu and ER were 0% for patients expressing high levels compared with 56% (±17%) for those with low levels (P=0.027). There were no significant correlations between the expression levels of the other proteins and clinical outcomes. In the present study, high levels of MIB-1 and low levels of NF-κB, HER2 and ER were shown to be good prognostic factors following definitive CCRT for

  16. Development of Novel p16INK4a Mimetics as Anticancer Therapy

    DTIC Science & Technology

    2014-10-01

    Initiative Schorer (PI) 10/1/12-8/1/14 Veterans Affairs’ Central Office Subproject Title: Palliative Care in Head and Neck Cancer Patients...The major goal of this subproject is to determine feasibility of incorporating palliative care for head and neck cancer patients undergoing active

  17. Targeted p16Ink4a epimutation causes tumorigenesis and reduces survival in mice

    USDA-ARS?s Scientific Manuscript database

    Cancer has long been viewed as a genetic disease; however, epigenetic silencing as the result of aberrant promoter DNA methylation is frequently associated with cancer development, suggesting an epigenetic component to the disease. Nonetheless, it has remained unclear whether an epimutation (an aber...

  18. Absence of p16INK4a and truncation of ARF tumor suppressors in chickens

    PubMed Central

    Kim, Soo-Hyun; Mitchell, Michael; Fujii, Hideta; Llanos, Susana; Peters, Gordon

    2003-01-01

    The INK4b-ARF-INK4a locus on human chromosome 9p21 (Human Genome Organization designation CDKN2B-CDKN2A), and the corresponding locus on mouse chromosome 4, encodes three distinct products: two members of the INK4 cyclin-dependent kinase inhibitor family and a completely unrelated protein, ARF, whose carboxyl-terminal half is specified by the second exon of INK4a but in an alternative reading frame. As INK4 proteins block the phosphorylation of the retinoblastoma gene product and ARF protects p53 from degradation, the locus plays a key role in tumor suppression and the control of cell proliferation. To gain further insights into the relative importance of INK4a and ARF in different settings, we have isolated and characterized the equivalent locus in chickens. Surprisingly, although we identified orthologues of INK4b and ARF, chickens do not encode an equivalent of INK4a. Moreover, the reading frame for chicken ARF does not extend into exon 2, because splicing occurs in a different register to that used in mammals. The resultant 60-aa product nevertheless shares functional attributes with its mammalian counterparts. As well as indicating that the locus has been subject to dynamic evolutionary pressures, these unexpected findings suggest that in chickens, the tumor-suppressor functions of INK4a have been compensated for by other genes. PMID:12506196

  19. Mechanisms of MEOX1 and MEOX2 regulation of the cyclin dependent kinase inhibitors p21 and p16 in vascular endothelial cells.

    PubMed

    Douville, Josette M; Cheung, David Y C; Herbert, Krista L; Moffatt, Teri; Wigle, Jeffrey T

    2011-01-01

    Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21(CIP1/WAF1) and p16(INK4a) govern the G(1)/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21(CIP1/WAF1) and p16(INK4a). MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21(CIP1/WAF1) and p16(INK4a) expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21(CIP1/WAF1) and p16(INK4a). In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16(INK4a) in a DNA binding dependent manner, whereas they induce p21(CIP1/WAF1) in a DNA binding independent manner.

  20. Role of quantitative p16(INK4A) mRNA assay and digital reading of p16(INK4A) immunostained sections in diagnosis of cervical intraepithelial neoplasia.

    PubMed

    Vasiljević, Nataša; Carter, Paul D; Reuter, Caroline; Warman, Rhian; Brentnall, Adam R; Carton, James R; Cuzick, Jack; Lorincz, Attila T

    2017-08-15

    Visual interpretation of cervical biopsies is subjective and variable, generally showing fair to moderate inter-reader agreement in distinguishing high from low grade cervical intraepithelial neoplasia (CIN). We investigated the performance of two objective p16 quantitative tests in comparison with visual assessment: (i) p16-mRNA assay and (ii) digital analysis of sections stained for p16 protein. The primary analysis considered 232 high-risk human papilloma virus positive (HPV+) samples from diagnostic cervical specimens. A p16 RT-qPCR (p16-mRNA assay) was run on mRNA extracted from formalin-fixed paraffin-embedded sections. Two p16 immunohistochemistry (IHC) readings, a visual read by a histopathologist (Visual IHC) and a digital read of a high-resolution scan (Digital IHC), were done on adjacent sections. The worst reviewed CIN grade (agreed by at least two histopathologists) from up to two biopsies and a loop excision was taken, with CIN2/3 as the primary endpoint. Visual IHC attained a specificity of 70% (95%CI 61-77) for 85% (95%CI 77-91%) sensitivity. The four-point Visual IHC staining area under the curve (AUC) was 0.77 (95%CI 0.71-0.82), compared with 0.71 (95%CI 0.64-0.77) for p16-mRNA and 0.67 (95%CI 0.60-0.74) for Digital IHC. Spearman rank-order correlations were: visual to p16-mRNA 0.41, visual to digital 0.49 and p16-mRNA to digital: 0.22. The addition of p16-mRNA assay to visual reading of p16 IHC improved the AUC from 0.77 to 0.84 (p = 0.0049). p16-mRNA testing may be complementary to visual IHC p16 staining for a more accurate diagnosis of CIN, or perhaps a substitute in locations with a lack of skilled pathologists. © 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

  1. Cooperativity of Oncogenic K-Ras and Downregulated p16/INK4A in Human Pancreatic Tumorigenesis

    PubMed Central

    Ling, Jianhua; Zhuang, Zhuonan; Li, Zhongkui; Wang, Huamin; Fleming, Jason B.; Freeman, James W.; Yu, Dihua; Huang, Peng; Chiao, Paul J.

    2014-01-01

    Activation of K-ras and inactivation of p16 are the most frequently identified genetic alterations in human pancreatic epithelial adenocarcinoma (PDAC). Mouse models engineered with mutant K-ras and deleted p16 recapitulate key pathological features of PDAC. However, a human cell culture transformation model that recapitulates the human pancreatic molecular carcinogenesis is lacking. In this study, we investigated the role of p16 in hTERT-immortalized human pancreatic epithelial nestin-expressing (HPNE) cells expressing mutant K-ras (K-rasG12V). We found that expression of p16 was induced by oncogenic K-ras in these HPNE cells and that silencing of this induced p16 expression resulted in tumorigenic transformation and development of metastatic PDAC in an orthotopic xenograft mouse model. Our results revealed that PI3K/Akt, ERK1/2 pathways and TGFα signaling were activated by K-ras and involved in the malignant transformation of human pancreatic cells. Also, p38/MAPK pathway was involved in p16 up-regulation. Thus, our findings establish an experimental cell-based model for dissecting signaling pathways in the development of human PDAC. This model provides an important tool for studying the molecular basis of PDAC development and gaining insight into signaling mechanisms and potential new therapeutic targets for altered oncogenic signaling pathways in PDAC. PMID:25029561

  2. Cooperativity of oncogenic K-ras and downregulated p16/INK4A in human pancreatic tumorigenesis.

    PubMed

    Chang, Zhe; Ju, Huaiqiang; Ling, Jianhua; Zhuang, Zhuonan; Li, Zhongkui; Wang, Huamin; Fleming, Jason B; Freeman, James W; Yu, Dihua; Huang, Peng; Chiao, Paul J

    2014-01-01

    Activation of K-ras and inactivation of p16 are the most frequently identified genetic alterations in human pancreatic epithelial adenocarcinoma (PDAC). Mouse models engineered with mutant K-ras and deleted p16 recapitulate key pathological features of PDAC. However, a human cell culture transformation model that recapitulates the human pancreatic molecular carcinogenesis is lacking. In this study, we investigated the role of p16 in hTERT-immortalized human pancreatic epithelial nestin-expressing (HPNE) cells expressing mutant K-ras (K-rasG12V). We found that expression of p16 was induced by oncogenic K-ras in these HPNE cells and that silencing of this induced p16 expression resulted in tumorigenic transformation and development of metastatic PDAC in an orthotopic xenograft mouse model. Our results revealed that PI3K/Akt, ERK1/2 pathways and TGFα signaling were activated by K-ras and involved in the malignant transformation of human pancreatic cells. Also, p38/MAPK pathway was involved in p16 up-regulation. Thus, our findings establish an experimental cell-based model for dissecting signaling pathways in the development of human PDAC. This model provides an important tool for studying the molecular basis of PDAC development and gaining insight into signaling mechanisms and potential new therapeutic targets for altered oncogenic signaling pathways in PDAC.

  3. Reactive oxygen-induced carcinogenesis causes hypermethylation of p16(Ink4a) and activation of MAP kinase.

    PubMed Central

    Govindarajan, Baskaran; Klafter, Robert; Miller, Mark Steven; Mansur, Claire; Mizesko, Melissa; Bai, Xianhe; LaMontagne, Kenneth; Arbiser, Jack L.

    2002-01-01

    BACKGROUND: Implantation of foreign materials into mice and humans has been noted to result in the appearance of soft tissue sarcomas at the site of implantation. These materials include metal replacement joints and Dacron vascular grafts. In addition, occupational exposure to nickel has been shown to result in an increased risk of carcinogenesis. The molecular mechanisms of foreign body-induced carcinogenesis are not fully understood. MATERIALS AND METHODS: In order to gain insight into these mechanisms, we implanted nickel sulfide into wild type C57BL/6 mice as well as a mouse heterozygous for the tumor suppressor gene, p53. Malignant fibrous histiocytomas arose in all mice, and we have characterized the profile of tumor suppressor genes and signal transduction pathways altered in these cells. RESULTS: All tumors demonstrated hypermethylation of the tumor suppressor gene p16, as well as activation of the mitogen activated protein kinase (MAP kinase) signaling pathway. This knowledge may be beneficial in the prevention and treatment of tumors caused by foreign body implantation. CONCLUSIONS: Oxidative stress induced by nickel sulfide appears to cause loss of p16 and activation of MAP kinase signaling. These findings support the hypothesis of synergistic interactions between MAP kinase activation and p16 loss in carcinogenesis. PMID:11984000

  4. p16INK4A inactivation mechanisms in non-small-cell lung cancer patients occupationally exposed to asbestos.

    PubMed

    Andujar, Pascal; Wang, Jinhui; Descatha, Alexis; Galateau-Sallé, Françoise; Abd-Alsamad, Issam; Billon-Galland, Marie-Annick; Blons, Hélène; Clin, Bénédicte; Danel, Claire; Housset, Bruno; Laurent-Puig, Pierre; Le Pimpec-Barthes, Françoise; Letourneux, Marc; Monnet, Isabelle; Régnard, Jean-François; Renier, Annie; Zucman-Rossi, Jessica; Pairon, Jean-Claude; Jaurand, Marie-Claude

    2010-01-01

    Epidemiological studies have shown that asbestos fibers constitute the major occupational risk factor and that asbestos acts synergistically with tobacco smoking to induce lung cancer. Although some somatic gene alterations in lung cancer have been linked to tobacco smoke, few data are available on the role of asbestos fibers. P16/CDKN2A is an important tumor suppressor gene that is frequently altered in lung cancer via promoter 5'-CpG island hypermethylation and homozygous deletion, and rarely via point mutation. Many studies suggest that tobacco smoking produces P16/CDKN2A promoter hypermethylation in lung cancer, but the status of this gene in relation to asbestos exposure has yet to be determined. The purpose of this study was to investigate the mechanism of P16/CDKN2A alterations in lung cancer in asbestos-exposed patients. P16/CDKN2A gene status was studied in 75 human non-small-cell lung cancer (NSCLC) cases with well-defined smoking habits, and detailed assessment of asbestos exposure, based on occupational questionnaire and determination of asbestos bodies in lung tissue. The results of this study confirm published data on the effect of tobacco smoke on P16/CDKN2A gene alterations, characterized by significantly higher P16/CDKN2A promoter hypermethylation in heavy smokers (more than 40 pack-years (P-Y)) than in smokers of less than 40 P-Y. These results also demonstrate a higher incidence of loss of heterozygosity and homozygous deletion in asbestos-exposed cases, after adjustment for age and cumulative tobacco consumption, than in unexposed cases (P=0.0062). This study suggests that P16/CDKN2A gene inactivation in asbestos-exposed NSCLC cases mainly occurs via deletion, a feature also found in malignant mesothelioma, a tumor independent of tobacco smoking but associated with asbestos exposure, suggesting a possible relationship with an effect of asbestos fibers.

  5. Early events in DNA replication require cyclin E and are blocked by p21CIP1

    PubMed Central

    1995-01-01

    Using immunodepletion of cyclin E and the inhibitor protein p21WAF/CIP1, we demonstrate that the cyclin E protein, in association with Cdk2, is required for the elongation phase of replication on single-stranded substrates. Although cyclin E/Cdk2 is likely to be the major target by which p21 inhibits the initiation of sperm DNA replication, p21 can inhibit single-stranded replication through a mechanism dependent on PCNA. While the cyclin E/Cdk2 complex appears to have a role in the initiation of DNA replication, another Cdk kinase, possibly cyclin A/Cdk, may be involved in a later step controlling the switch from initiation to elongation. The provision of a large maternal pool of cyclin E protein shows that regulators of replication are constitutively present, which explains the lack of a protein synthesis requirement for replication in the early embryonic cell cycle. PMID:7642695

  6. High prevalence of the G101W germline mutation in the CDKN2A (P16(ink4a)) gene in 62 Italian malignant melanoma families.

    PubMed

    Mantelli, Michela; Barile, Monica; Ciotti, Paola; Ghiorzo, Paola; Lantieri, Francesca; Pastorino, Lorenza; Catricalà, Caterina; Torre, Gabriella Della; Folco, Ugo; Grammatico, Paola; Padovani, Laura; Pasini, Barbara; Rovini, Dario; Queirolo, Paola; Rainero, Maria Luisa; Santi, Pier Luigi; Sertoli, Roberto M; Goldstein, Alisa M; Bianchi-Scarrà, Giovanna

    2002-01-22

    CDKN2A germline mutation frequency estimates are commonly based on families with several melanoma cases. When we started counseling in a research setting on gene susceptibility analysis in northern and central Italy, however, we mostly found small families with few cases. Here we briefly characterize those kindred, estimate CDKN2A/CDK4 mutation test yields, and provide indications on the possibility of implementing formal DNA testing for melanoma-prone families in Italy. In September 1995 we started genetic counseling in a research setting at our Medical Genetics Center. Screening for CDKN2A/CDK4 mutations was performed on families with two melanoma patients, one of whom was younger than 50 years at onset, the other complying with one of the following: 1) being a first-degree relative, 2) having an additional relative with pancreatic cancer, or 3) having multiple primary melanomas. Sixty-two of 67 (80%) melanoma cases met our criteria. Four previously described CDKN2A mutations (G101W, R24P, V126D, and N71S) were found in 21 of the 62 families (34%) with a high prevalence of G101W (18/21). The percentage of families with two melanoma cases/family harboring a mutation was low (7%, 2/27), but rose to 45% (9/20) if one of the melanoma patients carried multiple melanomas or if pancreatic cancer was present in that family. In the 15 families with three melanoma cases the presence of a mutation was higher (67%, 10/15) and reached 100% in the 4 families with four or more melanoma cases. Our results suggest that CDKN2A/CDK4 counseling-based mutational analysis may be reasonably efficient also for families with two melanoma cases, if one patient carries multiple melanomas or if pancreatic cancer is present in the family. Copyright 2002 Wiley-Liss, Inc.

  7. P16INK4a Immunostaining but Lack of Human Papilloma Virus Type 16 in Cutaneous Squamous Cell Carcinoma and Basal Cell Carcinoma: a Report from West Iran.

    PubMed

    Ramezani, Mazaher; Abdali, Elham; Khazaei, Sedigheh; Vaisi-Raygani, Asad; Sadeghi, Masoud

    2016-01-01

    The tumor suppressor p16 is a biomarker for transforming human papilloma virus (HPV) infections that can lead to contradictory results in skin carcinomas. The aim of this study was to evaluate p16 expression and HPV-16 infection in the cutaneous basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). This case-control study was performed on paraffin blocks of BCCs and SCCs and normal skin (53, 36, and 44 cases, respectively), between 2006 to 2015. Initial sections for groups were stained with hematoxylin and eosin (H and E). Immunohistochemistry was performed for p16 expression and human papilloma virus type 16 (HPV-16) infection. Normal group was skin of mammoplasty specimens and normal skin tissue in the periphery of tumors. The mean age at diagnosis was 42.1, 61.7 and 71.4 years for normal, BCC and SCC groups, respectively. P16 positivity was more in SCC and BCC groups compared to normal group (P<0.05) and HPV was negative in all patients in three groups. Also, the mean age at diagnosis and P16-positivity were higher for the SCC group than the BCC group (P<0.005). In conclusion, in non-melanoma skin cancers (SCC and BCC), p16-positivity can be a prognostic factor but there is no correlation between HPV-16 and p16 in these tumors.

  8. High-risk HPV genotypes and P16INK4a expression in a cohort of head and neck squamous cell carcinoma patients in Singapore.

    PubMed

    Tan, Louise Soo Yee; Fredrik, Petersson; Ker, Liang; Yu, Feng Gang; Wang, De Yun; Goh, Boon Cher; Loh, Kwok Seng; Lim, Chwee Ming

    2016-12-27

    Human papillomavirus (HPV), especially HPV16 genotype, is associated with oropharyngeal squamous cell carcinoma (OPSCC). We aim to determine the prevalence and characterize the high-risk (HR)-HPV genotypes in head and neck SCC (HNSCC) in a South-East Asian multi-ethnic society in Singapore and examine its prognostic significance.159 HNSCC archival tissue samples were retrieved and tumour DNA was screened for 18 HR-HPV genotypes using a PCR-based assay (Qiagen, digene HPV genotyping RH test). P16 protein overexpression was identified using immunohistochemistry (IHC). Statistical correlation between clinical outcomes were performed between HPV-positive and negative HNSCC patients.Six HR-HPVs (HPV16, 18, 31, 45, 56, 68) were detected in 90.6% of HNSCC; and 79.9% had multiple HPV genotypes detected. HPV31 and HPV45 were the most prevalent (79.2% and 87.4%, respectively); and HPV16 was predominantly found in OPSCC (p < 0.001). HPV-DNA PCR assay yielded a high sensitivity (96%) but low specificity (11%) when compared to p16 immunohistochemistry as the reference standard.P16-positive HNSCC was predominantly observed in OPSCC (73.7%; p = 0.005); and p16-positive OPSCC exhibited improved overall survival compared to p16-negative OPSCC (p = 0.022). Similarly, smoking and alcohol consumption were poor prognostic factors of overall survival (p = 0.007; p = 0.01) in OPSCC patients.HR-HPVs were identified in 90.6% of HNSCC patients using the HPV-DNA PCR assay. This test had a poor specificity when compared to p16 IHC; making it an unreliable detection technique in selecting patients for radiation dose de-escalation treatment protocol. P16-positive tumor was predominantly found in the oropharynx these patients demonstrated better overall survival than those with p16-negative OPSCC.

  9. Dissection of CDK4-binding and transactivation activities of p34(SEI-1) and comparison between functions of p34(SEI-1) and p16(INK4A).

    PubMed

    Li, Junan; Muscarella, Peter; Joo, Sang Hoon; Knobloch, Thomas J; Melvin, W Scott; Weghorst, Christopher M; Tsai, Ming-Daw

    2005-10-11

    Recent studies showed that p34(SEI-1), also known as TRIP-Br1 or SEI-1, plays a dual role in the regulation of cell-cycle progression. It exhibits the transactivation activity and regulates a number of genes required for G1/S transition, while it also binds and activates cyclin-dependent kinase 4 (CDK4) independent of the inhibitory activity of p16. The goals of this paper are to further dissect the two roles and to compare the functions between SEI-1 and p16. (i) Yeast one-hybrid-based random mutagenesis was first used to identify a number of SEI-1 residues important for LexA-mediated transactivation, including residues L51, K52, L53, H54, L57, and L69 located within the heptad repeat (residues 30-88), a domain required for LexA-mediated transactivation, and two residues M219 and L228 at the C-terminal segment that contributes to transactivation through modulating the heptad repeat. (ii) The functional significance of these residues was further confirmed by site-directed mutagenesis. It was also shown that the heptad repeat-involving transactivation is distinct from the well-known acidic region-involving transactivation. (iii) Yeast two-hybrid-based binding analysis was made possible with the transactivation-negative SEI-1 mutants, and the results showed that some of such mutants retain full ability to bind and activate CDK4. (iv) Site-specific mutants of CDK4 were used to show that there are notable differences among SEI-1, p16, and cyclin D2 in binding to CDK4. (v) The expression levels of SEI-1 and p16 were compared in 32 tumor specimens of human squamous cell carcinomas of the head and neck. The results indicate that SEI-1 was consistently overexpressed, while p16 was consistently underexpressed. These results provide important information on the molecular mechanism of the functions of SEI-1 and on the comparison between SEI-1 and p16 at both molecular and cellular levels.

  10. Epstein-Barr virus and p16INK4A methylation in squamous cell carcinoma and precancerous lesions of the cervix uteri.

    PubMed

    Kim, Na Rae; Lin, Zhenhua; Kim, Kyong Rae; Cho, Hyun Yee; Kim, Insun

    2005-08-01

    Methylation of p16 is an important mechanism in cervical carcinogenesis. However, the relationship between cervical squamous cell carcinoma (SCC) and Epstein-Barr virus (EBV) remains controversial. Here, we explored whether EBV infection and/or p16 gene inactivation would play any role in cervical carcinogenesis. Eighty-two specimens included 41 invasive SCCs, 30 cervical intraepithelial neoplasm (CIN; CIN 1, 11 cases, CIN II, 3 cases, CIN III 16 cases) and 11 nonneoplastic cervices. EBV was detected by polymerase chain reaction (PCR) for EBNA-1 and in situ hybridization for EBER-1. The p16 methylation-status and the expression of p16 protein were studied by methylation-specific PCR and immunohistochemistry, respectively. The materials were divided into four groups: 1) nonneoplastic cervices, 2) CIN I, 3) CIN II-III and 4) invasive SCCs. p16 methylation and p16 immunoexpressions increased in CIN and invasive SCCs than nonneoplastic tissue. p16-methylation and p16-immunoreactivities were higher in the EBV-positive group (p=0.009, p<0.001) than in the EBV-negative group. EBV was detected more frequently in CIN and SCCs than nonneoplastic cervices. In conclusion, a correlation between p16 methylation, p16 immunoreactivity and the detection of EBV strongly suggested that the cooperation of EBV and p16 gene may play a synergic effect on cell cycle deregulation.

  11. High risk HPV in situ hybridization, p16 INK 4A, and survivin expressions in cervical carcinomas and intraepithelial neoplasms: evaluation of prognostic factors.

    PubMed

    Demir, F; Kimiloglu, E; Igdem, A A; Ayanoglu, Y T; Erdogan, N

    2014-01-01

    Cervical carcinoma (CC) is one of the most important health problems of adult women in developing countries. CC is the second most common carcinoma of the women worldwide. Efficient screening and early therapeutic programmes are vital because of the higher burden of the disease. The authors included a total of 53 cases in this study. Distribution of diagnoses among cases was as follows: cervical intraepithelial neoplasm (CIN) (n=44), squamous cell carcinoma (SCC) (n=7), adenocarcinoma in situ (n=1), and condyloma accuminatum (n=l). Presence, density, and nuclear identification form of human papilloma virus (HPV) DNA in relation with host cell DNA were evaluated by in situ hybridization (ISH) and p16 and survivin by immunohistochem- ical methods (IHC). The authors determined that the presence, density, and nuclear identification form of high risk HPV DNA had diagnostic and prognostic importance in CC and CIN (p < 0.05). p16 and survivin also had diagnostic significance. Since p16 and survivin expressions signalled progressive oncogenic events, p16 and survivin were persistent HPV markers (for p16, p < 0.001, for survivin p < 0.01). The authors determined that expressions, density, and prevalence of all three markers showed correlation with increasing CIN grade (for p 16, p < 0.001, for survivin, p < 0.01, for HPV, p = 0.002). The episomal pattern which is the independent visit of Hr HPV DNA to host cell DNA, signalled early HPV infection (p = 0.001). When it is integrated into host cell DNA, especially if HPV DNA signal intensity and prevalence increases, then this signal signifies persistent HPV infection (p = 0.001). With the aid of these findings, the authors determined that HPV is infectious in CIN I and proliferative (neoplastic) in CIN II-CIN III lesions.

  12. The specific role of pRb in p16INK4A-mediated arrest of normal and malignant human breast cells

    PubMed Central

    Bazarov, Alexey V; Lee, Won Jae; Bazarov, Irina; Bosire, Moses; Hines, William C; Stankovich, Basha; Chicas, Agustin; Lowe, Scott W

    2012-01-01

    RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G1 was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation. PMID:22333593

  13. The specific role of pRb in p16 (INK4A) -mediated arrest of normal and malignant human breast cells.

    PubMed

    Bazarov, Alexey V; Lee, Won Jae; Bazarov, Irina; Bosire, Moses; Hines, William C; Stankovich, Basha; Chicas, Agustin; Lowe, Scott W; Yaswen, Paul

    2012-03-01

    RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G 1 was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.

  14. High-risk HPV genotypes and P16INK4a expression in a cohort of head and neck squamous cell carcinoma patients in Singapore

    PubMed Central

    Tan, Louise Soo Yee; Fredrik, Petersson; Ker, Liang; Yu, Feng Gang; Wang, De Yun; Goh, Boon Cher; Loh, Kwok Seng; Lim, Chwee Ming

    2016-01-01

    Human papillomavirus (HPV), especially HPV16 genotype, is associated with oropharyngeal squamous cell carcinoma (OPSCC). We aim to determine the prevalence and characterize the high-risk (HR)-HPV genotypes in head and neck SCC (HNSCC) in a South-East Asian multi-ethnic society in Singapore and examine its prognostic significance. 159 HNSCC archival tissue samples were retrieved and tumour DNA was screened for 18 HR-HPV genotypes using a PCR-based assay (Qiagen, digene HPV genotyping RH test). P16 protein overexpression was identified using immunohistochemistry (IHC). Statistical correlation between clinical outcomes were performed between HPV-positive and negative HNSCC patients. Six HR-HPVs (HPV16, 18, 31, 45, 56, 68) were detected in 90.6% of HNSCC; and 79.9% had multiple HPV genotypes detected. HPV31 and HPV45 were the most prevalent (79.2% and 87.4%, respectively); and HPV16 was predominantly found in OPSCC (p < 0.001). HPV-DNA PCR assay yielded a high sensitivity (96%) but low specificity (11%) when compared to p16 immunohistochemistry as the reference standard. P16-positive HNSCC was predominantly observed in OPSCC (73.7%; p = 0.005); and p16-positive OPSCC exhibited improved overall survival compared to p16-negative OPSCC (p = 0.022). Similarly, smoking and alcohol consumption were poor prognostic factors of overall survival (p = 0.007; p = 0.01) in OPSCC patients. HR-HPVs were identified in 90.6% of HNSCC patients using the HPV-DNA PCR assay. This test had a poor specificity when compared to p16 IHC; making it an unreliable detection technique in selecting patients for radiation dose de-escalation treatment protocol. P16-positive tumor was predominantly found in the oropharynx these patients demonstrated better overall survival than those with p16-negative OPSCC. PMID:27893418

  15. Activation of p21(CIP1/WAF1) in mammary epithelium accelerates mammary tumorigenesis and promotes lung metastasis.

    PubMed

    Cheng, Xiaoyun; Xia, Weiya; Yang, Jer-Yen; Hsu, Jennifer L; Chou, Chao-Kai; Sun, Hui-Lung; Wyszomierski, Shannon L; Mills, Gordon B; Muller, William J; Yu, Dihua; Hung, Mien-Chie

    2010-12-03

    While p21 is well known to inhibit cyclin-CDK activity in the nucleus and it has also been demonstrated to have oncogenic properties in different types of human cancers. In vitro studies showed that the oncogenic function of p21is closely related to its cytoplasmic localization. However, it is unclear whether cytoplasmic p21 contributes to tumorigenesis in vivo. To address this question, we generated transgenic mice expressing the Akt-phosphorylated form of p21 (p21T145D) in the mammary epithelium. The results showed that Akt-activated p21 was expressed in the cytoplasm of mammary epithelium. Overexpression of Akt-activated p21 accelerated tumor onset and promoted lung metastasis in MMTV/neu mice, providing evidence that p21, especially cytoplasmic phosphorylated p21, has an oncogenic role in promoting mammary tumorigenesis and metastasis.

  16. p21Cip-1/SDI-1/WAF-1 gene is involved in chondrogenic differentiation of ATDC5 cells in vitro.

    PubMed

    Negishi, Y; Ui, N; Nakajima, M; Kawashima, K; Maruyama, K; Takizawa, T; Endo, H

    2001-08-31

    Development of skeletal cartilage is characterized with coupling growth arrest and cell differentiation. Here, to understand the cyclin-dependent kinase inhibitors involved in the progression of chondrogenic differentiation, we examined changes in the expression levels of cyclin-dependent kinase inhibitor members using mouse ATDC5 prechondrocytes as a widely used in vitro model of cartilage differentiation. Up-regulation of p21 and p27 mRNA was observed following a decrease in growth rate of prechondrocytes, and both transcripts subsequently accumulated during chondrogenic differentiation; p15, p18, and p19 mRNA, in contrast, did not change during differentiation. Only the up-regulation of p21 mRNA during differentiation was prevented by the continuous treatment of early chondrogenic inhibitor, parathyroid hormone, indicating a close correlation between differentiation and p21 induction in ATDC5 cells. Therefore, to examine the role of p21 during chondrogenesis, we established stable cell lines overexpressing full-length p21 antisense RNA in ATDC5. The reduction of endogenous p21 in these cell lines caused inhibition of early chondrogenic differentiation in ATDC5, indicating that p21 gene plays an important role in this process of the cells in vitro. Furthermore, the level of p21 protein and p21.CDK2 complexes transiently increased during differentiation, but not in undifferentiated cells, leading to a decrease in CDK2-associated kinase. However, differentiation-dependent expressed p21 protein was degraded by a proteasome-dependent pathway. Thus, the progression of chondrogenic differentiation requires down-regulation of CDK2-associated kinase with an increase in p21 protein and subsequent degradation of this protein by a proteasomal pathway.

  17. Regulation of expression of the p21(CIP1) gene by the transcription factor ZNF217 and MDM2.

    PubMed

    Mantsou, Aglaia; Koutsogiannouli, Evangelia; Haitoglou, Costas; Papavassiliou, Athanasios G; Papanikolaou, Nikolaos A

    2016-12-01

    Using mouse double minute 2 (MDM2) protein-specific affinity chromatography and mass spectrometry, we have isolated the protein product of the oncogene znf217, which is a transcription factor and a component of a Hela-S-derived HDAC1 complex, as a novel MDM2-interacting protein. When co-expressed in cultured cancer cells, ZNF217 forms a complex with MDM2 and its ectopic over-expression reduces the steady-state levels of acetylated p53 in cell lines, suppressing its ability to activate the expression of a p21 promoter construct. In-silico analysis of the p21 promoter revealed the presence of several ZNF217-binding sites. These findings suggest that MDM2 controls p21 expression by at least 2 mechanisms: through ZNF217-mediated recruitment of HDAC1/MDM2 activity, which inhibits p53 acetylation; and through direct interaction with its binding site(s) on the p21 promoter.

  18. p21Cip1 plays a critical role in the physiological adaptation to fasting through activation of PPARα

    PubMed Central

    Lopez-Guadamillas, Elena; Fernandez-Marcos, Pablo J.; Pantoja, Cristina; Muñoz-Martin, Maribel; Martínez, Dolores; Gómez-López, Gonzalo; Campos-Olivas, Ramón; Valverde, Angela M.; Serrano, Manuel

    2016-01-01

    Fasting is a physiological stress that elicits well-known metabolic adaptations, however, little is known about the role of stress-responsive tumor suppressors in fasting. Here, we have examined the expression of several tumor suppressors upon fasting in mice. Interestingly, p21 mRNA is uniquely induced in all the tissues tested, particularly in liver and muscle (>10 fold), and this upregulation is independent of p53. Remarkably, in contrast to wild-type mice, p21-null mice become severely morbid after prolonged fasting. The defective adaptation to fasting of p21-null mice is associated to elevated energy expenditure, accelerated depletion of fat stores, and premature activation of protein catabolism in the muscle. Analysis of the liver transcriptome and cell-based assays revealed that the absence of p21 partially impairs the transcriptional program of PPARα, a key regulator of fasting metabolism. Finally, treatment of p21-null mice with a PPARα agonist substantially protects them from their accelerated loss of fat upon fasting. We conclude that p21 plays a relevant role in fasting adaptation through the positive regulation of PPARα. PMID:27721423

  19. A role for p38 in transcriptional elongation of p21CIP1 in response to Aurora B inhibition

    PubMed Central

    Kumari, Geeta; Ulrich, Tanja; Gaubatz, Stefan

    2013-01-01

    Aurora kinases play important functions in mitosis. They are overexpressed in many cancers and are targets for anticancer therapy. Inhibition of Aurora B results in cytokinesis failure and polyploidization, leading to activation of the p53 tumor suppressor and its target genes, including p21. The pathways that mediate p21 activation after Aurora B inhibition are not well understood. In this study, we identified a role for the p38 MAP kinase in activation of p21 when Aurora B is inhibited. We show that p38 is required for the acute cell cycle arrest in G1 and to prevent endoreduplication when Aurora B is inhibited. Stabilization of p53 occurs independently of p38, and recruitment of p53 to the p21 promoter also does not require p38. Instead, enrichment of the elongating form of RNA PolII at the distal region of the p21 gene is strongly reduced when p38 is blocked, indicating that p38 acts in transcriptional elongation of p21. Thus, our results identify an unexpected role of p38 in cell cycle regulation in response to Aurora B inhibition, by promoting the transcriptional elongation of the cell cycle inhibitor p21. PMID:23759594

  20. p21Cip1 Is Required for the Development of Monocytes and Their Response to Serum Transfer-induced Arthritis

    PubMed Central

    Scatizzi, John C.; Hutcheson, Jack; Bickel, Emily; Woods, James M.; Klosowska, Karolina; Moore, Terry L.; Haines, G. Kenneth; Perlman, Harris

    2006-01-01

    One of the central functions of cyclin-dependent kinase inhibitors, such as p21, p27, or p16, is to prevent entry into the cell cycle. However, the question remains as to whether they have other functions in the cell. We previously demonstrated that overexpression of p21 in fibroblasts isolated from patients with rheumatoid arthritis decreases the production of pro-inflammatory molecules. Overexpression of p21 has been also shown to reduce the development of experimental arthritis in mice and rats. To explore the role of endogenous p21 in the development of arthritis, we induced arthritis in p21−/− mice using the K/BxN serum transfer model of arthritis. Mice deficient in p21 were more resistant to serum transfer-induced arthritis (K/BxN) than wild-type (wt) control mice. Fewer macrophages were detected in p21−/− as compared to wt joints following transfer of K/BxN serum. Chemotaxis assays of bone marrow-derived macrophages from p21−/− and wt mice revealed no difference in migration. However, there was a substantial decrease in inflammatory monocytes circulating in peripheral blood and in monocyte precursors in bone marrow of p21−/− mice as compared to wt mice. Adoptive transfer of wt bone marrow-derived macrophages into p21−/− mice restored the sensitivity to serum transfer-induced arthritis. These data suggest a novel role for p21 in regulating the development and/or differentiation of monocytic populations that are crucial for the induction of inflammatory arthritis. PMID:16651620

  1. INK4 locus of the tumor-resistant rodent, the naked mole rat, expresses a functional p15/p16 hybrid isoform.

    PubMed

    Tian, Xiao; Azpurua, Jorge; Ke, Zhonghe; Augereau, Adeline; Zhang, Zhengdong D; Vijg, Jan; Gladyshev, Vadim N; Gorbunova, Vera; Seluanov, Andrei

    2015-01-27

    The naked mole rat (Heterocephalus glaber) is a long-lived and tumor-resistant rodent. Tumor resistance in the naked mole rat is mediated by the extracellular matrix component hyaluronan of very high molecular weight (HMW-HA). HMW-HA triggers hypersensitivity of naked mole rat cells to contact inhibition, which is associated with induction of the INK4 (inhibitors of cyclin dependent kinase 4) locus leading to cell-cycle arrest. The INK4a/b locus is among the most frequently mutated in human cancer. This locus encodes three distinct tumor suppressors: p15(INK4b), p16(INK4a), and ARF (alternate reading frame). Although p15(INK4b) has its own ORF, p16(INK4a) and ARF share common second and third exons with alternative reading frames. Here, we show that, in the naked mole rat, the INK4a/b locus encodes an additional product that consists of p15(INK4b) exon 1 joined to p16(INK4a) exons 2 and 3. We have named this isoform pALT(INK4a/b) (for alternative splicing). We show that pALT(INK4a/b) is present in both cultured cells and naked mole rat tissues but is absent in human and mouse cells. Additionally, we demonstrate that pALT(INK4a/b) expression is induced during early contact inhibition and upon a variety of stresses such as UV, gamma irradiation-induced senescence, loss of substrate attachment, and expression of oncogenes. When overexpressed in naked mole rat or human cells, pALT(INK4a/b) has stronger ability to induce cell-cycle arrest than either p15(INK4b) or p16(INK4a). We hypothesize that the presence of the fourth product, pALT(INK4a/b) of the INK4a/b locus in the naked mole rat, contributes to the increased resistance to tumorigenesis of this species.

  2. Inhibition of Breast Tumor Cell Growth by Ectopic Expression of p16/INK4A Via Combined Effects of Cell Cycle Arrest, Senescence and Apoptotic Induction, and Angiogenesis Inhibition.

    PubMed

    Lu, Yi; Zhang, Xiongwen; Zhang, Jun

    2012-01-01

    p16-mediated inhibition of cancer cell proliferation and tumor suppression have been studied before,; the common consensus is that p16's cell-cycle arrest function plays a primary role in these actions, with some additional apoptotic induction by p16. However, other effects of p16 that may potentially contribute to p16-mediated anti-tumor ability have not been well studied. The emerging data including ours indicated that p16 contributes its anti-cancer ability by inducing tumor cells to senescence. Moreover, we showed that p16 inhibits breast cancer cell growth by inhibiting the VEGF signaling pathway and angiogenesis. In this study, we used adenoviral-mediated p16 expression (AdRSVp16) and breast cancer cell line MDA-MB-231 as the model to simultaneously analyze all these p16's anti-tumor functions. We demonstrated that adenoviral-mediated p16 expression exhibited multiple anti-tumor functions by simultaneously suppressing in vitro growth and in vivo angiogenesis of breast cancer cells, blocking cell division, as well as inducing senescence and apoptosis. The in vivo study implies that p16's effect on anti-angiogenesis may play a more significant role than its anti-cell proliferation in the overall suppression of tumor growth. These results suggest, for the first time, that AdRSVp16-mediated tumor suppression results from a combination of p16's multiple anti-tumor functions including p16's well-known anti-proliferation/cell division function, apoptotic and senescence induction function, and its lesser-known/under-investigated anti-angiogenesis function. These combined results strongly indicate that p16 gene therapy has a multi-module platform with different anti-tumor functions; therefore, this study justifies and promotes the viral-mediated p16 gene therapy as a promising and powerful treatment approach for cancer patients due to p16's multiple anti-tumor functions.

  3. Distinctive association of p16INK4a overexpression with penile intraepithelial neoplasia depicting warty and/or basaloid features: a study of 141 cases evaluating a new nomenclature.

    PubMed

    Chaux, Alcides; Pfannl, Rolf; Lloveras, Belén; Alejo, María; Clavero, Omar; Lezcano, Cecilia; Muñoz, Nubia; de Sanjosé, Silvia; Bosch, Xavier; Hernández-Pérez, Marier; Velazquez, Elsa F; Cubilla, Antonio L

    2010-03-01

    From the pathogenic point of view, penile cancers may be grouped in human papillomavirus-related and unrelated tumors, each one of them with distinctive morphologic features. The former are predominantly composed of small, undifferentiated basaloid cells, with more or less prominent koilocytic changes, and the latter of keratinizing differentiated squamous cells. The same cellular types are observed in precancerous lesions. On the basis of these observations, we constructed a novel nomenclature for penile precancerous lesions and classified them as penile intraepithelial neoplasia (PeIN) of differentiated, warty, basaloid, and warty-basaloid types. The aim of this study was to test the usefulness of immunohistochemical p16 overexpression, considered as a surrogate for high-risk human papillomavirus infection, using this classification system. We pathologically evaluated 141 patients with PeIN, associated (123 cases) and unassociated (18 cases) with invasive cancer. Distribution of PeIN types was: differentiated, 72%; basaloid, 9%; warty-basaloid, 7%; warty, 4%; and mixed, 7%. There was a striking similarity in the morphology of in situ and invasive squamous cell carcinomas. Differentiated PeIN was commonly associated with usual, verrucous, papillary, and other low-grade keratinizing variants of squamous cell carcinoma whereas in basaloid and warty carcinomas the presence of in situ lesions with similar morphology was habitual. We evaluated p16 overexpression using a 4-tiered (0, 1, 2, and 3) pattern-based system. To properly distinguish differentiated PeIN from in situ lesions with warty and/or basaloid features only pattern 3, which requires full-thickness staining in all epithelial cells, was considered positive. Using this approach, there was a significant association of the negative patterns and differentiated PeIN and of the positive pattern and warty, basaloid, and warty-basaloid PeIN (P<0.0001). Basaloid variant had the strongest association. The sensitivity rate of p16 positivity for discriminating types of PeIN was of 82%, with a specificity of 100% and an accuracy of 95%. Lichen sclerosus was identified in 42 cases and their epithelial component was p16 negative in all cases. Although more studies are necessary to confirm these observations, p16 overexpression seems to be a useful tool for discriminating differentiated from warty, basaloid, and warty-basaloid PeIN.

  4. Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression

    SciTech Connect

    Yu, Zhendong; Wang, Hao; Zhang, Libin; Tang, Aifa; Zhai, Qinna; Wen, Jianxiang; Yao, Li; Li, Pengfei

    2009-09-04

    CDglyTK fusion suicide gene has been well characterized to effectively kill tumor cells. However, the exact mechanism and downstream target genes are not fully understood. In our study, we found that CDglyTK/prodrug treatment works more efficiently in p53 wild-type (HONE1) cells than in p53 mutant (CNE1) cells. We then used adenovirus-mediated gene delivery system to either knockdown or overexpress p53 and its target genes in these cells. Consistent results showed that both p53-PUMA/NOXA/Bcl2-Bax and p53-p21 pathways contribute to the CDglyTK induced tumor cell suppression. Our work for the first time addressed the role of p53 related genes in the CDglyTK/prodrug system.

  5. Induction of p21CIP1 protein and cell cycle arrest after inhibition of Aurora B kinase is attributed to aneuploidy and reactive oxygen species.

    PubMed

    Kumari, Geeta; Ulrich, Tanja; Krause, Michael; Finkernagel, Florian; Gaubatz, Stefan

    2014-06-06

    Cell cycle progression requires a series of highly coordinated events that ultimately lead to faithful segregation of chromosomes. Aurora B is an essential mitotic kinase, which is involved in regulation of microtubule-kinetochore attachments and cytokinesis. Inhibition of Aurora B results in stabilization of p53 and induction of p53-target genes such as p21 to inhibit proliferation. We have previously demonstrated that induction of p21 by p53 after inhibition of Aurora B is dependent on the p38 MAPK, which promotes transcriptional elongation of p21 by RNA Pol II. In this study, we show that a subset of p53-target genes are induced in a p38-dependent manner upon inhibition of Aurora B. We also demonstrate that inhibition of Aurora B results in down-regulation of E2F-mediated transcription and that the cell cycle arrest after Aurora B inhibition depends on p53 and pRB tumor suppressor pathways. In addition, we report that activation of p21 after inhibition of Aurora B is correlated with increased chromosome missegregation and aneuploidy but not with binucleation or tetraploidy. We provide evidence that p21 is activated in aneuploid cells by reactive oxygen species (ROS) and p38 MAPK. Finally, we demonstrate that certain drugs that act on aneuploid cells synergize with inhibitors of Aurora B to inhibit colony formation and oncogenic transformation. These findings provide an important link between aneuploidy and the stress pathways activated by Aurora B inhibition and also support the use of Aurora B inhibitors in combination therapy for treatment of cancer. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Expression of p21(Cip1/Waf1/Sdi1) and p27(Kip1) cyclin-dependent kinase inhibitors during human hematopoiesis.

    PubMed

    Taniguchi, T; Endo, H; Chikatsu, N; Uchimaru, K; Asano, S; Fujita, T; Nakahata, T; Motokura, T

    1999-06-15

    Expression of p21 and p27 cyclin-dependent kinase inhibitors is associated with induced differentiation and cell-cycle arrest in some hematopoietic cell lines. However, it is not clear how these inhibitors are expressed during normal hematopoiesis. We examined various human hematopoietic colonies derived from cord blood CD34(+) cells, bone marrow, and peripheral blood cells using a quantitative reverse transcription-polymerase chain reaction assay, immunochemistry, and/or Western blot analysis. p21 mRNA was expressed increasingly over time in all of the colonies examined (granulocytes, macrophages, megakaryocytes, and erythroblasts), whereas p27 mRNA levels remained low, except for erythroid bursts. Erythroid bursts expressed both p21 and p27 mRNAs with differentiation but expressed neither protein, whereas both proteins were expressed in megakaryocytes and peripheral blood monocytes. In bone marrow, p21 was immunostained almost exclusively in a subset of megakaryocytes and p27 protein was present in megakaryocytes, plasma cells, and endothelial cells. In megakaryocytes, reciprocal expression of p27 to Ki-67 was evident and an inverse relationship between p21 and Ki-67 positivities was also present, albeit less obvious. These observations suggest that a complex lineage-specific regulation is involved in p21 and p27 expression and that these inhibitors are involved in cell-cycle exit in megakaryocytes.

  7. Reduced abundance of the E3 ubiquitin ligase E6AP contributes to decreased expression of the INK4/ARF locus in non-small cell lung cancer.

    PubMed

    Gamell, Cristina; Gulati, Twishi; Levav-Cohen, Yaara; Young, Richard J; Do, Hongdo; Pilling, Pat; Takano, Elena; Watkins, Neil; Fox, Stephen B; Russell, Prudence; Ginsberg, Doron; Monahan, Brendon J; Wright, Gavin; Dobrovic, Alex; Haupt, Sue; Solomon, Ben; Haupt, Ygal

    2017-01-10

    The tumor suppressor p16(INK4a), one protein encoded by the INK4/ARF locus, is frequently absent in multiple cancers, including non-small cell lung cancer (NSCLC). Whereas increased methylation of the encoding gene (CDKN2A) accounts for its loss in a third of patients, no molecular explanation exists for the remainder. We unraveled an alternative mechanism for the silencing of the INK4/ARF locus involving the E3 ubiquitin ligase and transcriptional cofactor E6AP (also known as UBE3A). We found that the expression of three tumor suppressor genes encoded in the INK4/ARF locus (p15(INK4b), p16(INK4a), and p19(ARF)) was decreased in E6AP(-/-) mouse embryo fibroblasts. E6AP induced the expression of the INK4/ARF locus at the transcriptional level by inhibiting CDC6 transcription, a gene encoding a key repressor of the locus. Luciferase assays revealed that E6AP inhibited CDC6 expression by reducing its E2F1-dependent transcription. Chromatin immunoprecipitation analysis indicated that E6AP reduced the amount of E2F1 at the CDC6 promoter. In a subset of NSCLC samples, an E6AP-low/CDC6-high/p16(INK4a)-low protein abundance profile correlated with low methylation of the gene encoding p16(INK4a) (CDKN2A) and poor patient prognosis. These findings define a previously unrecognized tumor-suppressive role for E6AP in NSCLC, reveal an alternative silencing mechanism of the INK4/ARF locus, and reveal E6AP as a potential prognostic marker in NSCLC. Copyright © 2017, American Association for the Advancement of Science.

  8. A Case of T-cell Acute Lymphoblastic Leukemia Relapsed As Myeloid Acute Leukemia.

    PubMed

    Paganin, Maddalena; Buldini, Barbara; Germano, Giuseppe; Seganfreddo, Elena; Meglio, Annamaria di; Magrin, Elisa; Grillo, Francesca; Pigazzi, Martina; Rizzari, Carmelo; Cazzaniga, Giovanni; Khiabanian, Hossein; Palomero, Teresa; Rabadan, Raul; Ferrando, Adolfo A; Basso, Giuseppe

    2016-09-01

    A 4-year-old male with the diagnosis of T-cell acute lymphoblastic leukemia (T-ALL) relapsed after 19 months with an acute myeloid leukemia (AML). Immunoglobulin and T-cell receptor gene rearrangements analyses reveal that both leukemias were rearranged with a clonal relationship between them. Comparative genomic hybridization (Array-CGH) and whole-exome sequencing analyses of both samples suggest that this leukemia may have originated from a common T/myeloid progenitor. The presence of homozygous deletion of p16/INK4A, p14/ARF, p15/INK4B, and heterozygous deletion of WT1 locus remained stable in the leukemia throughout phenotypic switch, revealing that this AML can be genetically associated to T-ALL.

  9. Senescence and immortality in hepatocellular carcinoma.

    PubMed

    Ozturk, Mehmet; Arslan-Ergul, Ayca; Bagislar, Sevgi; Senturk, Serif; Yuzugullu, Haluk

    2009-12-01

    Cellular senescence is a process leading to terminal growth arrest with characteristic morphological features. This process is mediated by telomere-dependent, oncogene-induced and ROS-induced pathways, but persistent DNA damage is the most common cause. Senescence arrest is mediated by p16(INK4a)- and p21(Cip1)-dependent pathways both leading to retinoblastoma protein (pRb) activation. p53 plays a relay role between DNA damage sensing and p21(Cip1) activation. pRb arrests the cell cycle by recruiting proliferation genes to facultative heterochromatin for permanent silencing. Replicative senescence that occurs in hepatocytes in culture and in liver cirrhosis is associated with lack of telomerase activity and results in telomere shortening. Hepatocellular carcinoma (HCC) cells display inactivating mutations of p53 and epigenetic silencing of p16(INK4a). Moreover, they re-express telomerase reverse transcriptase required for telomere maintenance. Thus, senescence bypass and cellular immortality is likely to contribute significantly to HCC development. Oncogene-induced senescence in premalignant lesions and reversible immortality of cancer cells including HCC offer new potentials for tumor prevention and treatment.

  10. Reed-Sternberg cells in Hodgkin's lymphoma present features of cellular senescence

    PubMed Central

    Gopas, J; Stern, E; Zurgil, U; Ozer, J; Ben-Ari, A; Shubinsky, G; Braiman, A; Sinay, R; Ezratty, J; Dronov, V; Balachandran, S; Benharroch, D; Livneh, E

    2016-01-01

    Hodgkin's Lymphoma (HL) is one of the most prevailing malignancies in young adults. Reed–Sternberg (RS) cells in HL have distinctive large cell morphology, are characteristic of the disease and their presence is essential for diagnosis. Enlarged cells are one of the hallmarks of senescence, but whether RS cells are senescent has not been previously investigated. Here we show that RS cells have characteristics of senescent cells; RS cells in HL biopsies specifically express the senescence markers and cell cycle inhibitors p21Cip1 and p16INK4a and are negative for the proliferation marker Ki-67, suggesting that these cells have ceased to proliferate. Moreover, the RS-like cells in HL lines, stained specifically for senescence-associated β-galactosidase (SA-β-gal). Oxidative stress promoted senescence in these cells as demonstrated by their staining for p21Cip1, p16INK4a, p53 and γH2AX. Senescent cells produce copious amounts of inflammatory cytokines termed ‘senescence-associated secretory phenotype' (SASP), primarily regulated by Nuclear Factor κB (NF-κB). Indeed, we show that NF-κB activity and NF-κB-dependent cytokines production (e.g., IL-6, TNF-α, GM-CSF) were elevated in RS-like cells. Furthermore, NF-κB inhibitors, JSH-23 and curcumin reduced IL-6 secretion from RS-like cells. Thus, defining RS cells as senescent offers new insights on the origin of the proinflammatory microenvironment in HL. PMID:27831553

  11. Reed-Sternberg cells in Hodgkin's lymphoma present features of cellular senescence.

    PubMed

    Gopas, J; Stern, E; Zurgil, U; Ozer, J; Ben-Ari, A; Shubinsky, G; Braiman, A; Sinay, R; Ezratty, J; Dronov, V; Balachandran, S; Benharroch, D; Livneh, E

    2016-11-10

    Hodgkin's Lymphoma (HL) is one of the most prevailing malignancies in young adults. Reed-Sternberg (RS) cells in HL have distinctive large cell morphology, are characteristic of the disease and their presence is essential for diagnosis. Enlarged cells are one of the hallmarks of senescence, but whether RS cells are senescent has not been previously investigated. Here we show that RS cells have characteristics of senescent cells; RS cells in HL biopsies specifically express the senescence markers and cell cycle inhibitors p21(Cip1) and p16(INK4a) and are negative for the proliferation marker Ki-67, suggesting that these cells have ceased to proliferate. Moreover, the RS-like cells in HL lines, stained specifically for senescence-associated β-galactosidase (SA-β-gal). Oxidative stress promoted senescence in these cells as demonstrated by their staining for p21(Cip1), p16(INK4a), p53 and γH2AX. Senescent cells produce copious amounts of inflammatory cytokines termed 'senescence-associated secretory phenotype' (SASP), primarily regulated by Nuclear Factor κB (NF-κB). Indeed, we show that NF-κB activity and NF-κB-dependent cytokines production (e.g., IL-6, TNF-α, GM-CSF) were elevated in RS-like cells. Furthermore, NF-κB inhibitors, JSH-23 and curcumin reduced IL-6 secretion from RS-like cells. Thus, defining RS cells as senescent offers new insights on the origin of the proinflammatory microenvironment in HL.

  12. Progression in cutaneous malignant melanoma is associated with distinct expression profiles: a tissue microarray-based study.

    PubMed

    Alonso, Soledad R; Ortiz, Pablo; Pollán, Marina; Pérez-Gómez, Beatriz; Sánchez, Lydia; Acuña, Ma Jesús; Pajares, Raquel; Martínez-Tello, Francisco J; Hortelano, Carlos M; Piris, Miguel A; Rodríguez-Peralto, José L

    2004-01-01

    Cutaneous malignant melanoma remains the leading cause of skin cancer death in industrialized countries. Clinical and histological variables that predict survival, such as Breslow's index, tumor size, ulceration, or vascular invasion have been identified in malignant melanoma. Nevertheless, the potential relevance of biological variables still awaits an in-depth exploration. Using tissue microarrays (TMAs), we retrospectively analyzed 165 malignant melanoma samples from 88 patients corresponding to distinct histological progression phases, radial, vertical, and metastases. A panel of 39 different antibodies for cell cycle, apoptosis, melanoma antigens, transcription factors, DNA mismatch repair, and other proteins was used. Integrating the information, the study has identified expression profiles distinguishing specific melanoma progression stages. Most of the detected alterations were linked to the control of cell cycle G1/S transition; cyclin D1 was expressed in radial cases 48% (12 of 25) with significant lost of expression in vertical cases 14% (9 of 65), P = 0.002; whereas p16(INK4a) (89% in vertical versus 71% in metastatic cases, P = 0.009) and p27(KIP1) (76% in radial versus 45% in vertical cases, P = 0.010) were diminished in advanced stages. The study also defines a combination of biological markers associated with shorter overall survival in patients with vertical growth phase melanoma, that provided a predictor model with four antibodies (Ki67, p16(INK4a), p21(CIP1), and Bcl-6). This predictor model was validated using an independent series of 72 vertical growth phase melanoma patients.

  13. [Hypermethylation as a potential prognostic factor and a clue to a better understanding of the molecular pathogenesis of medulloblastoma--results of a genomewide methylation scan].

    PubMed

    Frühwald, M C; O'Dorisio, M S; Smith, L; Dai, Z; Wright, F A; Paulus, W; Jürgens, H; Plass, C

    2001-01-01

    The molecular mechanisms controlling initiation and progression of medulloblastomas are largely unclear. Changes in DNA methylation of promoter regions have been shown to disturb the expression of growth regulatory genes. We evaluated DNA methylation patterns in 17 medulloblastomas, 5 stPNETs and 5 medulloblastoma cell lines using Restriction Landmark Genomic Scanning (RLGS), a method displaying up to 2.000 potential gene loci in a single gene. To test whether previously characterized tumor suppressor genes are affected by hypermethylation we performed MS-PCR for p15INK4B, p16INK4A, VHL, TP53 and E-cadherin. The analysis of RLGS profiles from tumors revealed an abundance of hypermethylation in primary tumors and cell lines. Extrapolated to the human genome with its approximately 36,000 genes a total of 420 loci become hypermethylated in the tumor genomes. The previously characterized medulloblastoma breakpoint cluster in 17p11.2 appears to be a hotspot for aberrant methylation. Cox regression analysis of survival data identified seven CpG islands for which hypermethylation is suggestive of a poor prognosis. MS-PCR analysis of known genes demonstrated hypermethylation of p16INK4A in a limited number of tumors. The pattern of DNA hypermethylation was similar in medulloblastomas and stPNETs. However, some CpG islands were shown to be specific for a tumor type, while others were shared targets. Hypermethylation is a common abnormality in primary medulloblastomas and supratentorial PNETs. Several hundreds of CpG islands are potential targets for methylation in medulloblastomas including the breakpoint cluster in 17p11.2. The methylation status of certain gene sequences appears to be associated with the clinical outcome. Promoter hypermethylation has an outstanding potential as a marker for the identification of novel tumor suppressors as well as diagnostic and therapeutic targets in medulloblastomas.

  14. Mice Homozygous for a Deletion in the Glaucoma Susceptibility Locus INK4 Show Increased Vulnerability of Retinal Ganglion Cells to Elevated Intraocular Pressure.

    PubMed

    Gao, Shan; Jakobs, Tatjana C

    2016-04-01

    A genomic region located on chromosome 9p21 is associated with primary open-angle glaucoma and normal tension glaucoma in genome-wide association studies. The genomic region contains the gene for a long noncoding RNA called CDKN2B-AS, two genes that code for cyclin-dependent kinase inhibitors 2A and 2B (CDKN2A/p16(INK4A) and CDKN2B/p15(INK4B)) and an additional protein (p14(ARF)). We used a transgenic mouse model in which 70 kb of murine chromosome 4, syntenic to human chromosome 9p21, are deleted to study whether this deletion leads to a discernible phenotype in ocular structures implicated in glaucoma. Homozygous mice of this strain were previously reported to show persistent hyperplastic primary vitreous. Fundus photography and optical coherence tomography confirmed that finding but showed no abnormalities for heterozygous mice. Optokinetic response, eletroretinogram, and histology indicated that the heterozygous and mutant retinas were normal functionally and morphologically, whereas glial cells were activated in the retina and optic nerve head of mutant eyes. In quantitative PCR, CDKN2B expression was reduced by approximately 50% in the heterozygous mice and by 90% in the homozygous mice, which suggested that the CDKN2B knock down had no deleterious consequences for the retina under normal conditions. However, compared with wild-type and heterozygous animals, the homozygous mice are more vulnerable to retinal ganglion cell loss in response to elevated intraocular pressure.

  15. Reprogrammed keratinocytes from elderly type 2 diabetes patients suppress senescence genes to acquire induced pluripotency

    PubMed Central

    Ohmine, Seiga; Squillace, Karen A.; Hartjes, Katherine A.; Deeds, Michael C.; Armstrong, Adam S.; Thatava, Tayaramma; Sakuma, Toshie; Terzic, Andre; Kudva, Yogish; Ikeda, Yasuhiro

    2012-01-01

    Nuclear reprogramming enables patient-specific derivation of induced pluripotent stem (iPS) cells from adult tissue. Yet, iPS generation from patients with type 2 diabetes (T2D) has not been demonstrated. Here, we report reproducible iPS derivation of epidermal keratinocytes (HK) from elderly T2D patients. Transduced with human OCT4, SOX2, KLF4 and c-MYC stemness factors under serum-free and feeder-free conditions, reprogrammed cells underwent dedifferentiation with mitochondrial restructuring, induction of endogenous pluripotency genes - including NANOG, LIN28, and TERT, and down-regulation of cytoskeletal, MHC class I- and apoptosis-related genes. Notably, derived iPS clones acquired a rejuvenated state, characterized by elongated telomeres and suppressed senescence-related p15INK4b/p16INK4a gene expression and oxidative stress signaling. Stepwise guidance with lineage-specifying factors, including Indolactam V and GLP-1, redifferentiated HK-derived iPS clones into insulin-producing islet-like progeny. Thus, in elderly T2D patients, reprogramming of keratinocytes ensures a senescence-privileged status yielding iPS cells proficient for regenerative applications. PMID:22308265

  16. CBL enhances breast tumor formation by inhibiting tumor suppressive activity of TGF-β signaling.

    PubMed

    Kang, J M; Park, S; Kim, S J; Hong, H Y; Jeong, J; Kim, H-S; Kim, S-J

    2012-12-13

    Casitas B-lineage lymphoma (CBL) protein family functions as multifunctional adaptor proteins and E3 ubiquitin ligases that are implicated as regulators of signaling in various cell types. Recent discovery revealed mutations of proto-oncogenic CBL in the linker region and RING finger domain in human acute myeloid neoplasm, and these transforming mutations induced carcinogenesis. However, the adaptor function of CBL mediated signaling pathway during tumorigenesis has not been well characterized. Here, we show that CBL is highly expressed in breast cancer cells and significantly inhibits transforming growth factor-β (TGF-β) tumor suppressive activity. Knockdown of CBL expression resulted in the increased expression of TGF-β target genes, PAI-I and CDK inhibitors such as p15(INK4b) and p21(Cip1). Furthermore, we demonstrate that CBL is frequently overexpressed in human breast cancer tissues, and the loss of CBL decreases the tumorigenic activity of breast cancer cells in vivo. CBL directly binds to Smad3 through its proline-rich motif, thereby preventing Smad3 from interacting with Smad4 and blocking nuclear translocation of Smad3. CBL-b, one of CBL protein family, also interacted with Smad3 and knockdown of both CBL and CBL-b further enhanced TGF-β transcriptional activity. Our findings provide evidence for a previously undescribed mechanism by which oncogenic CBL can block TGF-β tumor suppressor activity.

  17. Premature expression of a muscle fibrosis axis in chronic HIV infection

    PubMed Central

    2012-01-01

    Background Despite the success of highly active antiretroviral therapy (HAART), HIV infected individuals remain at increased risk for frailty and declines in physical function that are more often observed in older uninfected individuals. This may reflect premature or accelerated muscle aging. Methods Skeletal muscle gene expression profiles were evaluated in three uninfected independent microarray datasets including young (19 to 29 years old), middle aged (40 to 45 years old) and older (65 to 85 years old) subjects, and a muscle dataset from HIV infected subjects (36 to 51 years old). Using Bayesian analysis, a ten gene muscle aging signature was identified that distinguished young from old uninfected muscle and included the senescence and cell cycle arrest gene p21/Cip1 (CDKN1A). This ten gene signature was then evaluated in muscle specimens from a cohort of middle aged (30 to 55 years old) HIV infected individuals. Expression of p21/Cip1 and related pathways were validated and further analyzed in a rodent model for HIV infection. Results We identify and replicate the expression of a set of muscle aging genes that were prematurely expressed in HIV infected, but not uninfected, middle aged subjects. We validated select genes in a rodent model of chronic HIV infection. Because the signature included p21/Cip1, a cell cycle arrest gene previously associated with muscle aging and fibrosis, we explored pathways related to senescence and fibrosis. In addition to p21/Cip1, we observed HIV associated upregulation of the senescence factor p16INK4a (CDKN2A) and fibrosis associated TGFβ1, CTGF, COL1A1 and COL1A2. Fibrosis in muscle tissue was quantified based on collagen deposition and confirmed to be elevated in association with infection status. Fiber type composition was also measured and displayed a significant increase in slow twitch fibers associated with infection. Conclusions The expression of genes associated with a muscle aging signature is prematurely

  18. Progression in Cutaneous Malignant Melanoma Is Associated with Distinct Expression Profiles

    PubMed Central

    Alonso, Soledad R.; Ortiz, Pablo; Pollán, Marina; Pérez-Gómez, Beatriz; Sánchez, Lydia; Acuña, Ma Jesús; Pajares, Raquel; Martínez-Tello, Francisco J.; Hortelano, Carlos M.; Piris, Miguel A.; Rodríguez-Peralto, José L.

    2004-01-01

    Cutaneous malignant melanoma remains the leading cause of skin cancer death in industrialized countries. Clinical and histological variables that predict survival, such as Breslow’s index, tumor size, ulceration, or vascular invasion have been identified in malignant melanoma. Nevertheless, the potential relevance of biological variables still awaits an in-depth exploration. Using tissue microarrays (TMAs), we retrospectively analyzed 165 malignant melanoma samples from 88 patients corresponding to distinct histological progression phases, radial, vertical, and metastases. A panel of 39 different antibodies for cell cycle, apoptosis, melanoma antigens, transcription factors, DNA mismatch repair, and other proteins was used. Integrating the information, the study has identified expression profiles distinguishing specific melanoma progression stages. Most of the detected alterations were linked to the control of cell cycle G1/S transition; cyclin D1 was expressed in radial cases 48% (12 of 25) with significant lost of expression in vertical cases 14% (9 of 65), P = 0.002; whereas p16INK4a (89% in vertical versus 71% in metastatic cases, P = 0.009) and p27KIP1 (76% in radial versus 45% in vertical cases, P = 0.010) were diminished in advanced stages. The study also defines a combination of biological markers associated with shorter overall survival in patients with vertical growth phase melanoma, that provided a predictor model with four antibodies (Ki67, p16INK4a, p21CIP1, and Bcl-6). This predictor model was validated using an independent series of 72 vertical growth phase melanoma patients. PMID:14695333

  19. Collagenase-resistant collagen promotes mouse aging and vascular cell senescence.

    PubMed

    Vafaie, Faran; Yin, Hao; O'Neil, Caroline; Nong, Zengxuan; Watson, Alanna; Arpino, John-Michael; Chu, Michael W A; Wayne Holdsworth, David; Gros, Robert; Pickering, J Geoffrey

    2014-02-01

    Collagen fibrils become resistant to cleavage over time. We hypothesized that resistance to type I collagen proteolysis not only marks biological aging but also drives it. To test this, we followed mice with a targeted mutation (Col1a1(r/r) ) that yields collagenase-resistant type I collagen. Compared with wild-type littermates, Col1a1(r/r) mice had a shortened lifespan and developed features of premature aging including kyphosis, weight loss, decreased bone mineral density, and hypertension. We also found that vascular smooth muscle cells (SMCs) in the aortic wall of Col1a1(r/r) mice were susceptible to stress-induced senescence, displaying senescence-associated ß-galactosidase (SA-ßGal) activity and upregulated p16(INK4A) in response to angiotensin II infusion. To elucidate the basis of this pro-aging effect, vascular SMCs from twelve patients undergoing coronary artery bypass surgery were cultured on collagen derived from Col1a1(r/r) or wild-type mice. This revealed that mutant collagen directly reduced replicative lifespan and increased stress-induced SA-ßGal activity, p16(INK4A) expression, and p21(CIP1) expression. The pro-senescence effect of mutant collagen was blocked by vitronectin, a ligand for αvß3 integrin that is presented by denatured but not native collagen. Moreover, inhibition of αvß3 with echistatin or with αvß3-blocking antibody increased senescence of SMCs on wild-type collagen. These findings reveal a novel aging cascade whereby resistance to collagen cleavage accelerates cellular aging. This interplay between extracellular and cellular compartments could hasten mammalian aging and the progression of aging-related diseases. © 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  20. Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation.

    PubMed

    Kilic Eren, Mehtap; Kilincli, Ayten; Eren, Özkan

    2015-01-01

    The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol's anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21CIP1 and p16INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21CIP1 and p16INK4A levels. Interestingly DNA damaging agent doxorubicin

  1. Protective Effect of Ginsenoside Rg1 on Hematopoietic Stem/Progenitor Cells through Attenuating Oxidative Stress and the Wnt/β-Catenin Signaling Pathway in a Mouse Model of d-Galactose-induced Aging.

    PubMed

    Li, Jing; Cai, Dachuan; Yao, Xin; Zhang, Yanyan; Chen, Linbo; Jing, Pengwei; Wang, Lu; Wang, Yaping

    2016-06-09

    Stem cell senescence is an important and current hypothesis accounting for organismal aging, especially the hematopoietic stem cell (HSC). Ginsenoside Rg1 is the main active pharmaceutical ingredient of ginseng, which is a traditional Chinese medicine. This study explored the protective effect of ginsenoside Rg1 on Sca-1⁺ hematopoietic stem/progenitor cells (HSC/HPCs) in a mouse model of d-galactose-induced aging. The mimetic aging mouse model was induced by continuous injection of d-gal for 42 days, and the C57BL/6 mice were respectively treated with ginsenoside Rg1, Vitamin E or normal saline after 7 days of d-gal injection. Compared with those in the d-gal administration alone group, ginsenoside Rg1 protected Sca-1⁺ HSC/HPCs by decreasing SA-β-Gal and enhancing the colony forming unit-mixture (CFU-Mix), and adjusting oxidative stress indices like reactive oxygen species (ROS), total anti-oxidant (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and malondialdehyde (MDA). In addition, ginsenoside Rg1 decreased β-catenin and c-Myc mRNA expression and enhanced the phosphorylation of GSK-3β. Moreover, ginsenoside Rg1 down-regulated advanced glycation end products (AGEs), 4-hydroxynonenal (4-HNE), phospho-histone H2A.X (r-H2A.X), 8-OHdG, p16(Ink4a), Rb, p21(Cip1/Waf1) and p53 in senescent Sca-1⁺ HSC/HPCs. Our findings indicated that ginsenoside Rg1 can improve the resistance of Sca-1⁺ HSC/HPCs in a mouse model of d-galactose-induced aging through the suppression of oxidative stress and excessive activation of the Wnt/β-catenin signaling pathway, and reduction of DNA damage response, p16(Ink4a)-Rb and p53-p21(Cip1/Waf1) signaling.

  2. Alterations of RB1, p53 and Wnt pathways in hepatocellular carcinomas associated with hepatitis C, hepatitis B and alcoholic liver cirrhosis.

    PubMed

    Edamoto, Yoshihiro; Hara, Akira; Biernat, Wojciech; Terracciano, Luigi; Cathomas, Gieri; Riehle, Hans-Martin; Matsuda, Masanori; Fujii, Hideki; Scoazec, Jean-Yves; Ohgaki, Hiroko

    2003-09-01

    Major etiologic factors associated with human hepatocellular carcinomas (HCCs) include infection with hepatitis C (HCV) and hepatitis B virus (HBV), excess alcohol intake and aflatoxin B(1) exposure. While the G-->T p53 mutation at codon 249 has been identified as a genetic hallmark of HCC caused by aflatoxin B(1), the genetic profile associated with other etiologic factors appears to be less distinctive. In our study, we screened HCCs resulting from HCV infection (51 cases), HBV infection (26 cases) or excess alcohol intake (23 cases) for alterations in genes involved in the RB1 pathway (p16(INK4a), p15(INK4b), RB1, CDK4 and cyclin D1), the p53 pathway (p53, p14(ARF) and MDM2) and the Wnt pathway (beta-catenin, APC). Alterations of the RB1 pathway, mainly p16(INK4a) methylation, loss of RB1 expression and cyclin D1 amplification, were most common (69-100% of cases). There was a significant correlation between loss of RB1 expression and RB1 methylation. All 24 HCCs with RB1 promoter methylation lacked RB1 expression, while none of the 67 cases with RB1 expression exhibited RB1 methylation (p < 0.0001), suggesting that promoter methylation is a major mechanism of loss of RB1 expression in HCCs. Alterations of the p53 pathway consisted mostly of p53 mutations or p14(ARF) promoter methylation (20-48%). Mutations of the p53 gene were found at a similar frequency (13-15%) in all etiologic groups, without any consistent base change or hot spot. Mutations of beta-catenin were found in 13-31% of cases, while no APC mutations were detected in any of the HCCs analyzed. With the exception of only 3 of 39 cases (8%), cyclin D1 amplification and beta-catenin mutations were mutually exclusive, supporting the view that cyclin D1 is a target of the Wnt signaling pathway. Overall, the RB1, p53 and Wnt pathways were commonly affected in HCCs of different etiology, probably reflecting common pathogenetic mechanisms, i.e., chronic liver injury and cirrhosis, but tumors associated with

  3. ZNF313 is a novel cell cycle activator with an E3 ligase activity inhibiting cellular senescence by destabilizing p21(WAF1.).

    PubMed

    Han, J; Kim, Y-L; Lee, K-W; Her, N-G; Ha, T-K; Yoon, S; Jeong, S-I; Lee, J-H; Kang, M-J; Lee, M-G; Ryu, B-K; Baik, J-H; Chi, S-G

    2013-08-01

    ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here we report that ZNF313 is an ubiquitin E3 ligase that has a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XIAP-associated factor 1 (XAF1)-interacting protein, which upregulates the stability and proapoptotic effect of XAF1. Intriguingly, we found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21(WAF1). ZNF313 ubiquitinates p21(WAF1) and also destabilizes p27(KIP1) and p57(KIP2), three members of the CDK-interacting protein (CIP)/kinase inhibitor protein (KIP) family of cyclin-dependent kinase inhibitors, whereas it does not affect the stability of the inhibitor of CDK (INK4) family members, such as p16(INK4A) and p15(INK4B). ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-to-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21(WAF1)-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is downregulated during cellular differentiation process in vitro and in vivo, while it is commonly upregulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues, but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21(WAF1), whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.

  4. Clinico-pathological features and somatic gene alterations in refractory ceramic fibre-induced murine mesothelioma reveal mineral fibre-induced mesothelioma identities

    PubMed Central

    Andujar, Pascal; Lecomte, Céline; Renier, Annie; Fleury-Feith, Jocelyne; Kheuang, Laurence; Daubriac, Julien; Janin, Anne; Jaurand, Marie-Claude

    2007-01-01

    Although human malignant mesothelioma (HMM) is mainly caused by asbestos exposure, refractory ceramic fibres (RCFs) have been classified as possibly carcinogenic to humans on the basis of their biological effects in rodents’ lung and pleura and in cultured cells. Hence, further investigations are needed to clarify the mechanism of fibre-induced carcinogenicity and to prevent use of harmful particles. In a previous study, mesotheliomas were found in hemizygous Nf2 (Nf2+/−) mice exposed to asbestos fibres, and showed similar alterations in genes at the Ink4 locus and in Trp53 as described in HMM. Here we found that Nf2+/− mice developed mesotheliomas after intra-peritoneal inoculation of a RCF sample (RCF1). Clinical features in exposed mice were similar to those observed in HMM, showing association between ascite and mesothelioma. Early passages of 12 mesothelioma cell cultures from ascites developed in RCF1-exposed Nf2+/− mice demonstrated frequent inactivation by deletion of genes at the Ink4 locus, and low rate of Trp53 point and insertion mutations. Nf2 gene was inactivated in all cultures. In most cases, co-inactivation of genes at the Ink4 locus and Nf2 was found and, at a lower rate, of Trp53 and Nf2. These results are the first to identify mutations in RCF-induced mesothelioma. They suggest that nf2 mutation is complementary of p15Ink4b, p16Ink4a and p19Arf or p53 mutations and show similar profile of gene alterations resulting from exposure to ceramic or asbestos fibres in Nf2+/− mice, also consistent with the one found in HMM. These somatic genetic changes define different pathways of mesothelial cell transformation. PMID:17272307

  5. Clinico-pathological features and somatic gene alterations in refractory ceramic fibre-induced murine mesothelioma reveal mineral fibre-induced mesothelioma identities.

    PubMed

    Andujar, Pascal; Lecomte, Céline; Renier, Annie; Fleury-Feith, Jocelyne; Kheuang, Laurence; Daubriac, Julien; Janin, Anne; Jaurand, Marie-Claude

    2007-07-01

    Although human malignant mesothelioma (HMM) is mainly caused by asbestos exposure, refractory ceramic fibres (RCFs) have been classified as possibly carcinogenic to humans on the basis of their biological effects in rodents' lung and pleura and in cultured cells. Hence, further investigations are needed to clarify the mechanism of fibre-induced carcinogenicity and to prevent use of harmful particles. In a previous study, mesotheliomas were found in hemizygous Nf2 (Nf2(+/-)) mice exposed to asbestos fibres, and showed similar alterations in genes at the Ink4 locus and in Trp53 as described in HMM. Here we found that Nf2(+/-) mice developed mesotheliomas after intra-peritoneal inoculation of a RCF sample (RCF1). Clinical features in exposed mice were similar to those observed in HMM, showing association between ascite and mesothelioma. Early passages of 12 mesothelioma cell cultures from ascites developed in RCF1-exposed Nf2(+/-) mice demonstrated frequent inactivation by deletion of genes at the Ink4 locus, and low rate of Trp53 point and insertion mutations. Nf2 gene was inactivated in all cultures. In most cases, co-inactivation of genes at the Ink4 locus and Nf2 was found and, at a lower rate, of Trp53 and Nf2. These results are the first to identify mutations in RCF-induced mesothelioma. They suggest that nf2 mutation is complementary of p15(Ink