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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. 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.

  3. 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.

  4. 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.

  5. 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)

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. Development of Novel p16INK4a Mimetics as Anticancer Therapy

    DTIC Science & Technology

    2015-10-01

    targeted therapy with the potential for fewer side effects and higher efficacy. 2. Key words peptide mesothelioma cell cycle cyclin-dependent...phosphorylation in vitro. (Months 19- 21) 90% accomplished d. Evaluation of efficacy of p16ink4a mimetics to prevent cell cycle progression into S-phase...proliferation assays, for effects on Rb phosphorylation and cell cycle arrest. Stabilizing peptides were demonstrated to be a viable option for

  19. Development of Novel p16INK4a Mimetics as Anticancer Therapy

    DTIC Science & Technology

    2014-10-01

    grown in RPMI-1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO, USA), 10 mM HEPES... antibiotic /antimycotic reagent (Gibco BRL, Grand Island, NY, USA) at 37 o C and 5% CO2. p16 INK4a Peptides and PD 0332991. Several peptides...20 (TBS-T) and incubated in blocking buffer (5% bovine serum albumin (BSA)in TBS-T) for 1 hour at 4 o C (Rb blots) or overnight (actin blots). Rb

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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).

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  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. 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.

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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.

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

    PubMed Central

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

    1999-01-01

    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.


Keywords: naevi; N-ras; p53; p16 PMID:10465111

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. [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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  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. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  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. 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.

  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. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. 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

  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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. Adding the p16(INK4a) marker to the traditional 3-marker (ER/Vim/CEA) panel engenders no supplemental benefit in distinguishing between primary endocervical and endometrial adenocarcinomas in a tissue microarray study.

    PubMed

    Han, Chih-Ping; Lee, Ming-Yung; Kok, Lai-Fong; Ruan, Alexandra; Wu, Tina S; Cheng, Ya-Wen; Tyan, Yeu-Sheng; Lin, Ching-Yi

    2009-09-01

    Endocervical adenocarcinomas (ECAs) and endometrial adenocarcinomas (EMAs) are malignancies that affect the uterus; however, their biologic behaviors are quite different. This distinction has clinical significance, because the appropriate therapy may depend on the site of tumor origin. In this study, we not only compare the individual expression status of 4 immunomarkers [estrogen receptor (ER), vimentin (Vim), carcinoembryonic antigen (CEA), and p16], but also evaluate whether p16 adds value to the ER/Vim/CEA panel characteristics and performance in distinguishing between primary ECA and EMA. A tissue microarray (TMA) was constructed using paraffin-embedded, formalin-fixed tissues from 38 hysterectomy specimens, including 14 ECAs and 24 EMAs. Tissue microarray sections were immunostained with 4 antibodies, by the avidin-biotin complex method for antigen visualization. The staining intensity and area extent of the immunohistochemical reactions were evaluated using the semiquantitative scoring system. The 3 markers (ER, Vim, CEA) and their respective panel expressions showed statistically significant (P<0.05) frequency differences in ECA and EMA tumors. The p16 marker also revealed a significant frequency difference (P<0.05) between ECA and EMA, but did not demonstrate any supplementary benefit to the traditional 3-marker panel. In conclusion, when histomorphologic and clinical doubt exist as to the primary site of origin, we suggest that the conventional 3-marker (ER/Vim/CEA) panel is appropriate. Ancillary p16-marker testing does not add value to the 3-marker panel in distinguishing between primary ECA and EMA.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. Involvement of ERK1/2, p38 MAPK, and PI3K/Akt signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells.

    PubMed

    Calvo, Natalia; Martín, María Julia; de Boland, Ana Russo; Gentili, Claudia

    2014-08-01

    Parathyroid hormone-related peptide (PTHrP) is distributed in most fetal and adult tissues, and its expression correlates with the severity of colon carcinoma. Recently we obtained evidence that in Caco-2 cells, a cell line from human colorectal adenocarcinoma, exogenous PTHrP increases the number of live cells, via ERK1/2, p38 MAPK, and PI3-kinase and induces the expression of cyclin D1, a cell cycle regulatory protein. In this study, we further investigated the role of PTHrP in the regulation of the cell cycle progression in these intestinal cells. Flow cytometry analysis revealed that PTHrP treatment diminishes the number of cells in the G0/G1 phase and increases the number in both S and G2/M phases. The hormone increases the expression of CDK6 and diminishes the amount of negative cell cycle regulators p27Kip1, p15INK4B, and p53. However, PTHrP does not modify the expression of cyclin D3, CDK4, and p16INK4A. In addition, inhibitors of ERK1/2 (PD98059), p38 MAPK (SB203580), and PI3Kinase (LY294002) reversed PTHrP response in Caco-2 cells. Taken together, our results suggest that PTHrP positively modulates cell cycle progression and changes the expression of proteins involved in cell cycle regulation via ERK1/2, p38 MAPK, and PI3K signaling pathways in Caco-2 cells.

  12. Influence of common genetic variation on lung cancer risk: meta-analysis of 14 900 cases and 29 485 controls

    PubMed Central

    Timofeeva, Maria N.; Hung, Rayjean J.; Rafnar, Thorunn; Christiani, David C.; Field, John K.; Bickeböller, Heike; Risch, Angela; McKay, James D.; Wang, Yufei; Dai, Juncheng; Gaborieau, Valerie; McLaughlin, John; Brenner, Darren; Narod, Steven A.; Caporaso, Neil E.; Albanes, Demetrius; Thun, Michael; Eisen, Timothy; Wichmann, H.-Erich; Rosenberger, Albert; Han, Younghun; Chen, Wei; Zhu, Dakai; Spitz, Margaret; Wu, Xifeng; Pande, Mala; Zhao, Yang; Zaridze, David; Szeszenia-Dabrowska, Neonilia; Lissowska, Jolanta; Rudnai, Peter; Fabianova, Eleonora; Mates, Dana; Bencko, Vladimir; Foretova, Lenka; Janout, Vladimir; Krokan, Hans E.; Gabrielsen, Maiken Elvestad; Skorpen, Frank; Vatten, Lars; Njølstad, Inger; Chen, Chu; Goodman, Gary; Lathrop, Mark; Benhamou, Simone; Vooder, Tõnu; Välk, Kristjan; Nelis, Mari; Metspalu, Andres; Raji, Olaide; Chen, Ying; Gosney, John; Liloglou, Triantafillos; Muley, Thomas; Dienemann, Hendrik; Thorleifsson, Gudmar; Shen, Hongbing; Stefansson, Kari; Brennan, Paul; Amos, Christopher I.; Houlston, Richard; Landi, Maria Teresa

    2012-01-01

    Recent genome-wide association studies (GWASs) have identified common genetic variants at 5p15.33, 6p21–6p22 and 15q25.1 associated with lung cancer risk. Several other genetic regions including variants of CHEK2 (22q12), TP53BP1 (15q15) and RAD52 (12p13) have been demonstrated to influence lung cancer risk in candidate- or pathway-based analyses. To identify novel risk variants for lung cancer, we performed a meta-analysis of 16 GWASs, totaling 14 900 cases and 29 485 controls of European descent. Our data provided increased support for previously identified risk loci at 5p15 (P = 7.2 × 10−16), 6p21 (P = 2.3 × 10−14) and 15q25 (P = 2.2 × 10−63). Furthermore, we demonstrated histology-specific effects for 5p15, 6p21 and 12p13 loci but not for the 15q25 region. Subgroup analysis also identified a novel disease locus for squamous cell carcinoma at 9p21 (CDKN2A/p16INK4A/p14ARF/CDKN2B/p15INK4B/ANRIL; rs1333040, P = 3.0 × 10−7) which was replicated in a series of 5415 Han Chinese (P = 0.03; combined analysis, P = 2.3 × 10−8). This large analysis provides additional evidence for the role of inherited genetic susceptibility to lung cancer and insight into biological differences in the development of the different histological types of lung cancer. PMID:22899653

  13. 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

  14. 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(Ink4b), p16(Ink4a) and p19(Arf) 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.

  15. SWI/SNF regulates a transcriptional program that induces senescence to prevent liver cancer

    PubMed Central

    Tordella, Luca; Khan, Sadaf; Hohmeyer, Anja; Banito, Ana; Klotz, Sabrina; Raguz, Selina; Martin, Nadine; Dhamarlingam, Gopuraja; Carroll, Thomas; González Meljem, José Mario; Deswal, Sumit; Martínez-Barbera, Juan Pedro; García-Escudero, Ramón; Zuber, Johannes; Zender, Lars; Gil, Jesús

    2016-01-01

    Oncogene-induced senescence (OIS) is a potent tumor suppressor mechanism. To identify senescence regulators relevant to cancer, we screened an shRNA library targeting genes deleted in hepatocellular carcinoma (HCC). Here, we describe how knockdown of the SWI/SNF component ARID1B prevents OIS and cooperates with RAS to induce liver tumors. ARID1B controls p16INK4a and p21CIP1a transcription but also regulates DNA damage, oxidative stress, and p53 induction, suggesting that SWI/SNF uses additional mechanisms to regulate senescence. To systematically identify SWI/SNF targets regulating senescence, we carried out a focused shRNA screen. We discovered several new senescence regulators, including ENTPD7, an enzyme that hydrolyses nucleotides. ENTPD7 affects oxidative stress, DNA damage, and senescence. Importantly, expression of ENTPD7 or inhibition of nucleotide synthesis in ARID1B-depleted cells results in re-establishment of senescence. Our results identify novel mechanisms by which epigenetic regulators can affect tumor progression and suggest that prosenescence therapies could be employed against SWI/SNF-mutated cancers. PMID:27737960

  16. Long Term Exposure to Polyphenols of Artichoke (Cynara scolymus L.) Exerts Induction of Senescence Driven Growth Arrest in the MDA-MB231 Human Breast Cancer Cell Line.

    PubMed

    Mileo, Anna Maria; Di Venere, Donato; Abbruzzese, Claudia; Miccadei, Stefania

    2015-01-01

    Polyphenolic extracts from the edible part of artichoke (Cynara scolymus L.) have been shown to be potential chemopreventive and anticancer dietary compounds. High doses of polyphenolic extracts (AEs) induce apoptosis and decrease the invasive potential of the human breast cancer cell line, MDA-MB231. However, the molecular mechanism underlying AEs antiproliferative effects is not completely understood. We demonstrate that chronic and low doses of AEs treatment at sublethal concentrations suppress human breast cancer cell growth via a caspases-independent mechanism. Furthermore, AEs exposure induces a significant increase of senescence-associated β-galactosidase (SA-β-gal) staining and upregulation of tumour suppressor genes, p16(INK4a) and p21(Cip1/Waf1) in MDA-MB231 cells. AEs treatment leads to epigenetic alterations in cancer cells, modulating DNA hypomethylation and lysine acetylation levels in total proteins. Cell growth arrest correlates with increased reactive oxygen species (ROS) production in AEs treated breast cancer cells. Inhibition of ROS generation by N-acetylcysteine (NAC) attenuates the antiproliferative effect. These findings demonstrate that chronic AEs treatment inhibits breast cancer cell growth via the induction of premature senescence through epigenetic and ROS-mediated mechanisms. Our results suggest that artichoke polyphenols could be a promising dietary tool either in cancer chemoprevention or/and in cancer treatment as a nonconventional, adjuvant therapy.

  17. Bone Marrow Mesenchymal Stem Cell Transplantation Retards the Natural Senescence of Rat Hearts

    PubMed Central

    Zhang, Mingyu; Liu, Di; Li, Shuang; Chang, Lingling; Zhang, Yu; Liu, Ruixue; Sun, Fei; Duan, Wenqi; Du, Weijie; Wu, Yanping; Zhao, Tianyang; Xu, Chaoqian

    2015-01-01

    Bone marrow mesenchymal stem cells (BMSCs) have been shown to offer a wide variety of cellular functions including the protective effects on damaged hearts. Here we investigated the antiaging properties of BMSCs and the underlying mechanism in a cellular model of cardiomyocyte senescence and a rat model of aging hearts. Neonatal rat ventricular cells (NRVCs) and BMSCs were cocultured in the same dish with a semipermeable membrane to separate the two populations. Monocultured NRVCs displayed the senescence-associated phenotypes, characterized by an increase in the number of β-galactosidase-positive cells and decreases in the degradation and disappearance of cellular organelles in a time-dependent manner. The levels of reactive oxygen species and malondialdehyde were elevated, whereas the activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase were decreased, along with upregulation of p53, p21Cip1/Waf1, and p16INK4a in the aging cardiomyocytes. These deleterious alterations were abrogated in aging NRVCs cocultured with BMSCs. Qualitatively, the same senescent phenotypes were consistently observed in aging rat hearts. Notably, BMSC transplantation significantly prevented these detrimental alterations and improved the impaired cardiac function in the aging rats. In summary, BMSCs possess strong antisenescence action on the aging NRVCs and hearts and can improve cardiac function after transplantation in aging rats. The present study, therefore, provides an alternative approach for the treatment of heart failure in the elderly population. PMID:25855590

  18. Loss of MT1-MMP causes cell senescence and nuclear defects which can be reversed by retinoic acid.

    PubMed

    Gutiérrez-Fernández, Ana; Soria-Valles, Clara; Osorio, Fernando G; Gutiérrez-Abril, Jesús; Garabaya, Cecilia; Aguirre, Alina; Fueyo, Antonio; Fernández-García, María Soledad; Puente, Xose S; López-Otín, Carlos

    2015-07-14

    MT1-MMP (MMP14) is a collagenolytic enzyme located at the cell surface and implicated in extracellular matrix (ECM) remodeling. Mmp14(-/-) mice present dwarfism, bone abnormalities, and premature death. We demonstrate herein that the loss of MT1-MMP also causes cardiac defects and severe metabolic changes, and alters the cytoskeleton and the nuclear lamina structure. Moreover, the absence of MT1-MMP induces a senescent phenotype characterized by up-regulation of p16(INK4a) and p21(CIP1/WAF) (1), increased activity of senescence-associated β-galactosidase, generation of a senescence-associated secretory phenotype, and somatotroph axis alterations. Consistent with the role of retinoic acid signaling in nuclear lamina stabilization, treatment of Mmp14(-/-) mice with all-trans retinoic acid reversed the nuclear lamina alterations, partially rescued the cell senescence phenotypes, ameliorated the pathological defects in bone, skin, and heart, and extended their life span. These results demonstrate that nuclear architecture and cell senescence can be modulated by a membrane protease, in a process involving the ECM as a key regulator of nuclear stiffness under cell stress conditions.

  19. Loss of MT1-MMP causes cell senescence and nuclear defects which can be reversed by retinoic acid

    PubMed Central

    Gutiérrez-Fernández, Ana; Soria-Valles, Clara; Osorio, Fernando G; Gutiérrez-Abril, Jesús; Garabaya, Cecilia; Aguirre, Alina; Fueyo, Antonio; Fernández-García, María Soledad; Puente, Xose S; López-Otín, Carlos

    2015-01-01

    MT1-MMP (MMP14) is a collagenolytic enzyme located at the cell surface and implicated in extracellular matrix (ECM) remodeling. Mmp14−/− mice present dwarfism, bone abnormalities, and premature death. We demonstrate herein that the loss of MT1-MMP also causes cardiac defects and severe metabolic changes, and alters the cytoskeleton and the nuclear lamina structure. Moreover, the absence of MT1-MMP induces a senescent phenotype characterized by up-regulation of p16INK4a and p21CIP1/WAF1, increased activity of senescence-associated β-galactosidase, generation of a senescence-associated secretory phenotype, and somatotroph axis alterations. Consistent with the role of retinoic acid signaling in nuclear lamina stabilization, treatment of Mmp14−/− mice with all-trans retinoic acid reversed the nuclear lamina alterations, partially rescued the cell senescence phenotypes, ameliorated the pathological defects in bone, skin, and heart, and extended their life span. These results demonstrate that nuclear architecture and cell senescence can be modulated by a membrane protease, in a process involving the ECM as a key regulator of nuclear stiffness under cell stress conditions. PMID:25991604

  20. Long Term Exposure to Polyphenols of Artichoke (Cynara scolymus L.) Exerts Induction of Senescence Driven Growth Arrest in the MDA-MB231 Human Breast Cancer Cell Line

    PubMed Central

    Mileo, Anna Maria; Di Venere, Donato; Abbruzzese, Claudia; Miccadei, Stefania

    2015-01-01

    Polyphenolic extracts from the edible part of artichoke (Cynara scolymus L.) have been shown to be potential chemopreventive and anticancer dietary compounds. High doses of polyphenolic extracts (AEs) induce apoptosis and decrease the invasive potential of the human breast cancer cell line, MDA-MB231. However, the molecular mechanism underlying AEs antiproliferative effects is not completely understood. We demonstrate that chronic and low doses of AEs treatment at sublethal concentrations suppress human breast cancer cell growth via a caspases-independent mechanism. Furthermore, AEs exposure induces a significant increase of senescence-associated β-galactosidase (SA-β-gal) staining and upregulation of tumour suppressor genes, p16INK4a and p21Cip1/Waf1 in MDA-MB231 cells. AEs treatment leads to epigenetic alterations in cancer cells, modulating DNA hypomethylation and lysine acetylation levels in total proteins. Cell growth arrest correlates with increased reactive oxygen species (ROS) production in AEs treated breast cancer cells. Inhibition of ROS generation by N-acetylcysteine (NAC) attenuates the antiproliferative effect. These findings demonstrate that chronic AEs treatment inhibits breast cancer cell growth via the induction of premature senescence through epigenetic and ROS-mediated mechanisms. Our results suggest that artichoke polyphenols could be a promising dietary tool either in cancer chemoprevention or/and in cancer treatment as a nonconventional, adjuvant therapy. PMID:26180585

  1. Butyrate, an HDAC inhibitor, stimulates interplay between different posttranslational modifications of histone H3 and differently alters G1-specific cell cycle proteins in vascular smooth muscle cells.

    PubMed

    Mathew, Omana P; Ranganna, Kasturi; Yatsu, Frank M

    2010-12-01

    HDACs and HATs regulate histone acetylation, an epigenetic modification that controls chromatin structure and through it, gene expression. Butyrate, a dietary HDAC inhibitor, inhibits VSMC proliferation, a crucial factor in atherogenesis, and the principle mechanism in arterial and in-stent restenosis. Here, the link between antiproliferation action of butyrate and the portraits of global covalent modifications of histone H3 that it induces are characterized to understand the mechanics of butyrate-arrested VSMC proliferation. Analysis of histone H3 modifications specific to butyrate arrested VSMC proliferation display induction of histone H3-Lysine9 acetylation, inhibition of histone H3-Serine10 phosphorylation, reduction of histone H3-Lysine9 dimethylation and stimulation of histone H3-Lysine4 di-methylation, which is linked to transcriptional activation, cell cycle/mitosis, transcriptional suppression and activation, respectively. Conversely, untreated VSMCs exhibit inhibition of H3-Lysine9 acetylation, induction of H3-Serine10 phosphorylation, stimulation of H3-Lysine9 di-methylation and reduction in H3-Lysine4 di-methylation. Butyrate's cooperative effects on H3-Lysine9 acetylation and H3-Serine10 phosphorylation, and contrasting effects on di-methylation of H3-Lysine9 and H3-Lysine4 suggests that the interplay between these site-specific modifications cause distinct chromatin alterations that allow cyclin D1 and D3 induction, G1-specific cdk4, cdk6 and cdk2 downregulation, and upregulation of cdk inhibitors, p15INK4b and p21Cip1. Regardless of butyrate's effect on D-type cyclins, downregulation of G1-specific cdks and upregulation of cdk inhibitors by butyrate prevents cell cycle progression by failing to inactivate Rb. Overall, through chromatin remodeling, butyrate appears to differentially alter G1-specific cell cycle proteins to ensure proliferation arrest of VSMCs, a crucial cellular component of blood vessel wall.

  2. Structural and dynamical characterization of the Miz-1 zinc fingers 5-8 by solution-state NMR.

    PubMed

    Bernard, David; Bédard, Mikaël; Bilodeau, Josée; Lavigne, Pierre

    2013-10-01

    Myc-interacting zinc finger protein-1 (Miz-1) is a BTB/POZ transcription factor that activates the transcription of cytostatic genes, such as p15(INK4B) or p21(CIP1). The C-terminus of Miz-1 contains 13 consensus C2H2 zinc finger domains (ZF). ZFs 1-4 have been shown to interact with SMAD3/4, while the remaining ZFs are expected to bind the promoters of target genes. We have noted unusual features in ZF 5 and the linker between ZFs 5 and 6. Indeed, a glutamate is found instead of the conserved basic residue two positions before the second zinc-coordinating histidine on the ZF 5 helix, and the linker sequence is DTDKE in place of the classical TGEKP sequence. In a canonical ββα fold, such unusual primary structure elements should cause severe electrostatic repulsions. In this context, we have characterized the structure and the dynamics of a Miz-1 construct comprising ZFs 5-8 (Miz 5-8) by solution-state NMR. Whilst ZFs 5, 7 and 8 were shown to adopt the classical ββα fold for C2H2 ZFs, the number of long-range NOEs was insufficient to define a classical fold for ZF 6. We show by using (15)N-relaxation dispersion experiments that this lack of NOEs is due to the presence of extensive motions on the μs-ms timescale. Since this negatively charged region would have to be located near the phosphodiester backbone in a DNA complex, we propose that in addition to promoting conformational searches, it could serve as a hinge region to keep ZFs 1-4 away from DNA.

  3. The anti-inflammatory effect of melatonin in SH-SY5Y neuroblastoma cells exposed to sublethal dose of hydrogen peroxide.

    PubMed

    Nopparat, Chutikorn; Chantadul, Varunya; Permpoonputtana, Kannika; Govitrapong, Piyarat

    2017-04-10

    Brain inflammaging is considered as one of the underlying factors of neurodegenerative diseases. The present study aimed to investigate the effects of melatonin, an endogenous indoleamine mainly synthesized by the pineal gland, on hydrogen peroxide (H2O2)-induced inflammaging state in SH-SY5Y cells. Our data showed that p21(Cip1) and p16(INK4a), cell cycle arrest markers, and the number of senescence-associated β-galactosidase (SA-βgal) staining increased significantly in H2O2-treated cells. Melatonin treatment could reverse this effect. Flow cytometry analysis showed a significantly higher percentage in the G0/G1 phase and a lower proportion in the S phase of H2O2 treated cells. Cells pretreated with H2O2 showed a dramatic decrease in the formation of Ki67 immunoactivity while the treatment with melatonin increased Ki67-positive cell. Both mRNA and protein expression levels of the pro-inflammatory cytokines, interleukin-1β (IL-1β), IL-6 and, tumor necrosis factor-α (TNF-α) which were increased after induction with H2O2, could be attenuated by melatonin. In addition, melatonin decreased the phospho-nuclear factor kappa B (pNF-κB) expression and prevented its nuclear translocation, as well as abrogated the reduction of nuclear factor erythroid 2-related factor 2 (Nrf2) in SH-SY5Y cells exposed to H2O2. The present data suggested the importance of melatonin on ameliorating inflammation in SH-SY5Y cells.

  4. Loss of heterozygosity at chromosome 9p21 is a frequent finding in enteropathy-type T-cell lymphoma.

    PubMed

    Obermann, E C; Diss, T C; Hamoudi, R A; Munson, P; Wilkins, B S; Camozzi, M L P; Isaacson, P G; Du, M Q; Dogan, A

    2004-02-01

    Enteropathy-type T-cell lymphoma (ETL) and ulcerative jejunitis (UJ) are rare disorders often occurring in patients with coeliac disease. The genetic events associated with the accumulation of intraepithelial lymphocytes in coeliac disease and tumour development are largely unknown. Deletions at chromosome 9p21, which harbours the tumour suppressor genes p14/ARF, p15/INK4b, and p16/INK4a, and 17p13, where p53 is located, are associated with the development and progression of lymphomas. To examine whether deletions at 9p21 and 17p13 play a role in ETL, 22 cases of ETL and seven cases of UJ were screened for loss of heterozygosity (LOH) by tissue microdissection and polymerase chain reaction (PCR) analysis for microsatellite markers. Furthermore, p53 and p16 protein expression was examined by immunohistochemistry. In addition, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis for detection of mutations in exons 5-8 of the p53 gene was performed in five cases of ETL and three cases of UJ. LOH was found in at least one microsatellite marker at the 9p21 locus in 8 of 22 (36%) ETLs, but not in UJ. Five of nine (56%) tumours composed of large cells showed LOH at 9p21, as opposed to two of eight (25%) tumours with small- or medium-sized cell morphology. The region spanning the p14/p15/p16 gene locus was most frequently affected (five cases); LOH at these markers coincided with loss of p16 protein expression in all of these cases. p53 overexpression was demonstrated in all ETLs examined and in four of seven cases of UJ. However, no alterations of the p53 gene were detected by LOH or PCR-SSCP analysis. The results of this study show that LOH at chromosome 9p21 is frequent in ETL, especially in tumours with large cell morphology; this finding suggests that gene loss at this locus may play a role in the development of ETL.

  5. High-resolution genomic copy number profiling of glioblastoma multiforme by single nucleotide polymorphism DNA microarray.

    PubMed

    Yin, Dong; Ogawa, Seishi; Kawamata, Norihiko; Tunici, Patrizia; Finocchiaro, Gaetano; Eoli, Marica; Ruckert, Christian; Huynh, Thien; Liu, Gentao; Kato, Motohiro; Sanada, Masashi; Jauch, Anna; Dugas, Martin; Black, Keith L; Koeffler, H Phillip

    2009-05-01

    Glioblastoma multiforme (GBM) is an extremely malignant brain tumor. To identify new genomic alterations in GBM, genomic DNA of tumor tissue/explants from 55 individuals and 6 GBM cell lines were examined using single nucleotide polymorphism DNA microarray (SNP-Chip). Further gene expression analysis relied on an additional 56 GBM samples. SNP-Chip results were validated using several techniques, including quantitative PCR (Q-PCR), nucleotide sequencing, and a combination of Q-PCR and detection of microsatellite markers for loss of heterozygosity with normal copy number [acquired uniparental disomy (AUPD)]. Whole genomic DNA copy number in each GBM sample was profiled by SNP-Chip. Several signaling pathways were frequently abnormal. Either the p16(INK4A)/p15(INK4B)-CDK4/6-pRb or p14(ARF)-MDM2/4-p53 pathways were abnormal in 89% (49 of 55) of cases. Simultaneous abnormalities of both pathways occurred in 84% (46 of 55) samples. The phosphoinositide 3-kinase pathway was altered in 71% (39 of 55) GBMs either by deletion of PTEN or amplification of epidermal growth factor receptor and/or vascular endothelial growth factor receptor/platelet-derived growth factor receptor alpha. Deletion of chromosome 6q26-27 often occurred (16 of 55 samples). The minimum common deleted region included PARK2, PACRG, QKI, and PDE10A genes. Further reverse transcription Q-PCR studies showed that PARK2 expression was decreased in another collection of GBMs at a frequency of 61% (34 of 56) of samples. The 1p36.23 region was deleted in 35% (19 of 55) of samples. Notably, three samples had homozygous deletion encompassing this site. Also, a novel internal deletion of a putative tumor suppressor gene, LRP1B, was discovered causing an aberrant protein. AUPDs occurred in 58% (32 of 55) of the GBM samples and five of six GBM cell lines. A common AUPD was found at chromosome 17p13.3-12 (included p53 gene) in 13 of 61 samples and cell lines. Single-strand conformational polymorphism and nucleotide

  6. A steroid like phytochemical Antcin M is an anti-aging reagent that eliminates hyperglycemia-accelerated premature senescence in dermal fibroblasts by direct activation of Nrf2 and SIRT-1

    PubMed Central

    Senthil, Kumar K.J.; Gokila, Vani M.; Mau, Jeng-Leun; Lin, Chin-Chung; Chu, Fang-Hua; Wei, Chia-Cheng; Liao, Vivian Hsiu-Chuan; Wang, Sheng-Yang

    2016-01-01

    The present study revealed the anti-aging properties of antcin M (ANM) and elucidated the molecular mechanism underlying the effects. We found that exposure of human normal dermal fibroblasts (HNDFs) to high-glucose (HG, 30 mM) for 3 days, accelerated G0/G1 phase arrest and senescence. Indeed, co-treatment with ANM (10 μM) significantly attenuated HG-induced growth arrest and promoted cell proliferation. Further molecular analysis revealed that ANM blocked the HG-induced reduction in G1-S transition regulatory proteins such as cyclin D, cyclin E, CDK4, CDK6, CDK2 and protein retinoblastoma (pRb). In addition, treatment with ANM eliminated HG-induced reactive oxygen species (ROS) through the induction of anti-oxidant genes, HO-1 and NQO-1 via transcriptional activation of Nrf2. Moreover, treatment with ANM abolished HG-induced SIPS as evidenced by reduced senescence-associated β-galactosidase (SA-β-gal) activity. This effect was further confirmed by reduction in senescence-associated marker proteins including, p21CIP1, p16INK4A, and p53/FoxO1 acetylation. Also, the HG-induced decline in aging-related marker protein SMP30 was rescued by ANM. Furthermore, treatment with ANM increased SIRT-1 expression, and prevented SIRT-1 depletion. This protection was consistent with inhibition of SIRT-1 phosphorylation at Ser47 followed by blocking its upstream kinases, p38 MAPK and JNK/SAPK. Further analysis revealed that ANM partially protected HG-induced senescence in SIRT-1 silenced cells. A similar effect was also observed in Nrf2 silenced cells. However, a complete loss of protection was observed in both Nrf2 and SIRT-1 knockdown cells suggesting that both induction of Nrf2-mediated anti-oxidant defense and SIRT-1-mediated deacetylation activity contribute to the anti-aging properties of ANM in vitro. Result of in vivo studies shows that ANM-treated C. elegens exhibits an increased survival rate during HG-induced oxidative stress insult. Furthermore, ANM significantly

  7. A steroid like phytochemical Antcin M is an anti-aging reagent that eliminates hyperglycemia-accelerated premature senescence in dermal fibroblasts by direct activation of Nrf2 and SIRT-1.

    PubMed

    Senthil, Kumar K J; Gokila, Vani M; Mau, Jeng-Leun; Lin, Chin-Chung; Chu, Fang-Hua; Wei, Chia-Cheng; Liao, Vivian Hsiu-Chuan; Wang, Sheng-Yang

    2016-09-27

    The present study revealed the anti-aging properties of antcin M (ANM) and elucidated the molecular mechanism underlying the effects. We found that exposure of human normal dermal fibroblasts (HNDFs) to high-glucose (HG, 30 mM) for 3 days, accelerated G0/G1 phase arrest and senescence. Indeed, co-treatment with ANM (10 µM) significantly attenuated HG-induced growth arrest and promoted cell proliferation. Further molecular analysis revealed that ANM blocked the HG-induced reduction in G1-S transition regulatory proteins such as cyclin D, cyclin E, CDK4, CDK6, CDK2 and protein retinoblastoma (pRb). In addition, treatment with ANM eliminated HG-induced reactive oxygen species (ROS) through the induction of anti-oxidant genes, HO-1 and NQO-1 via transcriptional activation of Nrf2. Moreover, treatment with ANM abolished HG-induced SIPS as evidenced by reduced senescence-associated β-galactosidase (SA-β-gal) activity. This effect was further confirmed by reduction in senescence-associated marker proteins including, p21CIP1, p16INK4A, and p53/FoxO1 acetylation. Also, the HG-induced decline in aging-related marker protein SMP30 was rescued by ANM. Furthermore, treatment with ANM increased SIRT-1 expression, and prevented SIRT-1 depletion. This protection was consistent with inhibition of SIRT-1 phosphorylation at Ser47 followed by blocking its upstream kinases, p38 MAPK and JNK/SAPK. Further analysis revealed that ANM partially protected HG-induced senescence in SIRT-1 silenced cells. A similar effect was also observed in Nrf2 silenced cells. However, a complete loss of protection was observed in both Nrf2 and SIRT-1 knockdown cells suggesting that both induction of Nrf2-mediated anti-oxidant defense and SIRT-1-mediated deacetylation activity contribute to the anti-aging properties of ANM in vitro. Result of in vivo studies shows that ANM-treated C. elegens exhibits an increased survival rate during HG-induced oxidative stress insult. Furthermore, ANM significantly

  8. Dysregulation of the Bmi-1/p16(Ink⁴a) pathway provokes an aging-associated decline of submandibular gland function.

    PubMed

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

    2015-08-01

    Bmi-1 prevents stem cell aging, at least partly, by blocking expression of the cyclin-dependent kinase inhibitor p16(Ink4a) . Therefore, dysregulation of the Bmi-1/p16(Ink4a) pathway is considered key to the loss of tissue homeostasis and development of associated degenerative diseases during aging. However, because Bmi-1 knockout (KO) mice die within 20 weeks after birth, it is difficult to determine exactly where and when dysregulation of the Bmi-1/p16(Ink4a) pathway occurs during aging in vivo. Using real-time in vivo imaging of p16(Ink4a) expression in Bmi-1-KO mice, we uncovered a novel function of the Bmi-1/p16(Ink4a) pathway in controlling homeostasis of the submandibular glands (SMGs), which secrete saliva into the oral cavity. This pathway is dysregulated during aging in vivo, leading to induction of p16(Ink4a) expression and subsequent declined SMG function. These findings will advance our understanding of the molecular mechanisms underlying the aging-related decline of SMG function and associated salivary gland hypofunction, which is particularly problematic among the elderly.

  9. Correlation of cell cycle regulatory proteins (p53 and p16(ink)⁴(a)) and bcl-2 oncoprotein with mitotic index and thickness of primary cutaneous malignant melanoma.

    PubMed

    Kostov, Miloš; Mijović, Zaklina; Mihailović, Dragan; Cerović, Snežana; Stojanović, Miroslav; Jelić, Marija

    2010-11-01

    The purpose of the study was to determine the frequency of expression p53 and p16INK4a proteins and bcl-2 oncoprotein in malignant skin melanoma and to determine their correlation with the proliferative index and tumor thickness. The study involved 53 patients: 27 (51%) male and 26 (49%) female. Mitotic index showed a correlation with p53 protein expression, a negative correlation with p16INK4a protein expression. Statistically significant correlations were determined between the Breslow tumor thickness, Clark invasion level and p53 protein expression, as well as Breslow tumor thickness and bcl-2 oncoprotein expression (p<0.05), whereas there was no correlation between the p16INK4a protein expression and melanoma thicknes and Clark invasion level. Overexpression p53 protein and bcl-2 oncoprotein, with the loss p16INK4a protein of expression in the nodular melanoma, confirms a frequent loss of function of these tumor suppressor gene and oncogene, and indicates a vertical tumor growth phase. The loss of tumor suppression function the p53 protein and bcl-2 oncoprotein overexpression in cutaneous melanoma correlates with larger tumor thickness, whereas the overexpression of mutated p53 protein and loss p16INK4a protein of expression indicate a higher proliferative tumour potential. Therefore, these evaluated proteins may be the aggressive biological tumour activity markers.

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

    SciTech Connect

    Zhao Yanhua; Zhang Yan; Yang Zhen; Li, Albert; Dong Jianli

    2008-06-06

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

  11. Stromal-Epithelial Interactions and Tamoxifen-Sensitivity: A Bench-to-Bedside Model of Chemoprevention

    DTIC Science & Technology

    2008-05-01

    Immunohistochemistry. ERa protein expression was tested in RPFNA cytology specimens by immuno - histochemistry (IHC) by a modification of published methods (32...6870 – 5. 3. Holst CR, Nuovo GJ, Esteller M, et al. Methylation of p16 (INK4a) promoters occurs in vivo in histologically normal human mammary epithelia

  12. Oncogenic Functions of cdK4 and cdK6

    DTIC Science & Technology

    1998-07-01

    frequently leads to dysregulated cdk4/cdk6 activity in human tumors, as do mutations in cdk4 that prevent its association with p16INK4a ( Motokura et al...novel cyclin D partner. Mol. Cell. Biol. 14: 2077-2086. Morgan, D. 1995. Principals of CDK regulation. Nature 474: 131-134. Motokura , T., T. Bloom

  13. Impact of HPV infection on oral squamous cell carcinoma

    PubMed Central

    Götz, Carolin; Drecoll, Enken; Straub, Melanie; Bissinger, Oliver; Wolff, Klaus-Dietrich; Kolk, Andreas

    2016-01-01

    Background Head and neck squamous cell carcinomas (HNSCC) are often divided by their aetiology. Noxae associated collectives are compared with the human papilloma virus (HPV)-associated group, whereas different localisations of oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinomas are mostly discussed as one single group. Our aim was to show that classification by aetiology is not appropriate for OSCC. Results HPV DNA was detected by PCR in 7 (3.47%) patients, and we identified 12 (5.94%) positive (+) cases by p16INK4a immunostaining. Only 4 (1.98%) of the p16INK4a+ cases were + for HPV using PCR. Our homogenous collective of OSCC allowed us to compare HPV+ and HPV negative (−) patients without creating bias for tumour localisation, age, gender or tumour stage. Materials and methods After testing OSCC samples for HPV positivity, we compared the results of two commonly used HPV detection methods, p16INK4a immunostaining and HPV DNA-related PCR, on 202 OSCC patients. HPV subtypes were determined with an HPV LCD Array Kit. Clinicopathological features of the patients were analysed, and the disease specific survival rates (DSS) for HPV+ and HPV− patients were obtained. Conclusions p16INK4a immunostaining is a not a reliable HPV detection method for OSCC. Positive p16INK4a immunostaining did not agree with + results from PCR of HPV DNA. Furthermore, the influence of HPV-related oncogenic transformation in OSCC is overestimated. The significance of HPV infection remains clinically unclear, and its influence on survival rates is not relevant to OSCC cases. PMID:27732948

  14. Prevalence of human papillomavirus in oral squamous cell carcinomas in northern Spain.

    PubMed

    Rodríguez-Santamarta, Tania; Rodrigo, Juan Pablo; García-Pedrero, Juana M; Álvarez-Teijeiro, Saúl; Ángeles Villaronga, M; Suárez-Fernández, Laura; Alvarez-Argüelles, Marta E; Astudillo, Aurora; de Vicente, Juan Carlos

    2016-12-01

    The aim of this study was to analyze the prevalence of high-risk HPV in oral squamous cell carcinoma (OSCC) in a northern Spanish population, as well as to ascertain the prognostic role of p16(INK4a) expression. The examination samples were collected from paraffin tissue blocks, from 125 patients surgically treated between 1996 and 2007. All cases were histologically evaluated, and the presence of HPV was assessed by p16 and p53immunohistochemistry followed by DNA detection by in situ hybridization (ISH) and polymerase chain reaction (PCR) amplification using the combination of consensus primers MY11/GP6 + . Fourteen cases (11 %) were p16-immunopositive, and p53 was scored positive in 73 cases (58 %). Five cases (4 %) showed a simultaneous p16-positive and p53-negative immunostaining. ISH was negative in all the cases. Among the p16INK4a-immunopositive cases, PCR amplification failed to reveal HPV DNA in any tumor samples. There were no statistically significant differences in any clinical or pathological characteristics of the patients regarding p16(INK4a) expression. T classification, neck-node metastasis, and clinical stage showed outcome relevance. However, no significant differences in cause-specific survival based on p16INK4a were observed. We did not find any high-risk HPV types in our patients, thus, are unlikely that HPV has an important role in the etiology of OSCC. p16INK4a protein was neither an accurate marker of HPV infection nor a prognosis marker in OSCC.

  15. Delayed cell cycle progression in selenoprotein W depleted cells is regulated by a mitogen-activated protein kinase kinase 4–p38–p53 pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a p53- and p21Cip1-dependent G1-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser33 in p53, which is associated with decreased p53...

  16. Altered expression of G1/S phase cell cycle regulators in placental mesenchymal stromal cells derived from preeclamptic pregnancies with fetal-placental compromise

    PubMed Central

    Giuffrida, Domenica; Masturzo, Bianca; Eva, Carola; Todros, Tullia

    2017-01-01

    ABSTRACT Herein, we evaluated whether Placental Mesenchymal Stromal Cells (PDMSCs) derived from normal and Preeclamptic (PE) placentae presented differences in the expression of G1/S-phase regulators p16INK4A, p18INK4C, CDK4 and CDK6. Finally, we investigated normal and PE-PDMSCs paracrine effects on JunB, Cyclin D1, p16INK4A, p18INK4C, CDK4 and CDK6 expressions in physiological term villous explants. PDMSCs were isolated from physiological (n = 20) and PE (n = 24) placentae. Passage three normal and PE-PDMSC and conditioned media (CM) were collected after 48h. Physiological villous explants (n = 60) were treated for 72h with normal or PE-PDMSCs CM. Explants viability was assessed by Lactate Dehydrogenase Cytotoxicity assay. Cyclin D1 localization was evaluated by Immuofluorescence (IF) while JunB, Cyclin-D1 p16INK4A, p18INK4C, CDK4 and CDK6 levels were assessed by Real Time PCR and Western Blot assay. We reported significantly increased p16INK4A and p18INK4C expression in PE- relative to normal PDMSCs while no differences in CDK4 and CDK6 levels were detected. Explants viability was not affected by normal or PE-PDMSCs CM. Normal PDMSCs CM increased JunB, p16INK4 and p18INK4C and decreased Cyclin-D1 in placental tissues. In contrast, PE-PDMSCs CM induced JunB downregulation and Cyclin D1 increase in placental explants. Cyclin D1 IF staining showed that CM treatment targeted mainly the syncytiotrophoblast. We showed Cyclin D1-p16INK4A/p18INK4C altered pathway in PE-PDMSCs demonstrating an aberrant G1/S phase transition in these pathological cells. The abnormal Cyclin D1-p16INK4A/p18INK4C expression in explants conditioned by PE-PDMSCs media suggest a key contribution of mesenchymal cells to the altered trophoblast cell cycle regulation typical of PE pregnancies with fetal-placental compromise. PMID:27937072

  17. Inhibition of histone deacetylases by chlamydocin induces apoptosis and proteasome-mediated degradation of survivin.

    PubMed

    De Schepper, Stefanie; Bruwiere, Hélène; Verhulst, Tinne; Steller, Ulf; Andries, Luc; Wouters, Walter; Janicot, Michel; Arts, Janine; Van Heusden, Jim

    2003-02-01

    The naturally occurring cyclic tetrapeptide chlamydocin is a very potent inhibitor of cell proliferation. Here we show that chlamydocin is a highly potent histone deacetylase (HDAC) inhibitor, inhibiting HDAC activity in vitro with an IC(50) of 1.3 nM. Like other HDAC inhibitors, chlamydocin induces the accumulation of hyperacetylated histones H3 and H4 in A2780 ovarian cancer cells, increases the expression of p21(cip1/waf1), and causes an accumulation of cells in G(2)/M phase of the cell cycle. In addition, chlamydocin induces apoptosis by activating caspase-3, which in turn leads to the cleavage of p21(cip1/waf1) into a 15-kDa breakdown product and drives cells from growth arrest into apoptosis. Concomitant with the activation of caspase-3 and cleavage of p21(cip1/waf1), chlamydocin decreases the protein level of survivin, a member of the inhibitor of apoptosis protein family that is selectively expressed in tumors. Although our data indicate a potential link between degradation of survivin and activation of the apoptotic pathway induced by HDAC inhibitors, stable overexpression of survivin does not suppress the activation of caspase-3 or cleavage of p21(cip1/waf1) induced by chlamydocin treatment. The decrease of survivin protein level is mediated by degradation via proteasomes since it can be inhibited by specific proteasome inhibitors. Taken together, our results show that induction of apoptosis by chlamydocin involves caspase-dependent cleavage of p21(cip1/waf1), which is strikingly associated with proteasome-mediated degradation of survivin.

  18. Mucosal alpha-papillomaviruses are not associated with esophageal squamous cell carcinomas: Lack of mechanistic evidence from South Africa, China and Iran and from a world-wide meta-analysis.

    PubMed

    Halec, Gordana; Schmitt, Markus; Egger, Sam; Abnet, Christian C; Babb, Chantal; Dawsey, Sanford M; Flechtenmacher, Christa; Gheit, Tarik; Hale, Martin; Holzinger, Dana; Malekzadeh, Reza; Taylor, Philip R; Tommasino, Massimo; Urban, Margaret I; Waterboer, Tim; Pawlita, Michael; Sitas, Freddy

    2016-07-01

    Epidemiological and mechanistic evidence on the causative role of human papillomaviruses (HPV) in esophageal squamous cell carcinoma (ESCC) is unclear. We retrieved alcohol- and formalin-fixed paraffin-embedded ESCC tissues from 133 patients seropositive for antibodies against HPV early proteins, from high-incidence ESCC regions: South Africa, China and Iran. With rigorous care to prevent nucleic acid contamination, we analyzed these tissues for the presence of 51 mucosotropic human alpha-papillomaviruses by two sensitive, broad-spectrum genotyping methods, and for the markers of HPV-transformed phenotype: (i) HPV16/18 viral loads by quantitative real-time PCR, (ii) type-specific viral mRNA by E6*I/E6 full-length RT-PCR assays and (iii) expression of cellular protein p16(INK4a). Of 118 analyzable ESCC tissues, 10 (8%) were positive for DNA of HPV types: 16 (4 tumors); 33, 35, 45 (1 tumor each); 11 (2 tumors) and 16, 70 double infection (1 tumor). Inconsistent HPV DNA+ findings by two genotyping methods and negativity in qPCR indicated very low viral loads. A single HPV16 DNA+ tumor additionally harbored HPV16 E6*I mRNA but was p16(INK4a) negative (HPV16 E1 seropositive patient). Another HPV16 DNA+ tumor from an HPV16 E6 seropositive patient showed p16(INK4a) upregulation but no HPV16 mRNA. In the tumor tissues of these serologically preselected ESCC patients, we did not find consistent presence of HPV DNA, HPV mRNA or p16(INK4a) upregulation. These results were supported by a meta-analysis of 14 other similar studies regarding HPV-transformation of ESCC. Our study does not support the etiological role of the 51 analyzed mucosotropic HPV types in the ESCC carcinogenesis.

  19. Molecular analysis of the Ink4a/Rb1-Arf/Tp53 pathways in radon-induced rat lung tumors.

    PubMed

    Bastide, Kristell; Guilly, Marie-Noëlle; Bernaudin, Jean-François; Joubert, Christophe; Lectard, Bruno; Levalois, Céline; Malfoy, Bernard; Chevillard, Sylvie

    2009-03-01

    Inhalation of radon is closely associated with an increased risk of lung cancers. While the involvement of Ink4a in lung tumor development has been widely described, the tumor suppressor gene has not been studied in radon-induced lung tumors. In this study, loss of heterozygosity (LOH) analysis of the Cdkn2a locus, common to the Ink4a and Arf genes, was performed on 33 radon-induced rat lung tumors and showed a DNA loss in 50% of cases. The analysis of p16(Ink4a) protein expression by immunohistochemistry revealed that 50% of the tumors were negative for this protein. Looking for the origin of this lack of expression, we observed a low frequency of homozygous deletion (6%), a lack of mutation, an absence of correlation between promoter methylation and Ink4a mRNA expression and no correlation between LOH and protein expression. However, a tendency for an inverse correlation between p16(Ink4a) and pRb protein expression was observed. The expressions of p19Arf, Mmd2 and Mdm4 were not deregulated and only 14% of the tumors were mutated for Tp53. These results indicated that Ink4a/Cdk4/Rb1 pathway deregulation, more than Arf/Mdm2/Tp53 pathway, has a major role in the development of these tumors through p16(Ink4a) deregulation. However, all known mechanisms of inactivation of the pathway do not play a recurrent role in these tumors and the actual origin of the lack of p16(Ink4a) protein expression remains to be established.

  20. FOXP1 controls mesenchymal stem cell commitment and senescence during skeletal aging.

    PubMed

    Li, Hanjun; Liu, Pei; Xu, Shuqin; Li, Yinghua; Dekker, Joseph D; Li, Baojie; Fan, Ying; Zhang, Zhenlin; Hong, Yang; Yang, Gong; Tang, Tingting; Ren, Yongxin; Tucker, Haley O; Yao, Zhengju; Guo, Xizhi

    2017-04-03

    A hallmark of aged mesenchymal stem/progenitor cells (MSCs) in bone marrow is the pivot of differentiation potency from osteoblast to adipocyte coupled with a decrease in self-renewal capacity. However, how these cellular events are orchestrated in the aging progress is not fully understood. In this study, we have used molecular and genetic approaches to investigate the role of forkhead box P1 (FOXP1) in transcriptional control of MSC senescence. In bone marrow MSCs, FOXP1 expression levels declined with age in an inverse manner with those of the senescence marker p16INK4A. Conditional depletion of Foxp1 in bone marrow MSCs led to premature aging characteristics, including increased bone marrow adiposity, decreased bone mass, and impaired MSC self-renewal capacity in mice. At the molecular level, FOXP1 regulated cell-fate choice of MSCs through interactions with the CEBPβ/δ complex and recombination signal binding protein for immunoglobulin κ J region (RBPjκ), key modulators of adipogenesis and osteogenesis, respectively. Loss of p16INK4A in Foxp1-deficient MSCs partially rescued the defects in replication capacity and bone mass accrual. Promoter occupancy analyses revealed that FOXP1 directly represses transcription of p16INK4A. These results indicate that FOXP1 attenuates MSC senescence by orchestrating their cell-fate switch while maintaining their replicative capacity in a dose- and age-dependent manner.

  1. Promoter Methylation of RASSF1A Associates to Adult Secondary Glioblastomas and Pediatric Glioblastomas

    PubMed Central

    Muñoz, Jorge; Inda, María del Mar; Lázcoz, Paula; Zazpe, Idoya; Fan, Xing; Alfaro, Jorge; Tuñón, Teresa; Rey, Juan A.; Castresana, Javier S.

    2012-01-01

    While allelic losses and mutations of tumor suppressor genes implicated in the etiology of astrocytoma have been widely assessed, the role of epigenetics is still a matter of study. We analyzed the frequency of promoter hypermethylation by methylation-specific PCR (MSP) in five tumor suppressor genes (PTEN, MGMT, RASSF1A, p14ARF, and p16INK4A), in astrocytoma samples and cell lines. RASSF1A was the most frequently hypermethylated gene in all grades of astrocytoma samples, in cell lines, and in adult secondary GBM. It was followed by MGMT. PTEN showed a slight methylation signal in only one GBM and one pilocytic astrocytoma, and in two cell lines; while p14ARF and p16INK4A did not show any evidence of methylation in primary tumors or cell lines. In pediatric GBM, RASSF1A was again the most frequently altered gene, followed by MGMT; PTEN, p14 and p16 showed no alterations. Lack or reduced expression of RASSF1A in cell lines was correlated with the presence of methylation. RASSF1A promoter hypermethylation might be used as a diagnostic marker for secondary GBM and pediatric GBM. Promoter hypermethylation might not be an important inactivation mechanism in other genes like PTEN, p14ARF and p16INK4A, in which other alterations (mutations, homozygous deletions) are prevalent. PMID:22389839

  2. Fully automated screening of immunocytochemically stained specimens for early cancer detection

    NASA Astrophysics Data System (ADS)

    Bell, André A.; Schneider, Timna E.; Müller-Frank, Dirk A. C.; Meyer-Ebrecht, Dietrich; Böcking, Alfred; Aach, Til

    2007-03-01

    Cytopathological cancer diagnoses can be obtained less invasive than histopathological investigations. Cells containing specimens can be obtained without pain or discomfort, bloody biopsies are avoided, and the diagnosis can, in some cases, even be made earlier. Since no tissue biopsies are necessary these methods can also be used in screening applications, e.g., for cervical cancer. Among the cytopathological methods a diagnosis based on the analysis of the amount of DNA in individual cells achieves high sensitivity and specificity. Yet this analysis is time consuming, which is prohibitive for a screening application. Hence, it will be advantageous to retain, by a preceding selection step, only a subset of suspicious specimens. This can be achieved using highly sensitive immunocytochemical markers like p16 ink4a for preselection of suspicious cells and specimens. We present a method to fully automatically acquire images at distinct positions at cytological specimens using a conventional computer controlled microscope and an autofocus algorithm. Based on the thus obtained images we automatically detect p16 ink4a-positive objects. This detection in turn is based on an analysis of the color distribution of the p16 ink4a marker in the Lab-colorspace. A Gaussian-mixture-model is used to describe this distribution and the method described in this paper so far achieves a sensitivity of up to 90%.

  3. Interphase fish analysis of cell cycle genes in asbestos-treated human mesothelial cells (HMC), SV40-transformed HMC (MeT-5A) and mesothelioma cells (COLO).

    PubMed

    Dopp, Elke; Poser, Ina; Papp, Thilo

    2002-01-01

    The epidemiologic association between asbestos exposure and human malignant mesothelioma is well established. However, the molecular mechanisms linking asbestos exposure of humans and the subsequent mesothelioma formation is not well understood. The most frequent genetic changes found so far in human malignant mesothelioma (HMM) are deletions and point mutations in the tumor suppressor genes p16INK4a and NF2. Whereas homozygous deletions appear to be the predominant mechanism leading to p16/CDKN2A inactivation, inactivating point mutations coupled with allelic loss mainly occur at the NF2 locus. In the present study, asbestos-treated human mesothelial cells (HMC), SV40-transformed human mesothelial cells (MeT-5A) and a human mesothelioma cell line (COLO) were investigated for genetic changes of cell cycle genes (cyclin D1, p16INK4a, RB1, CDK2) using multicolor fluorescence in situ hybridization (mFISH) in interphase cells. The results show that cyclin D1 is unaffected in all investigated cells. The p16INK4a gene locus was shown to be mutated in COLO cells but not in HMC. After labeling of CDK2 and RB1, hemizygous loss of one allele of each gene was observed in asbestos-treated HMC whereas gene amplification of these genes was detectable in MeT-5A and COLO cells. Our data indicate that disarrangement of the RB1 dependent pathway seems to be involved in mesothelioma formation.

  4. Detection of hemizygous deletions in genomic DNA from leukaemia specimens for the diagnosis of patients.

    PubMed

    Kees, Ursula R; Terry, Philippa A; Ford, Jette; Everett, Janet; Murch, Ashleigh; de Klerk, Nick; Baker, David L

    2005-02-01

    Hemizygous deletions in genomic DNA appear to play an important role in tumorigenesis. The loss or inactivation of tumour suppressor genes (TSGs) is of critical importance in most malignancies, and has been shown to affect response to therapy. Here, we report a quantitative real-time polymerase chain reaction (qPCR) designed to detect two TSGs at the CDKN2A locus, p16(INK4A) and p14(ARF) that allows the detection of hemizygous deletions. Testing by qPCR of 18 bone marrow specimens from paediatric acute lymphoblastic leukaemia (ALL) patients at diagnosis revealed nine to be GG, six to be GD and three to be DD for exon 2 of p14(ARF)/p16(INK4A), concordant with Southern blotting analysis. A panel of 13 ALL cell lines was investigated for deletions at the CDKN2A locus and one of the lines, typed as GD for all exons, was further assessed by fluorescence in situ hybridisation, confirming the qPCR findings. The expression levels of p16(INK4A) and p14(ARF) were measured in all cell lines and these quantitative reverse transcriptase PCR results also agreed with the typing by qPCR. The qPCR method described is suitable for detection of hemizygous loss in primary patient material and the accuracy of the method was verified by three independent techniques.

  5. The ability of antigen, but not interleukin-2, to promote n-butyrate-induced T helper 1 cell anergy is associated with increased expression and altered association patterns of cyclin-dependent kinase inhibitors

    PubMed Central

    Jackson, Stephanie K; DeLoose, Annick; Gilbert, Kathleen M

    2002-01-01

    The ability of the cell cycle inhibitor n-butyrate to induce T helper 1 (Th1) cell anergy is dependent upon its ability to block the cell cycle progression of activated Th1 cells in G1. Results reported here show that although both interleukin (IL)-2 and antigen (Ag) push Th1 cells into G1 where they are blocked by n-butyrate, only the Ag-activated Th1 cells demonstrate functional anergy once the n-butyrate has been removed from the culture. Because n-butyrate-induced Th1 cell anergy has been linked to increased expression of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, mechanistic experiments focused on the role of these inhibitors. It was found that when Th1 cells were reincubated in Ag-stimulated secondary cultures, the Th1 cells previously exposed to Ag and n-butyrate (anergic Th1 cells) demonstrated a cumulative increase in p21Cip1 and p27Kip1 when compared with Th1 cells previously exposed to recombinant (r)IL-2 and n-butyrate (non-anergic Th1 cells). p27Kip1 in the anergic Th1 cells from the secondary cultures was associated with cyclin-dependent kinases (cdks). In contrast, p21Cip1 in the anergic Th1 cells, although present at high levels, did not associate significantly with cdks, suggesting that p21Cip1 may target some other protein in the anergic Th1 cells. Taken together, these findings suggest that Th1 cell exposure to Ag and n-butyrate, rather than IL-2 and n-butyrate, is needed to induce the cumulative increase in p21Cip1 and p27Kip1 that is associated with the proliferative unresponsiveness in anergic Th1 cells. In addition, p21Cip1 may inhibit proliferation in the anergic Th1 cells by some mechanism other than suppression of cdks that is unique to the induction of Th1 cell anergy. PMID:12153511

  6. The ability of antigen, but not interleukin-2, to promote n-butyrate-induced T helper 1 cell anergy is associated with increased expression and altered association patterns of cyclin-dependent kinase inhibitors.

    PubMed

    Jackson, Stephanie K; DeLoose, Annick; Gilbert, Kathleen M

    2002-08-01

    The ability of the cell cycle inhibitor n-butyrate to induce T helper 1 (Th1) cell anergy is dependent upon its ability to block the cell cycle progression of activated Th1 cells in G1. Results reported here show that although both interleukin (IL)-2 and antigen (Ag) push Th1 cells into G1 where they are blocked by n-butyrate, only the Ag-activated Th1 cells demonstrate functional anergy once the n-butyrate has been removed from the culture. Because n-butyrate-induced Th1 cell anergy has been linked to increased expression of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, mechanistic experiments focused on the role of these inhibitors. It was found that when Th1 cells were reincubated in Ag-stimulated secondary cultures, the Th1 cells previously exposed to Ag and n-butyrate (anergic Th1 cells) demonstrated a cumulative increase in p21Cip1 and p27Kip1 when compared with Th1 cells previously exposed to recombinant (r)IL-2 and n-butyrate (non-anergic Th1 cells). p27Kip1 in the anergic Th1 cells from the secondary cultures was associated with cyclin-dependent kinases (cdks). In contrast, p21Cip1 in the anergic Th1 cells, although present at high levels, did not associate significantly with cdks, suggesting that p21Cip1 may target some other protein in the anergic Th1 cells. Taken together, these findings suggest that Th1 cell exposure to Ag and n-butyrate, rather than IL-2 and n-butyrate, is needed to induce the cumulative increase in p21Cip1 and p27Kip1 that is associated with the proliferative unresponsiveness in anergic Th1 cells. In addition, p21Cip1 may inhibit proliferation in the anergic Th1 cells by some mechanism other than suppression of cdks that is unique to the induction of Th1 cell anergy.

  7. Nitric oxide-donating aspirin inhibits the growth of pancreatic cancer cells through redox-dependent signaling

    PubMed Central

    Zhou, Hui; Huang, Liqun; Sun, Yu; Rigas, Basil

    2009-01-01

    The novel chemopreventive nitric oxide-donating aspirin (NO-ASA) prevents nearly 90% of ductal adenocarcinomas in a animal tumor model. To decipher the mechanism of this effect, we studied in BxPC-3 human pancreatic cancer cells the sequence of signaling events leading from NO-ASA treatment to cell growth inhibition. NO-ASA inhibited the growth of BxPC-3 cells (IC50 = 13 μM), by inhibiting proliferation modestly and inducing apoptosis, necrosis and G1/S cell cycle block. At 15 min of treatment with NO-ASA, the intracellular levels of reactive oxygen species (ROS) began increasing (peak at 8 h, baseline levels by 24 h). ROS activated almost immediately in a time- and concentration-dependent manner the MAPK pathways p38, ERK, and JNK (their activation was abrogated by the antioxidant N-acetylcysteine). MAPK activation induced p21cip-1, which suppressed the levels of cyclin D1 that controls G1/S cell cycle transition. NO-ASA induced COX-2 expression starting 90 min after p21cip-1 was induced. When COX-2 expression was knocked-down using siRNA against cox-2, the expression of p21cip-1 was induced by NO-ASA, regardless of the level of expression of COX-2, suggesting a marginal, if any, role for COX-2 in the growth inhibitory effect of NO-ASA. These findings along with the temporal sequence of individual changes indicate a signaling sequence that involves ROS → MAPKs → p21cip-1 → cyclin D1 → cell death. Our findings establish the critical role of ROS as proximal signaling molecules in the action of anticancer compounds and may be useful in designing mechanism-driven approaches to cancer control. PMID:18805632

  8. Transcription factor LSF (TFCP2) inhibits melanoma growth

    PubMed Central

    Goto, Yuji; Yajima, Ichiro; Kumasaka, Mayuko; Ohgami, Nobutaka; Tanaka, Asami; Tsuzuki, Toyonori; Inoue, Yuji; Fukushima, Satoshi; Ihn, Hironobu; Kyoya, Mikiko; Ohashi, Hiroyuki; Kawakami, Tamihiro; Bennett, Dorothy C.; Kato, Masashi

    2016-01-01

    Late SV40 factor 3 (LSF), a transcription factor, contributes to human hepatocellular carcinoma (HCC). However, decreased expression level of LSF in skin melanoma compared to that in benign melanocytic tumors and nevi in mice and humans was found in this study. Anchorage-dependent and -independent growth of melanoma cells was suppressed by LSF overexpression through an increased percentage of G1 phase cells and an increased p21CIP1 expression level in vitro and in vivo. Anchorage-dependent growth in LSF-overexpressed melanoma cells was promoted by depletion of LSF in the LSF-overexpressed cells. Integrated results of our EMSA and chromatin immunoprecipitation assays showed binding of LSF within a 150-bp upstream region of the transcription start site of p21CIP1 in melanoma cells. Taken together, our results suggest potential roles of LSF as a growth regulator through control of the transcription of p21CIP1 in melanocytes and melanoma cells as well as a biomarker for nevus. PMID:26506241

  9. Structure-activity relationship between carboxylic acids and T cell cycle blockade.

    PubMed

    Gilbert, Kathleen M; DeLoose, Annick; Valentine, Jimmie L; Fifer, E Kim

    2006-04-04

    This study was designed to examine the potential structure-activity relationship between carboxylic acids, histone acetylation and T cell cycle blockade. Toward this goal a series of structural homologues of the short-chain carboxylic acid n-butyrate were studied for their ability to block the IL-2-stimulated proliferation of cloned CD4+ T cells. The carboxylic acids were also tested for their ability to inhibit histone deacetylation. In addition, Western blotting was used to examine the relative capacity of the carboxlic acids to upregulate the cyclin kinase-dependent inhibitor p21cip1 in T cells. As shown earlier n-butyrate effectively inhibited histone deacetylation. The increased acetylation induced by n-butyrate was associated with the upregulation of the cyclin-dependent kinase inhibitor p21cip1 and the cell cycle blockade of CD4+ T cells. Of the other carboxylic acids studied, the short chain acids, C3-C5, without branching were the best inhibitors of histone deacetylase. This inhibition correlated with increased expression of the cell cycle blocker p21cip1, and the associated suppression of CD4+ T cell proliferation. The branched-chain carboxylic acids tested were ineffective in all the assays. These results underline the relationship between the ability of a carboxylic acid to inhibit histone deacetylation, and their ability to block T cell proliferation, and suggests that branching inhibits these effects.

  10. Delineating transcriptional networks of prognostic gene signatures refines treatment recommendations for lymph node-negative breast cancer patients.

    PubMed

    Lanigan, Fiona; Brien, Gerard L; Fan, Yue; Madden, Stephen F; Jerman, Emilia; Maratha, Ashwini; Aloraifi, Fatima; Hokamp, Karsten; Dunne, Eiseart J; Lohan, Amanda J; Flanagan, Louise; Garbe, James C; Stampfer, Martha R; Fridberg, Marie; Jirstrom, Karin; Quinn, Cecily M; Loftus, Brendan; Gallagher, William M; Geraghty, James; Bracken, Adrian P

    2015-09-01

    The majority of women diagnosed with lymph node-negative breast cancer are unnecessarily treated with damaging chemotherapeutics after surgical resection. This highlights the importance of understanding and more accurately predicting patient prognosis. In the present study, we define the transcriptional networks regulating well-established prognostic gene expression signatures. We find that the same set of transcriptional regulators consistently lie upstream of both 'prognosis' and 'proliferation' gene signatures, suggesting that a central transcriptional network underpins a shared phenotype within these signatures. Strikingly, the master transcriptional regulators within this network predict recurrence risk for lymph node-negative breast cancer better than currently used multigene prognostic assays, particularly in estrogen receptor-positive patients. Simultaneous examination of p16(INK4A) expression, which predicts tumours that have bypassed cellular senescence, revealed that intermediate levels of p16(INK4A) correlate with an intact pRB pathway and improved survival. A combination of these master transcriptional regulators and p16(INK4A), termed the OncoMasTR score, stratifies tumours based on their proliferative and senescence capacity, facilitating a clearer delineation of lymph node-negative breast cancer patients at high risk of recurrence, and thus requiring chemotherapy. Furthermore, OncoMasTR accurately classifies over 60% of patients as 'low risk', an improvement on existing prognostic assays, which has the potential to reduce overtreatment in early-stage patients. Taken together, the present study provides new insights into the transcriptional regulation of cellular proliferation in breast cancer and provides an opportunity to enhance and streamline methods of predicting breast cancer prognosis.

  11. Activation of p53 by Nutlin-3a, an antagonist of MDM2, induces apoptosis and cellular senescence in adult T-cell leukemia cells.

    PubMed

    Hasegawa, H; Yamada, Y; Iha, H; Tsukasaki, K; Nagai, K; Atogami, S; Sugahara, K; Tsuruda, K; Ishizaki, A; Kamihira, S

    2009-11-01

    It has been reported that the induction of cellular senescence through p53 activation is an effective strategy in tumor regression. Unfortunately, however, tumors including adult T-cell leukemia/lymphoma (ATL) have disadvantages such as p53 mutations and a lack of p16(INK4a) and/or p14(ARF). In this study we characterized Nutlin-3a-induced cell death in 16 leukemia/lymphoma cell lines. Eight cell lines, including six ATL-related cell lines, had wild-type p53 and Nutlin-3a-activated p53, and the cell lines underwent apoptosis or cell-cycle arrest, whereas eight cell lines with mutated p53 were resistant. Interestingly, senescence-associated-beta-galactosidase (SA-beta-gal) staining revealed that only ATL-related cell lines with wild-type p53 showed cellular senescence, although they lack both p16(INK4a) and p14(ARF). These results indicate that cellular senescence is an important event in p53-dependent cell death in ATL cells and is inducible without p16(INK4a) and p14(ARF). Furthermore, knockdown of Tp53-induced glycolysis and apoptosis regulator (TIGAR), a novel target gene of p53, by small interfering RNA(siRNA) indicated its important role in the induction of cellular senescence. As many patients with ATL carry wild-type p53, our study suggests that p53 activation by Nutlin-3a is a promising strategy in ATL. We also found synergism with a combination of Nutlin-3a and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), suggesting the application of Nutlin-3a-based therapy to be broader than expected.

  12. Hypersensitivity to contact inhibition provides a clue to cancer resistance of naked mole-rat.

    PubMed

    Seluanov, Andrei; Hine, Christopher; Azpurua, Jorge; Feigenson, Marina; Bozzella, Michael; Mao, Zhiyong; Catania, Kenneth C; Gorbunova, Vera

    2009-11-17

    The naked mole-rat is the longest living rodent with a maximum lifespan exceeding 28 years. In addition to its longevity, naked mole-rats have an extraordinary resistance to cancer as tumors have never been observed in these rodents. Furthermore, we show that a combination of activated Ras and SV40 LT fails to induce robust anchorage-independent growth in naked mole-rat cells, while it readily transforms mouse fibroblasts. The mechanisms responsible for the cancer resistance of naked mole-rats were unknown. Here we show that naked mole-rat fibroblasts display hypersensitivity to contact inhibition, a phenomenon we termed "early contact inhibition." Contact inhibition is a key anticancer mechanism that arrests cell division when cells reach a high density. In cell culture, naked mole-rat fibroblasts arrest at a much lower density than those from a mouse. We demonstrate that early contact inhibition requires the activity of p53 and pRb tumor suppressor pathways. Inactivation of both p53 and pRb attenuates early contact inhibition. Contact inhibition in human and mouse is triggered by the induction of p27(Kip1). In contrast, early contact inhibition in naked mole-rat is associated with the induction of p16(Ink4a). Furthermore, we show that the roles of p16(Ink4a) and p27(Kip1) in the control of contact inhibition became temporally separated in this species: the early contact inhibition is controlled by p16(Ink4a), and regular contact inhibition is controlled by p27(Kip1). We propose that the additional layer of protection conferred by two-tiered contact inhibition contributes to the remarkable tumor resistance of the naked mole-rat.

  13. HPV DNA prevalence and type distribution in anal carcinomas worldwide

    PubMed Central

    Alemany, L; Saunier, M; Alvarado, I; Quirós, B; Salmeron, J; Shin, HR; Pirog, E; Guimerà, N; Hernández, GA; Felix, A; Clavero, O; Lloveras, B; Kasamatsu, E; Goodman, MT; Hernandez, BY; Laco, J; Tinoco, L; Geraets, DT; Lynch, CF; Mandys, V; Poljak, M; Jach, R; Verge, J; Clavel, C; Ndiaye, C; Klaustermeier, J; Cubilla, A; Castellsagué, X; Bravo, IG; Pawlita, M; Quint, W; Muñoz, N; Bosch, FX; Sanjosé, S

    2014-01-01

    Knowledge about the human papillomaviruses (HPV) types in anal cancers in some world regions is scanty. Here we describe the HPV DNA prevalence and type distribution in a series of invasive anal cancers and anal intraepithelial neoplasias (AIN) grades 2/3 from 24 countries. We analyzed 43 AIN 2/3 cases and 496 anal cancers diagnosed from 1986 to 2011. After histopathological evaluation of formalin-fixed paraffin-embedded samples, HPV DNA detection and genotyping was performed using SPF-10/DEIA/LiPA25 system (version 1). A subset of 116 cancers was further tested for p16INK4a expression, a cellular surrogate marker for HPV-associated transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance in cancer dataset. HPV DNA was detected in 88.3% of anal cancers (95%CI:85.1–91.0%) and in 95.4% of AIN 2/3 (95%CI:84.2–99.4%). Among cancers, the highest prevalence was observed in warty-basaloid subtype of squamous cell carcinomas, in younger patients and in North American geographical region. There were no statistically significant differences in prevalence by gender. HPV16 was the most frequent HPV type detected in both cancers (80.7%) and AIN 2/3 lesions (75.4%). HPV18 was the second most common type in invasive cancers (3.6%). p16INK4a overexpression was found in 95% of HPV DNA positive anal cancers. In view of HPV DNA results and high proportion of p16INK4a overexpression, infection by HPV is most likely to be a necessary cause for anal cancers in both men and women. The large contribution of HPV16 reinforces the potential impact of HPV vaccines in the prevention of these lesions. PMID:24817381

  14. Fatty acid-binding protein FABP4 mechanistically links obesity with aggressive AML by enhancing aberrant DNA methylation in AML cells.

    PubMed

    Yan, F; Shen, N; Pang, J X; Zhang, Y W; Rao, E Y; Bode, A M; Al-Kali, A; Zhang, D E; Litzow, M R; Li, B; Liu, S J

    2016-12-02

    Obesity is becoming more prevalent worldwide and is a major risk factor for cancer development. Acute myeloid leukemia (AML), the most common acute leukemia in adults, remains a frequently fatal disease. Here we investigated the molecular mechanisms by which obesity favors AML growth and uncovered the fatty acid-binding protein 4 (FABP4) and DNA methyltransferase 1 (DNMT1) regulatory axis that mediates aggressive AML in obesity. We showed that leukemia burden was much higher in high-fat diet-induced obese mice, which had higher levels of FABP4 and interleukin (IL)-6 in the sera. Upregulation of environmental and cellular FABP4 accelerated AML cell growth in both a cell-autonomous and cell-non-autonomous manner. Genetic disruption of FABP4 in AML cells or in mice blocked cell proliferation in vitro and induced leukemia regression in vivo. Mechanistic investigations showed that FABP4 upregulation increased IL-6 expression and signal transducer and activator of transcription factor 3 phosphorylation leading to DNMT1 overexpression and further silencing of the p15(INK4B) tumor-suppressor gene in AML cells. Conversely, FABP4 ablation reduced DNMT1-dependent DNA methylation and restored p15(INK4B) expression, thus conferring substantial protection against AML growth. Our findings reveal the FABP4/DNMT1 axis in the control of AML cell fate in obesity and suggest that interference with the FABP4/DNMT1 axis might be a new strategy to treat leukemia.Leukemia advance online publication, 2 December 2016; doi:10.1038/leu.2016.349.

  15. Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway.

    PubMed

    Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji; Lee, Hui-Young; Hong, Suntaek; Kim, Seong-Jin; Kim, Byung-Chul

    2013-06-14

    Hydrogen peroxide (H2O2) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H2O2 are less understood. Here we report an important mechanism for antagonistic effects of H2O2 on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H2O2 (0.05-0.2 mM) completely blocked TGF-β1-mediated induction of p15(INK4B) expression and increase of its promoter activity. Interestingly, H2O2 selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H2O2 increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H2O2 on TGF-β1-induced increase of p15(INK4B)-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H2O2 as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing a potential mechanism whereby H2O2 antagonizes the cytostatic function of TGF-β1.

  16. Using biomarkers as objective standards in the diagnosis of cervical biopsies.

    PubMed

    Galgano, Mary T; Castle, Philip E; Atkins, Kristen A; Brix, William K; Nassau, Sarah R; Stoler, Mark H

    2010-08-01

    Histopathologic diagnosis of cervical biopsies determines clinical management of patients with an abnormal cervical cancer-screening test yet is prone to poor interobserver reproducibility. Immunohistochemical staining for biomarkers related to the different stages of cervical carcinogenesis may provide objective standards to reduce diagnostic variability of cervical biopsy evaluations but systematic, rigorous evaluations of their potential clinical utility are lacking. To address diagnostic utility of human papillomavirus (HPV) L1, p16(INK4a), and Ki-67 immunohistochemical staining for improving diagnostic accuracy, we conducted a community-based and population-based evaluation using 1455 consecutive cervical biopsies submitted to the Department of Pathology at the University of Virginia during a period of 14 months. Thin-sections of each biopsy from 1451 of 1455 (99.7%) biopsies underwent evaluation of immunohistochemical stains for the 3 biomarkers, masked to the original diagnosis, and the results were compared with an adjudicated, consensus diagnosis by 3 pathologists. p16 immunostaining, using the strongest staining as the cutpoint, was 86.7% sensitive and 82.8% specific for cervical intraepithelial neoplasia (CIN) grade 2 or more severe (CIN2(+)) diagnoses. The performance of p16(INK4a) was more sensitive (P<0.001), less specific (P<0.001), and of similar overall accuracy for CIN2(+) compared with the combined performance of all pathologist reviews in routine clinical diagnostic service (sensitivity=68.9%, specificity=97.2%). Ki-67 immunostaining was also strongly associated with a CIN2(+) diagnosis but its performance at all staining intensities was inferior to p16 immunostaining, and did not increase the accuracy of CIN2(+) diagnosis when combined with p16(INK4a) immunostaining compared with p16(INK4a) immunostaining alone. We found no utility for L1 immunostaining in distinguishing between CIN and non-CIN. In conclusion, with a rigorous evaluation, we

  17. Aberrant DNA methylation profile in pleural fluid for differential diagnosis of malignant pleural mesothelioma.

    PubMed

    Fujii, Masanori; Fujimoto, Nobukazu; Hiraki, Akio; Gemba, Kenichi; Aoe, Keisuke; Umemura, Shigeki; Katayama, Hideki; Takigawa, Nagio; Kiura, Katsuyuki; Tanimoto, Mitsune; Kishimoto, Takumi

    2012-03-01

    Malignant pleural mesothelioma (MPM) usually develops pleural fluid. We investigated the value of DNA methylation in the pleural fluid for differentiating MPM from lung cancer (LC). Pleural fluid was collected from 39 patients with MPM, 46 with LC, 25 with benign asbestos pleurisy (BAP) and 30 with other causes. The methylation of O(6)-methylguanine-DNA methyltransferase (MGMT), p16(INK4a) , ras association domain family 1A (RASSF1A), death-associated protein kinase (DAPK), and retinoic acid receptor β (RARβ) was examined using quantitative real-time PCR. DNA methylation of RASSF1A, p16(INK4a), RARβ, MGMT and DAPK was detected in 12 (30.8%), 3 (7.7%), 11 (28.2%), 0 (0.0%) and five patients (12.8%) with MPM, and in 22 (47.8%), 14 (30.4%), 24 (52.2%), 1 (2.2%) and six patients (13.0%) with LC, respectively. The mean methylation ratios of RASSF1A, p16(INK4a) and RARβ were 0.37 (range 0.0-2.84), 0.11 (0.0-2.67) and 0.44 (0.0-3.32) in MPM, and 0.87 (0.0-3.14), 1.16 (0.0-5.35) and 1.69 (0.0-6.49) in LC, respectively. The methylation ratios for the three genes were significantly higher in LC than in MPM (RASSF1A, P = 0.039; p16(INK4a), P = 0.005; and RARβ, P = 0.002). Patients with methylation in at least one gene were 3.51 (95% confidence interval, 1.09-11.34) times more likely to have LC. Hypermethylation seemed no greater with MPM than with BAP. Extended exposure to asbestos (≧30 years) was correlated with an increased methylation frequency (P = 0.020). Hypermethylation of tumor suppressor genes in pleural fluid DNA has the potential to be a valuable marker for differentiating MPM from LC.

  18. Biological relevance of human papillomaviruses in vulvar cancer.

    PubMed

    Halec, Gordana; Alemany, Laia; Quiros, Beatriz; Clavero, Omar; Höfler, Daniela; Alejo, Maria; Quint, Wim; Pawlita, Michael; Bosch, Francesc X; de Sanjose, Silvia

    2017-04-01

    The carcinogenic role of high-risk human papillomavirus (HR-HPV) types in the increasing subset of vulvar intraepithelial neoplasia and vulvar cancer in young women has been established. However, the actual number of vulvar cancer cases attributed to HPV is still imprecisely defined. In an attempt to provide a more precise definition of HPV-driven vulvar cancer, we performed HPV-type-specific E6*I mRNA analyses available for 20 HR-/possible HR (pHR)-HPV types, on tissue samples from 447 cases of vulvar cancer. HPV DNA genotyping was performed using SPF10-LiPA25 assay due to its high sensitivity in formalin-fixed paraffin-embedded tissues. Data on p16(INK4a) expression was available for comparative analysis via kappa statistics. The use of highly sensitive assays covering the detection of HPV mRNA in a broad spectrum of mucosal HPV types resulted in the detection of viral transcripts in 87% of HPV DNA+ vulvar cancers. Overall concordance between HPV mRNA+ and p16(INK4a) upregulation (strong, diffuse immunostaining in >25% of tumor cells) was 92% (K=0.625, 95% confidence interval (CI)=0.531-0.719). Among these cases, 83% were concordant pairs of HPV mRNA+ and p16(INK4a)+ and 9% were concordant pairs of HPV mRNA- and p16(INK4a)-. Our data confirm the biological role of HR-/pHR-HPV types in the great majority of HPV DNA+ vulvar cancers, resulting in an HPV-attributable fraction of at least 21% worldwide. Most HPV DNA+ vulvar cancers were associated with HPV16 (85%), but a causative role for other, less frequently occurring mucosal HPV types (HPV26, 66, 67, 68, 70 and 73) was also confirmed at the mRNA level for the first time. These findings should be taken into consideration for future screening options as HPV-associated vulvar preneoplastic lesions have increased in incidence in younger women and require different treatment than vulvar lesions that develop from rare autoimmune-related mechanisms in older women.

  19. Targets for molecular therapy of skin cancer.

    PubMed

    Green, Cheryl L; Khavari, Paul A

    2004-02-01

    Cancers of the skin encompass the first and second most common neoplasms in the United States, epidermal basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), respectively, as well as the melanocytic malignancy, malignant melanoma (MM). Recently identified alterations in the function of specific genes in these cancers provide new potential therapeutic targets. These alterations affect conserved regulators of cellular proliferation and viability, including the Sonic Hedgehog, Ras/Raf, ARF/p53, p16(INK4A)/CDK4/Rb and NF-kappaB pathways. New modalities designed to target these specific proteins may represent promising approaches to therapy of human skin cancers.

  20. Unique Polycomb Gene Expression Pattern in Hodgkin’s Lymphoma and Hodgkin’s Lymphoma-Derived Cell Lines

    PubMed Central

    Dukers, Danny F.; van Galen, Joost C.; Giroth, Cindy; Jansen, Patty; Sewalt, Richard G.A.B.; Otte, Arie P.; Kluin-Nelemans, Hanneke C.; Meijer, Chris J.L.M.; Raaphorst, Frank M.

    2004-01-01

    Human Polycomb-group (PcG) genes play a crucial role in the regulation of embryonic development and regulation of the cell cycle and hematopoiesis. PcG genes encode proteins that form two distinct PcG complexes, involved in maintenance of cell identity and gene silencing patterns. We recently showed that expression of the BMI-1 and EZH2 PcG genes is separated during normal B-cell development in germinal centers, whereas Hodgkin/Reed-Sternberg (H/RS) cells co-express BMI-1 and EZH2. In the current study, we used immunohistochemistry and immunofluorescence to determine whether the binding partners of these PcG proteins are also present in H/RS cells and H/RS-derived cell lines. PcG expression profiles were analyzed in combination with expression of the cell cycle inhibitor p16INK4a, because experimental model systems indicate that p16 is a downstream target of Bmi-1. We found that H/RS cells and HL-derived cell lines co-express all core proteins of the two known PcG complexes, including BMI-1, MEL-18, RING1, HPH1, HPC1, and -2, EED, EZH2, YY1, and the HPC2 binding partner, CtBP. Expression of HPC1 has not been found in normal mature B cells and other malignant lymphomas of B-cell origin, suggesting that the PcG expression profile of H/RS is unique. In contrast to Bmi-1 transgenic mice where p16INK4a is down-regulated, 27 of 52 BMI-1POS cases of HL revealed strong nuclear expression of p16INK4a. We propose that abnormal expression of BMI-1 and its binding partners in H/RS cells contributes to development of HL. However, abnormal expression of BMI-1 in HL is not necessarily associated with down-regulation of p16INK4a. PMID:14982841

  1. DNA Hypermethylation Patterns Detected in Serum as a Tool for Early Breast Cancer Diagnosis

    DTIC Science & Technology

    2009-09-01

    23 201 genes (CCND2, RASSF1A, APC, and HIN1) was able 202 to distinguish between invasive carcinomas (n = 66), 203 fibroadenomas (n = 31) and normal...studies have estimated that one in two women 227develops fibrocystic disease and one in five fibroadenoma 228during her lifetime [56]. Mammography is...of genes including, BRCA1, p16 INK4A, ESR1, 245GSTP1, TRb1, RARb2, HIC1, APC, CCND2, and CDH1, it 246was found that fibroadenomas (n = 10) had patterns

  2. [Importance of immunohistochemical studies in the diagnosis and the prognostic evaluation of cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the uterine cervix. Review].

    PubMed

    García-Tamayo, Jorge; Molina, Julia; Blasco-Olaetxea, Eduardo

    2009-06-01

    Immunohistochemical studies in cervical intraepithelial neoplasia and cervical carcinoma are evaluated in this review. A variety of proteins like p53, bcl2, C-Myc, Ki 67, Cyclines, P16 INK4a, p21, p27, beta-catenin, Wnt and MCM, have been related to the development of cervical neoplasia and human papilloma virus infection. It is described how transcriptional factors of genes induce loss of heterozygosity, numerical chromosome abnormality and inactivation of gene products or the partial loss of some membrane glycoproteins induced by oncogenic human papillomaviruses (HPV).

  3. [Papillomavirus in the genesis of oral leukoplakia].

    PubMed

    Babichenko, I I; Rabinovich, O F; Ivina, A A; Rabinovich, I M; Togonidze, A A

    2014-01-01

    Immunohistochemical examination of the proliferative activity of cells was made investigating the expression of Ki-67 protein and the location of proteins associated with epithelial cell papillomavirus infection involving P16(INK4a) and HPV16 proteins in different cell areas of the intact mucosa, in leukoplakia with the signs of hyperplasia and dysplasia, and in squamous cell carcinoma. There was a positive correlation between the proliferative activity of cells in the parabasal cell areas and the expression of P16(INK4a) protein in oral leukoplakia with the signs of hyperplasia (r(s)=0.397; p=0.018). In oral leukoplakia with dysplastic changes, there was a positive correlation between the proliferation of cells in the parabasal and prickle cell layers and the location of HPV type 16 antigens (r(s)=0.515; p=0.041 and r(s)=0.651; p=0.006). Detection of papillomavirus infection in leukoplakia can solve not only the problems with its genesis, but this is also a morphological basis for the effective prevention and treatment of this common oral mucosal disease.

  4. NONO couples the circadian clock to the cell cycle.

    PubMed

    Kowalska, Elzbieta; Ripperger, Juergen A; Hoegger, Dominik C; Bruegger, Pascal; Buch, Thorsten; Birchler, Thomas; Mueller, Anke; Albrecht, Urs; Contaldo, Claudio; Brown, Steven A

    2013-01-29

    Mammalian circadian clocks restrict cell proliferation to defined time windows, but the mechanism and consequences of this interrelationship are not fully understood. Previously we identified the multifunctional nuclear protein NONO as a partner of circadian PERIOD (PER) proteins. Here we show that it also conveys circadian gating to the cell cycle, a connection surprisingly important for wound healing in mice. Specifically, although fibroblasts from NONO-deficient mice showed approximately normal circadian cycles, they displayed elevated cell doubling and lower cellular senescence. At a molecular level, NONO bound to the p16-Ink4A cell cycle checkpoint gene and potentiated its circadian activation in a PER protein-dependent fashion. Loss of either NONO or PER abolished this activation and circadian expression of p16-Ink4A and eliminated circadian cell cycle gating. In vivo, lack of NONO resulted in defective wound repair. Because wound healing defects were also seen in multiple circadian clock-deficient mouse lines, our results therefore suggest that coupling of the cell cycle to the circadian clock via NONO may be useful to segregate in temporal fashion cell proliferation from tissue organization.

  5. Failure of cell cleavage induces senescence in tetraploid primary cells.

    PubMed

    Panopoulos, Andreas; Pacios-Bras, Cristina; Choi, Justin; Yenjerla, Mythili; Sussman, Mark A; Fotedar, Rati; Margolis, Robert L

    2014-10-15

    Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen-transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.

  6. Segmental Aging Underlies the Development of a Parkinson Phenotype in the AS/AGU Rat

    PubMed Central

    Khojah, Sohair M.; Payne, Anthony P.; McGuinness, Dagmara; Shiels, Paul G.

    2016-01-01

    There is a paucity of information on the molecular biology of aging processes in the brain. We have used biomarkers of aging (SA β-Gal, p16Ink4a, Sirt5, Sirt6, and Sirt7) to demonstrate the presence of an accelerated aging phenotype across different brain regions in the AS/AGU rat, a spontaneous Parkinsonian mutant of PKCγ derived from a parental AS strain. P16INK4a expression was significantly higher in AS/AGU animals compared to age-matched AS controls (p < 0.001) and displayed segmental expression across various brain regions. The age-related expression of sirtuins similarly showed differences between strains and between brain regions. Our data clearly show segmental aging processes within the rat brain, and that these are accelerated in the AS/AGU mutant. The accelerated aging, Parkinsonian phenotype, and disruption to dopamine signalling in the basal ganglia in AS/AGU rats, suggests that this rat strain represents a useful model for studies of development and progression of Parkinson’s disease in the context of biological aging and may offer unique mechanistic insights into the biology of aging. PMID:27763519

  7. Promoter CpG methylation of multiple genes in pituitary adenomas: frequent involvement of caspase-8.

    PubMed

    Bello, M Josefa; De Campos, Jose M; Isla, Alberto; Casartelli, Cacilda; Rey, Juan A

    2006-02-01

    The epigenetic changes in pituitary adenomas were identified by evaluating the methylation status of nine genes (RB1, p14(ARF), p16(INK4a), p73, TIMP-3, MGMT, DAPK, THBS1 and caspase-8) in a series of 35 tumours using methylation-specific PCR analysis plus sequencing. The series included non-functional adenomas (n=23), prolactinomas (n=6), prolactinoma plus thyroid-stimulating hormone adenoma (n=1), growth hormone adenomas (n=4), and adrenocorticotropic adenoma (n=1). All of the tumours had methylation of at least one of these genes and 40% of samples (14 of 35) displayed concurrent methylation of at least three genes. The frequencies of aberrant methylation were: 20% for RB1, 17% for p14(ARF), 34% for p16(INK4a), 29% for p73, 11% for TIMP-3, 23% for MGMT, 6% for DAPK, 43% for THBS1 and 54% for caspase-8. No aberrant methylation was observed in two non-malignant pituitary samples from healthy controls. Although some differences in the frequency of gene methylation between functional and non-functional adenomas were detected, these differences did not reach statistical significance. Our results suggest that promoter methylation is a frequent event in pituitary adenoma tumourigenesis, a process in which inactivation of apoptosis-related genes (DAPK, caspase-8) might play a key role.

  8. NONO couples the circadian clock to the cell cycle

    PubMed Central

    Kowalska, Elzbieta; Ripperger, Juergen A.; Hoegger, Dominik C.; Bruegger, Pascal; Buch, Thorsten; Birchler, Thomas; Mueller, Anke; Albrecht, Urs; Contaldo, Claudio; Brown, Steven A.

    2013-01-01

    Mammalian circadian clocks restrict cell proliferation to defined time windows, but the mechanism and consequences of this interrelationship are not fully understood. Previously we identified the multifunctional nuclear protein NONO as a partner of circadian PERIOD (PER) proteins. Here we show that it also conveys circadian gating to the cell cycle, a connection surprisingly important for wound healing in mice. Specifically, although fibroblasts from NONO-deficient mice showed approximately normal circadian cycles, they displayed elevated cell doubling and lower cellular senescence. At a molecular level, NONO bound to the p16-Ink4A cell cycle checkpoint gene and potentiated its circadian activation in a PER protein-dependent fashion. Loss of either NONO or PER abolished this activation and circadian expression of p16-Ink4A and eliminated circadian cell cycle gating. In vivo, lack of NONO resulted in defective wound repair. Because wound healing defects were also seen in multiple circadian clock-deficient mouse lines, our results therefore suggest that coupling of the cell cycle to the circadian clock via NONO may be useful to segregate in temporal fashion cell proliferation from tissue organization. PMID:23267082

  9. Elimination of p19ARF-expressing cells enhances pulmonary function in mice

    PubMed Central

    Hashimoto, Michihiro; Asai, Azusa; Kawagishi, Hiroyuki; Mikawa, Ryuta; Iwashita, Yuji; Kanayama, Kazuki; Sugimoto, Kazushi; Sato, Tadashi; Maruyama, Mitsuo

    2016-01-01

    Senescent cells accumulate in many tissues as animals age and are considered to underlie several aging-associated pathologies. The tumor suppressors p19ARF and p16INK4a, both of which are encoded in the CDKN2A locus, play critical roles in inducing and maintaining permanent cell cycle arrest during cellular senescence. Although the elimination of p16INK4a-expressing cells extends the life span of the mouse, it is unclear whether tissue function is restored by the elimination of senescent cells in aged animals and whether and how p19ARF contributes to tissue aging. The aging-associated decline in lung function is characterized by an increase in compliance as well as pathogenic susceptibility to pulmonary diseases. We herein demonstrated that pulmonary function in 12-month-old mice was reversibly restored by the elimination of p19ARF-expressing cells. The ablation of p19ARF-expressing cells using a toxin receptor-mediated cell knockout system ameliorated aging-associated lung hypofunction. Furthermore, the aging-associated gene expression profile was reversed after the elimination of p19ARF. Our results indicate that the aging-associated decline in lung function was, at least partly, attributed to p19ARF and was recovered by eliminating p19ARF-expressing cells. PMID:27699227

  10. Use of human tissue to assess the oncogenic activity of melanoma-associated mutations.

    PubMed

    Chudnovsky, Yakov; Adams, Amy E; Robbins, Paul B; Lin, Qun; Khavari, Paul A

    2005-07-01

    Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Induction of Ras and Raf can be caused by active N-Ras and B-Raf mutants as well as by gene amplification. Activation of PI3K pathway components occurs by PTEN loss and by AKT3 amplification. Melanomas also commonly show impairment of the p16(INK4A)-CDK4-Rb and ARF-HDM2-p53 tumor suppressor pathways. CDKN2A mutations can produce p16(INK4A) and ARF protein loss. Rb bypass can also occur through activating CDK4 mutations as well as by CDK4 amplification. In addition to ARF deletion, p53 pathway disruption can result from dominant negative TP53 mutations. TERT amplification also occurs in melanoma. The extent to which these mutations can induce human melanocytic neoplasia is unknown. Here we characterize pathways sufficient to generate human melanocytic neoplasia and show that genetically altered human tissue facilitates functional analysis of mutations observed in human tumors.

  11. Expression of the polycomb‐group gene BMI1 is related to an unfavourable prognosis in primary nodal DLBCL

    PubMed Central

    van Galen, Joost C; Muris, Jettie J F; Oudejans, Joost J; Vos, Wim; Giroth, Cindy P E; Ossenkoppele, Gert J; Otte, Arie P; Raaphorst, Frank M; Meijer, Chris J L M

    2007-01-01

    Background Clinical outcome in patients with diffuse large B cell lymphomas (DLBCL) is highly variable and poorly predictable. Microarray studies showed that patients with DLBCL with a germinal centre B cell‐like (GCB) phenotype have a better prognosis than those with an activated B cell‐like (ABC) phenotype. The BMI1 proto‐oncogene was identified as one of the genes present in the signature of the ABC type of DLBCL, associated with a poor prognosis. Objectives (1) To investigate, in primary nodal DLBCL, the expression of BMI1 and its association with clinical outcome and DLBCL signature; (2) to look for an association between BMI1 expression and the expression of its putative downstream targets p14ARF and p16INK4a. Results BMI1 expression was found to be associated with poor clinical outcome, but not clearly with an ABC‐like phenotype of DLBCL. Expression of BMI1 was frequently, but not always, related to low levels of expression of p14ARF and p16INK4a. Conclusion Expression of BMI1 is associated with an unfavourable clinical outcome of primary nodal DLBCL. PMID:16837630

  12. Population Pharmacokinetics Modeling of Unbound Efavirenz, Atazanavir, and Ritonavir in HIV‐Infected Subjects With Aging Biomarkers

    PubMed Central

    Chen, J; Cottrell, M; Trezza, CR; Prince, HMA; Sykes, C; Torrice, C; White, N; Malone, S; Wang, R; Patterson, KB; Sharpless, NE; Forrest, A

    2016-01-01

    Unbound drug is the pharmacodynamically relevant concentration. This study aimed to determine if chronologic age or markers of biologic aging, such as the frailty phenotype and p16INK4a gene expression, altered unbound pharmacokinetics (PKs) of efavirenz (EFV) and atazanavir/ritonavir (ATV/RTV). Sixty human immunodeficiency virus (HIV)‐infected participants receiving EFV and 31 receiving ATV/RTV provided 1 to 11 samples to quantify total and unbound plasma concentrations. Population PK models with total and unbound concentrations simultaneously described are developed for each drug. The unbound fractions for EFV, ATV, and RTV are 0.65%, 5.67%, and 0.63%, respectively. Covariate analysis suggests RTV unbound PK is sensitive to body size; unbound fraction of RTV is 34% lower with body mass index (BMI) above 30 kg/m2. No alterations in drug clearance or unbound fraction with age, frailty, or p16INK4a expression were observed. Assessing functional and physiologic aging markers to inform potential PK changes is necessary to determine if drug/dosing changes are warranted in the aging population. PMID:28032946

  13. Oxidative stress-associated senescence in dermal papilla cells of men with androgenetic alopecia.

    PubMed

    Upton, James H; Hannen, Rosalind F; Bahta, Adiam W; Farjo, Nilofer; Farjo, Bessam; Philpott, Michael P

    2015-05-01

    Dermal papilla cells (DPCs) taken from male androgenetic alopecia (AGA) patients undergo premature senescence in vitro in association with the expression of p16(INK4a), suggesting that DPCs from balding scalp are more sensitive to environmental stress than nonbalding cells. As one of the major triggers of senescence in vitro stems from the cell "culture shock" owing to oxidative stress, we have further investigated the effects of oxidative stress on balding and occipital scalp DPCs. Patient-matched DPCs from balding and occipital scalp were cultured at atmospheric (21%) or physiologically normal (2%) O2. At 21% O2, DPCs showed flattened morphology and a significant reduction in mobility, population doubling, increased levels of reactive oxygen species and senescence-associated β-Gal activity, and increased expression of p16(INK4a) and pRB. Balding DPCs secreted higher levels of the negative hair growth regulators transforming growth factor beta 1 and 2 in response to H2O2 but not cell culture-associated oxidative stress. Balding DPCs had higher levels of catalase and total glutathione but appear to be less able to handle oxidative stress compared with occipital DPCs. These in vitro findings suggest that there may be a role for oxidative stress in the pathogenesis of AGA both in relation to cell senescence and migration but also secretion of known hair follicle inhibitory factors.

  14. Bmi1 limits dilated cardiomyopathy and heart failure by inhibiting cardiac senescence.

    PubMed

    Gonzalez-Valdes, I; Hidalgo, I; Bujarrabal, A; Lara-Pezzi, E; Padron-Barthe, L; Garcia-Pavia, P; Gómez-del Arco, P; Gomez, P; Redondo, J M; Ruiz-Cabello, J M; Jimenez-Borreguero, L J; Enriquez, J A; de la Pompa, J L; Hidalgo, A; Gonzalez, S

    2015-03-09

    Dilated cardiomyopathy (DCM) is the most frequent cause of heart failure and the leading indication for heart transplantation. Here we show that epigenetic regulator and central transcriptional instructor in adult stem cells, Bmi1, protects against DCM by repressing cardiac senescence. Cardiac-specific Bmi1 deletion induces the development of DCM, which progresses to lung congestion and heart failure. In contrast, Bmi1 overexpression in the heart protects from hypertrophic stimuli. Transcriptome analysis of mouse and human DCM samples indicates that p16(INK4a) derepression, accompanied by a senescence-associated secretory phenotype (SASP), is linked to severely impaired ventricular dimensions and contractility. Genetic reduction of p16(INK4a) levels reverses the pathology of Bmi1-deficient hearts. In parabiosis assays, the paracrine senescence response underlying the DCM phenotype does not transmit to healthy mice. As senescence is implicated in tissue repair and the loss of regenerative potential in aging tissues, these findings suggest a source for cardiac rejuvenation.

  15. Adrenal Androgen Dehydroepiandrosterone Sulfate Inhibits Vascular Remodeling Following Arterial Injury

    PubMed Central

    Ii, Masaaki; Hoshiga, Masaaki; Negoro, Nobuyuki; Fukui, Ryosuke; Nakakoji, Takahiro; Kohbayashi, Eiko; Shibata, Nobuhiko; Furutama, Daisuke; Ishihara, Tadashi; Hanafusa, Toshiaki; Losordo, Douglas W.; Ohsawa, Nakaaki

    2009-01-01

    Recent epidemiologic studies have suggested that serum dehydroepiandrosterone sulfate (DHEAS) levels have a significant inverse correlation with the incidence of cardiovascular diseases. However, direct evidence for the association with DHEAS and vascular disorders has not yet been explored. DHEAS significantly reduced neointima formation 28 days after surgery without altering other serum metabolite levels in a rabbit carotid balloon injury model. Immunohistochemical analyses revealed the reduction of proliferating cell nuclear antigen (PCNA) index and increase of TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) index, expressing differentiated vascular smooth muscle cell (VSMC) markers in the media 7 days after surgery. In vitro, DHEAS exhibited inhibitory effects on VSMC proliferation and migration activities, inducing G1 cell cycle arrest with upregulation of one of the cyclin dependent kinase (CDK) inhibitors p16INK4a and apoptosis with activating peroxisome proliferator-activated receptor (PPAR)-α in VSMCs. DHEAS inhibits vascular remodeling reducing neointima formation after vascular injury via its effects on VSMC phenotypic modulation, functions and apoptosis upregulating p16INK4a/activating PPARα. DHEAS may play a pathophysiological role for vascular remodeling in cardiovascular disease. PMID:19298964

  16. CDK2 activation in mouse epidermis induces keratinocyte proliferation but does not affect skin tumor development.

    PubMed

    Macias, Everardo; Miliani de Marval, Paula L; De Siervi, Adriana; Conti, Claudio J; Senderowicz, Adrian M; Rodriguez-Puebla, Marcelo L

    2008-08-01

    It has been widely assumed that elevated CDK2 kinase activity plays a contributory role in tumorigenesis. We have previously shown that mice overexpressing CDK4 under control of the keratin 5 promoter (K5CDK4 mice) develop epidermal hyperplasia and increased susceptibility to squamous cell carcinomas. In this model, CDK4 overexpression results in increased CDK2 activity associated with the noncatalytic function of CDK4, sequestration of p21(Cip1) and p27(Kip1). Furthermore, we have shown that ablation of Cdk2 reduces Ras-Cdk4 tumorigenesis, suggesting that increased CDK2 activity plays an important role in Ras-mediated tumorigenesis. To investigate this hypothesis, we generated two transgenic mouse models of elevated CDK2 kinase activity, K5Cdk2 and K5Cdk4(D158N) mice. The D158N mutation blocks CDK4 kinase activity without interfering with its binding capability. CDK2 activation via overexpression of CDK4(D158N), but not of CDK2, resulted in epidermal hyperplasia. We observed elevated levels of p21(Cip1) in K5Cdk2, but not in K5Cdk4(D158N), epidermis, suggesting that CDK2 overexpression elicits a p21(Cip1) response to maintain keratinocyte homeostasis. Surprisingly, we found that neither CDK2 overexpression nor the indirect activation of CDK2 enhanced skin tumor development. Thus, although the indirect activation of CDK2 is sufficient to induce keratinocyte hyperproliferation, activation of CDK2 alone does not induce malignant progression in Ras-mediated tumorigenesis.

  17. Immunohistochemical cellular distribution of proteins related to M phase regulation in early proliferative lesions induced by tumor promotion in rat two-stage carcinogenesis models.

    PubMed

    Yafune, Atsunori; Taniai, Eriko; Morita, Reiko; Akane, Hirotoshi; Kimura, Masayuki; Mitsumori, Kunitoshi; Shibutani, Makoto

    2014-01-01

    We have previously reported that 28-day treatment with hepatocarcinogens increases liver cells expressing p21(Cip1), a G1/S checkpoint protein, and M phase proteins, i.e., nuclear Cdc2, Aurora B, phosphorylated-Histone H3 (p-Histone H3) and heterochromatin protein 1α (HP1α), in rats. To examine the roles of these markers in the early stages of carcinogenesis, we investigated their cellular distribution in several carcinogenic target organs using rat two-stage carcinogenesis models. Promoting agents targeting the liver (piperonyl butoxide and methapyrilene hydrochloride), thyroid (sulfadimethoxine), urinary bladder (phenylethyl isothiocyanate), and forestomach and glandular stomach (catechol) were administered to rats after initiation treatment for the liver with N-diethylnitrosamine, thyroid with N-bis(2-hydroxypropyl)nitrosamine, urinary bladder with N-butyl-N-(4-hydroxybutyl)nitrosamine, and forestomach and glandular stomach with N-methyl-N'-nitro-N-nitrosoguanidine. Numbers of cells positive for nuclear Cdc2, Aurora B, p-Histone H3 and HP1α increased within preneoplastic lesions as determined by glutathione S-transferase placental form in the liver or phosphorylated p44/42 mitogen-activated protein kinase in the thyroid, and hyperplastic lesions having no known preneoplastic markers in the urinary bladder, forestomach and glandular stomach. Immunoreactive cells for p21(Cip1) were decreased within thyroid preneoplastic lesions; however, they were increased within liver preneoplastic lesions and hyperplastic lesions in other organs. These results suggest that M phase disruption commonly occur during the formation of preneoplastic lesions and hyperplastic lesions. Differences in the expression patterns of p21(Cip1) between thyroid preneoplastic and proliferative lesions in other organs may reflect differences in cell cycle regulation involving G1/S checkpoint function between proliferative lesions in each organ.

  18. Targeting prostate cancer based on signal transduction and cell cycle pathways

    PubMed Central

    Lee, John T.; Lehmann, Brian D.; Terrian, David M.; Chappell, William H.; Stivala, Franca; Libra, Massimo; Martelli, Alberto M.; Steelman, Linda S.

    2008-01-01

    Prostate cancer remains a leading cause of death in men despite increased capacity to diagnose at earlier stages. After prostate cancer has become hormone independent, which often occurs after hormonal ablation therapies, it is difficult to effectively treat. Prostate cancer may arise from mutations and dysregulation of various genes involved in regulation signal transduction (e.g., PTEN, Akt, etc.,) and the cell cycle (e.g., p53, p21Cip1, p27Kip1, Rb, etc.,). This review focuses on the aberrant interactions of signal transduction and cell cycle genes products and how they can contribute to prostate cancer and alter therapeutic effectiveness. PMID:18594202

  19. Oxazole-bridged combretastatin A analogues with improved anticancer properties.

    PubMed

    Biersack, Bernhard; Effenberger, Katharina; Schobert, Rainer; Ocker, Matthias

    2010-03-01

    Three new oxazole-bridged combretastatin A analogues with additional functional groups at the B-ring [-SMe, -OH, p-quinone] were tested for antiproliferative activity and specificity on human HL-60 leukemia, 518A2 melanoma, and colon carcinomas HCT-116 (wt)/(p53(-/-)) and HT-29 cells. While all oxazoles, except quinone 8, were efficacious against HCT-116 cells at submicromolar IC(50) values (48 h incubation), only thioanisole 5 achieved this potency in combretastatin-refractory HT-29 cells by significant upregulation of p21(cip1/waf1) associated with an S/G(2) cell-cycle arrest.

  20. Effects of a novel carbocyclic analog of pyrrolo[2,3-d]pyrimidine nucleoside on pleiotropic induction of cell death in prostate cancer cells with different androgen responsiveness.

    PubMed

    Suh, Hyewon; Choi, Ko-woon; Lee, Jongbok; Ryou, Chongsuk; Rhee, Hakjune; Lee, Chul-Hoon

    2016-02-15

    Prostate cancer is the most frequently diagnosed cancer and is one of the leading causes of male cancer death in the world. Recently, in the course of our screening for a novel anticancer compound, we synthesized carbocyclic analogs of pyrrolo[2,3-d]pyrimidine nucleoside; compounds 5, and 6. In the current study, we report the effects of compound 5 on pleiotropic induction of cell death via up-regulation of AR-associated p21(Cip1) protein in prostate cancer cells with different androgen responsiveness, such as LNCaP (androgen-dependent and -sensitive), LNCaP(C4-2) (androgen-independent and -sensitive; androgen-refractory), and DU145 (androgen-independent and -insensitive) cells. The treatment of LNCaP cells with 6 μM compound 5 for 24 h stimulated the androgen receptor (AR) activity and dramatically up-regulated transcription (56-fold) of p21(Cip1), which, in turn, induces typical apoptosis in the cells. However, induction of apoptosis through up-regulation (23-fold) of AR-associated p21(Cip1) achieved in LNCaP(C4-2) cells was possible by intensive cell treatment with compound 5 (9 μM, 48 h), because the cells are less sensitive and independent to androgen than LNCaP cells. Furthermore, 6 μM compound 5-treated DU145 cells, which exhibit extremely low AR activation due to no androgen responsiveness and dependency, showed neither up-regulation of p21(Cip1) nor apoptotic induction. Instead, a different type of cell death, autophagy-like death through the LC3B-associated autophagosome formation, was obviously induced in DU145 cells. Taken together, our results suggest that pleiotropic induction of prostate cancer cell death by compound 5 is determined by how efficiently and how abundantly androgen-dependent activation of the AR occurs, whereas compound 6 shows no induction of apoptosis in LNCaP cells.

  1. Sesamin Inhibits PDGF-Mediated Proliferation of Vascular Smooth Muscle Cells by Upregulating p21 and p27.

    PubMed

    Han, Joo-Hui; Lee, Sang-Gil; Jung, Sang-Hyuk; Lee, Jung-Jin; Park, Hyun-Soo; Kim, Young Ho; Myung, Chang-Seon

    2015-08-26

    Sesamin, an active ingredient of Asiasarum heterotropoides, is known to exhibit many bioactive functions, but the effect thereof on vascular smooth muscle cell (VSMC) proliferation remains poorly understood. Hence, we explored the antiproliferative action of sesamin on VSMCs and the underlying mechanism thereof, focusing on possible effects of sesamin on cell cycle progression. Sesamin significantly inhibited platelet-derived growth factor (PDGF)-induced VSMC proliferation (inhibition percentage at 1, 5, and 10 μM sesamin was 49.8 ± 22.0%, 74.6 ± 19.9%, and 87.8 ± 13.0%, respectively) in the absence of cytotoxicity and apoptosis, and PDGF-induced DNA synthesis; and arrested cell cycle progression in the G0/G1-to-S phase. Sesamin potently inhibited cyclin D1 and CDK4 expression, pRb phosphorylation, and expression of the proliferating cell nuclear antigen (PCNA); and upregulated p27(KIP1), p21(CIP1), and p53. The results thus indicate that the antiproliferative effect of sesamin on PDGF-stimulated VSMCs is attributable to arrest of the cell cycle in G0/G1 caused, in turn, by upregulation of p27(KIP1), p21(CIP1), and p53, and inhibition of cyclin E-CDK2 and cyclin D1-CDK4 expression.

  2. Thrombomodulin regulates monocye differentiation via PKCδ and ERK1/2 pathway in vitro and in atherosclerotic artery

    PubMed Central

    Tsai, Chien-Sung; Lin, Yi-Wen; Huang, Chun-Yao; Shih, Chun-Min; Tsai, Yi-Ting; Tsao, Nai-Wen; Lin, Chin-Sheng; Shih, Chun-Che; Jeng, Hellen; Lin, Feng-Yen

    2016-01-01

    Thrombomodulin (TM) modulates the activation of protein C and coagulation. Additionally, TM regulates monocyte migration and inflammation. However, its role on monocyte differentiation is still unknown. We investigated the effects of TM on monocyte differentiation. First, we found that TM was increased when THP-1 cells were treated with phorbol-12-myristate-13-acetate (PMA). Overexpression of TM enhanced the macrophage markers, CD14 and CD68 expression in PMA-induced THP-1. TM siRNA depressed the PMA-induced increase of p21Cip1/WAF1 via ERK1/2-NF-kB p65 signaling. TM regulated cytoskeletal reorganization via its interaction with paxillin, cofilin, LIMK1, and PYK2. In addition, PMA-induced p21Cip1/WAF1 expression, CD14-positive cell labeling intensity and ERK1/2 phosphorylation were markedly inhibited when protein kinase C-δ (PKCδ) was knocked down. We identified that TM directly interacts with PKCδ. PKCδ was highly expressed in human atherosclerotic arteries and colocalized with TM in CD68-positive infiltrated macrophages of plaques, indicating that the coordination between TM and PKCδ in macrophages participated in atherogenesis. TM may act as a scaffold for PKCδ docking, which keeps PKCδ in the region close to the monocyte membrane to promote the activation of ERK1/2. Taken together, our findings suggest that TM-PKCδ interaction may contribute to cardiovascular disorders by affecting monocye differentiation, which may develop future therapeutic applications. PMID:27910925

  3. The Spindle Assembly Checkpoint Safeguards Genomic Integrity of Skeletal Muscle Satellite Cells.

    PubMed

    Kollu, Swapna; Abou-Khalil, Rana; Shen, Carl; Brack, Andrew S

    2015-06-09

    To ensure accurate genomic segregation, cells evolved the spindle assembly checkpoint (SAC), whose role in adult stem cells remains unknown. Inducible perturbation of a SAC kinase, Mps1, and its downstream effector, Mad2, in skeletal muscle stem cells shows the SAC to be critical for normal muscle growth, repair, and self-renewal of the stem cell pool. SAC-deficient muscle stem cells arrest in G1 phase of the cell cycle with elevated aneuploidy, resisting differentiation even under inductive conditions. p21(CIP1) is responsible for these SAC-deficient phenotypes. Despite aneuploidy's correlation with aging, we find that aged proliferating muscle stem cells display robust SAC activity without elevated aneuploidy. Thus, muscle stem cells have a two-step mechanism to safeguard their genomic integrity. The SAC prevents chromosome missegregation and, if it fails, p21(CIP1)-dependent G1 arrest limits cellular propagation and tissue integration. These mechanisms ensure that muscle stem cells with compromised genomes do not contribute to tissue homeostasis.

  4. Type I collagen aging impairs discoidin domain receptor 2-mediated tumor cell growth suppression.

    PubMed

    Saby, Charles; Buache, Emilie; Brassart-Pasco, Sylvie; El Btaouri, Hassan; Courageot, Marie-Pierre; Van Gulick, Laurence; Garnotel, Roselyne; Jeannesson, Pierre; Morjani, Hamid

    2016-05-03

    Tumor cells are confronted to a type I collagen rich environment which regulates cell proliferation and invasion. Biological aging has been associated with structural changes of type I collagen. Here, we address the effect of collagen aging on cell proliferation in a three-dimensional context (3D).We provide evidence for an inhibitory effect of adult collagen, but not of the old one, on proliferation of human fibrosarcoma HT-1080 cells. This effect involves both the activation of the tyrosine kinase Discoidin Domain Receptor 2 (DDR2) and the tyrosine phosphatase SHP-2. DDR2 and SHP-2 were less activated in old collagen. DDR2 inhibition decreased SHP-2 phosphorylation in adult collagen and increased cell proliferation to a level similar to that observed in old collagen.In the presence of old collagen, a high level of JAK2 and ERK1/2 phosphorylation was observed while expression of the cell cycle negative regulator p21CIP1 was decreased. Inhibition of DDR2 kinase function also led to an increase in ERK1/2 phosphorylation and a decrease in p21CIP1 expression. Similar signaling profile was observed when DDR2 was inhibited in adult collagen. Altogether, these data suggest that biological collagen aging could increase tumor cell proliferation by reducingthe activation of the key matrix sensor DDR2.

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

    PubMed Central

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

    2016-01-01

    Cell-cycle inhibitors of the Ink4 and Cip/Kip families are involved in cellular senescence and tumor suppression. These inhibitors are individually dispensable for the cell cycle and inactivation of specific family members results in increased proliferation and enhanced susceptibility to tumor development. We have now analyzed the consequences of eliminating a substantial part of the cell-cycle inhibitory activity in the cell by generating a mouse model, which combines the absence of both p21Cip1 and p27Kip1 proteins with the endogenous expression of a Cdk4 R24C mutant insensitive to Ink4 inhibitors. Pairwise combination of Cdk4 R24C, p21-null and p27-null alleles results in frequent hyperplasias and tumors, mainly in cells of endocrine origin such as pituitary cells and in mesenchymal tissues. Interestingly, complete abrogation of p21Cip1 and p27Kip1 in Cdk4 R24C mutant mice results in a different phenotype characterized by perinatal death accompanied by general hypoplasia in most tissues. This phenotype correlates with increased replicative stress in developing tissues such as the nervous system and subsequent apoptotic cell death. Partial inhibition of Cdk4/6 rescues replicative stress signaling as well as p53 induction in the absence of cell-cycle inhibitors. We conclude that one of the major physiological activities of cell-cycle inhibitors is to prevent replicative stress during development. PMID:26292757

  6. Effect of Crocin on Cell Cycle Regulators in N-Nitroso-N-Methylurea-Induced Breast Cancer in Rats.

    PubMed

    Ashrafi, Mahboobeh; Bathaie, S Zahra; Abroun, Saeid; Azizian, Mahshid

    2015-11-01

    We previously showed the anticancer effect of crocin, a saffron carotenoid, in both breast and gastric cancers in animal models, but its mechanism of action is not clearly known, yet. In this study, the effect of crocin on cell cycle regulators is investigated. Female Wistar Albino rats were divided into two groups, with or without N-nitroso-N-methylurea (NMU) injection. After tumor formation, each group of rats was divided into two subgroups, receiving crocin or vehicle only. After 5 weeks, the rats were sacrificed and the tumors were retained for pathologic investigation and determination of the parameters. Before crocin treatment, the tumor volumes were 13.27±3.77 and 12.37±1.88, but at the end of the experiment, they were 23.66±8.82 and 11.91±2.27 in the control and crocin-treated groups, respectively. Pathologic investigation indicated the adenocarcinoma induction by NMU. Reverse transcription-polymerase chain reaction and Western blot analysis showed overexpression of cyclin D1 and p21(Cip1) in the NMU-induced breast tumors; however, the expression of both of them suppressed by crocin treatment. The previous studies indicated that crocin induces apoptosis in tumor tissue. In this study, we show that it also suppresses tumor growth and induces cell cycle arrest by downregulation of cyclin D1. In addition, crocin suppressed p21(Cip1) in a p53-dependent manner.

  7. The Insect Peptide CopA3 Increases Colonic Epithelial Cell Proliferation and Mucosal Barrier Function to Prevent Inflammatory Responses in the Gut*

    PubMed Central

    Kim, Dae Hong; Hwang, Jae Sam; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng; Lu, Li Fang; Lee, Junguee; Seok, Heon; Pothoulakis, Charalabos; Lamont, John Thomas; Kim, Ho

    2016-01-01

    The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. EGF enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide CopA3 promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin As-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the dextran sulfate sodium-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21Cip1/Waf1, and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21Cip1/Waf1. Taken together, our findings indicate that the insect peptide CopA3 prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function. PMID:26655716

  8. The Insect Peptide CopA3 Increases Colonic Epithelial Cell Proliferation and Mucosal Barrier Function to Prevent Inflammatory Responses in the Gut.

    PubMed

    Kim, Dae Hong; Hwang, Jae Sam; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng; Lu, Li Fang; Lee, Junguee; Seok, Heon; Pothoulakis, Charalabos; Lamont, John Thomas; Kim, Ho

    2016-02-12

    The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. EGF enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide CopA3 promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin As-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the dextran sulfate sodium-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21(Cip1/Waf1), and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21(Cip1/Waf1). Taken together, our findings indicate that the insect peptide CopA3 prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function.

  9. A retroviral mutagenesis screen reveals strong cooperation between Bcl11a overexpression and loss of the Nf1 tumor suppressor gene

    PubMed Central

    Yin, Bin; Delwel, Ruud; Valk, Peter J.; Wallace, Margaret R.; Loh, Mignon L.; Shannon, Kevin M.

    2009-01-01

    NF1 inactivation occurs in specific human cancers, including juvenile myelomonocytic leukemia, an aggressive myeloproliferative disorder of childhood. However, evidence suggests that Nf1 loss alone does not cause leukemia. We therefore hypothesized that inactivation of the Nf1 tumor suppressor gene requires cooperating mutations to cause acute leukemia. To search for candidate genes that cooperate with Nf1 deficiency in leukemogenesis, we performed a forward genetic screen using retroviral insertion mutagenesis in Nf1 mutant mice. We identified 43 common proviral insertion sites that contain candidate genes involved in leukemogenesis. One of these genes, Bcl11a, confers a growth advantage in cultured Nf1 mutant hematopoietic cells and causes early onset of leukemia of either myeloid or lymphoid lineage in mice when expressed in Nf1-deficient bone marrow. Bcl11a-expressing cells display compromised p21Cip1 induction, suggesting that Bcl11a's oncogenic effects are mediated, in part, through suppression of p21Cip1. Importantly, Bcl11a is expressed in human chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia samples. A subset of AML patients, who had poor outcomes, of 16 clusters, displayed high levels of BCL11A in leukemic cells. These findings suggest that deregulated Bcl11a cooperates with Nf1 in leukemogenesis, and a therapeutic strategy targeting the BCL11A pathway may prove beneficial in the treatment of leukemia. PMID:18948576

  10. Correlation of EGFR, pEGFR and p16INK4 expressions and high risk HPV infection in HIV/AIDS-related squamous cell carcinoma of conjunctiva

    PubMed Central

    2014-01-01

    Background Squamous cell carcinoma of conjunctiva has increased tenfold in the era of HIV/AIDS. The disease pattern has also changed in Africa, affecting young persons, with peak age-specific incidence of 30-39 years, similar to that of Kaposi sarcoma, a well known HIV/AIDS defining neoplasm. In addition, the disease has assumed more aggressive clinical course. The contributing role of exposure to high risk HPV in the development of SCCC is still emerging. Objective The present study aimed to investigate if immunohistochemical expressions of EGFR, pEGFR and p16, could predict infection with high risk HPV in HIV-related SCCC. Methods FFPE tissue blocks of fifty-eight cases diagnosed on hematoxylin and eosin with SCCC between 2005-2011, and subsequently confirmed from medical records to be HIV positive at the department of human pathology, UoN/KNH, were used for the study. Immunohistochemistry was performed to assess the expressions of p16INK4A, EGFR and pEGFR. This was followed with semi-nested PCR based detection and sequencing of HPV genotypes. The sequences were compared with the GenBank database, and data analyzed for significant statistical correlations using SPSS 16.0. Ethical approval to conduct the study was obtained from KNH-ERC. Results Out of the fifty-eight cases of SCCC analyzed, twenty-nine (50%) had well differentiated (grade 1), twenty one (36.2%) moderately differentiated (grade 2) while eight (13.8%) had poorly differentiated (grade 3) tumours. Immunohistochemistry assay was done in all the fifty eight studied cases, of which thirty nine cases (67.2%) were positive for p16INK4A staining, forty eight cases (82.8%) for EGFR and fifty one cases (87.9%) showed positivity for p-EGFR. HPV DNA was detected in 4 out of 40 SCCC cases (10%) in which PCR was performed, with HPV16 being the only HPV sub-type detected. Significant statistical association was found between HPV detection and p16INK4 (p=0.000, at 99% C.I) and EGFR (p=0.028, at 95% C.I) expressions

  11. The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein.

    PubMed Central

    Amellem, O.; Sandvik, J. A.; Stokke, T.; Pettersen, E. O.

    1998-01-01

    We have studied the role of the oxygen-dependent pyrimidine metabolism in the regulation of cell cycle progression under moderate hypoxia in human cell lines containing functional (T-47D) or non-functional (NHIK 3025, SAOS-2) retinoblastoma gene product (pRB). Under aerobic conditions, pRB exerts its growth-regulatory effects during early G1 phase of the cell cycle, when all pRB present has been assumed to be in the underphosphorylated form and bound in the nucleus. We demonstrate that pRB is dephosphorylated and re-bound in the nucleus in approximately 90% of T-47D cells located in S and G2 phases under moderately hypoxic conditions. Under these conditions, no T-47D cells entered S-phase, and no progression through S-phase was observed. Progression of cells through G2 and mitosis seems independent of their functional pRB status. The p21WAF1/CIP1 protein level was significantly reduced by moderate hypoxia in p53-deficient T-47D cells, whereas p16(INK4a) was not expressed in these cells, suggesting that the hypoxia-induced cell cycle arrest is independent of these cyclin-dependent kinase inhibitors. The addition of pyrimidine deoxynucleosides did not release T-47D cells, containing mainly underphosphorylated pRB, from the cell cycle arrest induced by moderate hypoxia. However, NHIK 3025 cells, in which pRB is abrogated by expression of the HPV18 E7 oncoprotein, and SAOS-2 cells, which lack pRB expression, continued cell cycle progression under moderate hypoxia provided that excess pyrimidine deoxynucleosides were present. NHIK 3025 cells express high levels of p16INK4a under both aerobic and moderately hypoxic conditions, suggesting that the inhibitory function of p16(INK4a) would not be manifested in such pRB-deficient cells. Thus, pRB, a key member of the cell cycle checkpoint network, seems to play a major role by inducing growth arrest under moderate hypoxia, and it gradually overrides hypoxia-induced suppression of pyrimidine metabolism in the regulation of

  12. Epstein-Barr Virus Nuclear Antigen 3A Promotes Cellular Proliferation by Repression of the Cyclin-Dependent Kinase Inhibitor p21WAF1/CIP1

    PubMed Central

    Tursiella, Melissa L.; Bowman, Emily R.; Wanzeck, Keith C.; Throm, Robert E.; Liao, Jason; Zhu, Junjia; Sample, Clare E.

    2014-01-01

    Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14ARF and p16INK4a expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14ARF and p16INK4a. By contrast, p16INK4a was not detectably expressed in Wp-R BL and the low-level expression of p14ARF was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21WAF1/CIP1, a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21WAF1/CIP1 expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the proliferation of

  13. SLIT/ROBO2 Signaling Promotes Mammary Stem Cell Senescence by Inhibiting Wnt Signaling

    PubMed Central

    Harburg, Gwyndolen; Compton, Jennifer; Liu, Wei; Iwai, Naomi; Zada, Shahrzad; Marlow, Rebecca; Strickland, Phyllis; Zeng, Yi Arial; Hinck, Lindsay

    2014-01-01

    Summary WNT signaling stimulates the self-renewal of many types of adult stem cells, including mammary stem cells (MaSCs), but mechanisms that limit this activity are poorly understood. Here, we demonstrate that SLIT2 restricts stem cell renewal by signaling through ROBO2 in a subset of basal cells to negatively regulate WNT signaling. The absence of SLIT/ROBO2 signaling leads to increased levels of nuclear β-catenin. Robo2 loss does not increase the number of stem cells; instead, stem cell renewal is enhanced in the absence of SLIT/ROBO2 signaling. This is due to repressed expression of p16INK4a, which, in turn, delays MaSC senescence. Together, our studies support a model in which SLITs restrict the expansion of MaSCs by countering the activity of WNTs and limiting self-renewal. PMID:25241737

  14. p14 — EDRN Public Portal

    Cancer.gov

    CDKN2A locus gives rise to 2 distinct transcripts from different promoters: p16 (INK4A) and p14 (ARF). Each has a 5-prime exon which is spliced into common exons 2 and 3 but in some sequences in a different reading frame. The beta transcript of CDKN2A encodes p14(ARF). P14/ARF has the ability to elicit a p53 response, which results in increased expression of both CDKN1A, also called CIP1, and MDM2, resulting in a cell cycle arrest in both the G1 and G2/M phases. p14/ARF binds to MDM2 and promotes the rapid degradation of MDM2. The deletion of the ARF-INK4A locus simultaneously impairs the INK4A--cyclin D/CDK4--RB and the ARF--MDM2--p53 pathways.

  15. Role of protein biomarkers in the detection of high-grade disease in cervical cancer screening programs.

    PubMed

    Brown, Charlotte A; Bogers, Johnannes; Sahebali, Shaira; Depuydt, Christophe E; De Prins, Frans; Malinowski, Douglas P

    2012-01-01

    Since the Pap test was introduced in the 1940s, there has been an approximately 70% reduction in the incidence of squamous cell cervical cancers in many developed countries by the application of organized and opportunistic screening programs. The efficacy of the Pap test, however, is hampered by high interobserver variability and high false-negative and false-positive rates. The use of biomarkers has demonstrated the ability to overcome these issues, leading to improved positive predictive value of cervical screening results. In addition, the introduction of HPV primary screening programs will necessitate the use of a follow-up test with high specificity to triage the high number of HPV-positive tests. This paper will focus on protein biomarkers currently available for use in cervical cancer screening, which appear to improve the detection of women at greatest risk for developing cervical cancer, including Ki-67, p16(INK4a), BD ProEx C, and Cytoactiv HPV L1.

  16. Skin hypoxia: a promoting environmental factor in melanomagenesis.

    PubMed

    Bedogni, Barbara; Powell, Marianne Broome

    2006-06-01

    Melanomagenesis is a complex phenomenon in which environmental, genetic and host factors play a role. Sun burns in early childhood are a known risk factor in melanoma development. Alteration of prosurvival genes such as Ras and Akt and loss of function of the p16(INK4a)-CDK4/6-pRb and p14(ARF)-HDM2-p53 pathways are strongly associated with human melanoma. We have demonstrated that normally occurring skin hypoxia represents a previously unappreciated host promoting factor in melanomagenesis. Melanocytes that express oncogenes such as Akt, and are therefore genetically unstable, show a transform phenotype only in a mild hypoxic environment that resembles the hypoxic status of the skin. Hypoxia, therefore, is not just a prerogative of advanced neoplasia; physiologic tissue hypoxia, through the activity of HIF1alpha, can function as a promoting factor in tumorigenesis.

  17. Pancreatitis-induced Inflammation Contributes to Pancreatic Cancer by Inhibiting Oncogene-Induced Senescence

    PubMed Central

    Guerra, Carmen; Collado, Manuel; Navas, Carolina; Schuhmacher, Alberto J; Hernández-Porras, Isabel; Cañamero, Marta; Rodriguez-Justo, Manuel; Serrano, Manuel; Barbacid, Mariano

    2016-01-01

    Pancreatic acinar cells of adult mice (≥P60) are resistant to transformation by some of the most robust oncogenic insults including expression of K-Ras oncogenes and loss of p16Ink4a/p19Arf or Trp53 tumor suppressors. Yet, these acinar cells yield pancreatic intraepithelial neoplasias (mPanIN) and ductal adenocarcinomas (mPDAC) if exposed to limited bouts of non-acute pancreatitis, providing they harbor K-Ras oncogenes. Pancreatitis contributes to tumor progression by abrogating the senescence barrier characteristic of low-grade mPanINs. Attenuation of pancreatitis-induced inflammation also accelerates tissue repair and thwarts mPanIN expansion. Patients with chronic pancreatitis display senescent PanINs, if they have received anti-inflammatory drugs. These results put forward the concept that anti-inflammatory treatment of people diagnosed with pancreatitis may reduce their risk of developing PDAC. PMID:21665147

  18. EGF RECEPTOR SIGNALING IS ESSENTIAL FOR K-RAS ONCOGENE-DRIVEN PANCREATIC DUCTAL ADENOCARCINOMA

    PubMed Central

    Navas, Carolina; Hernández-Porras, Isabel; Schuhmacher, Alberto J; Sibilia, Maria; Guerra, Carmen; Barbacid, Mariano

    2013-01-01

    Clinical evidence indicates that mutation/activation of EGF receptors (EGFRs) is mutually exclusive with the presence of K-RAS oncogenes in lung and colon tumors. We have validated these observations using genetically engineered mouse models. However, pancreatic ductal adenocarcinomas driven by K-Ras oncogenes are totally dependent on EGFR signaling. Similar results were obtained using human pancreatic tumor cell lines. EGFRs were also essential even in the context of pancreatic injury and absence of p16Ink4a/p19Arf. Only loss of p53 made pancreatic tumors independent of EGFR signaling. Additional inhibition of PI3K and STAT3 effectively prevented proliferation of explants derived from these p53–defective pancreatic tumors. These findings may provide the bases for more rational approaches to treat pancreatic tumors in the clinic. PMID:22975375

  19. American cranberry (Vaccinium macrocarpon) extract affects human prostate cancer cell growth via cell cycle arrest by modulating expression of cell cycle regulators.

    PubMed

    Déziel, Bob; MacPhee, James; Patel, Kunal; Catalli, Adriana; Kulka, Marianna; Neto, Catherine; Gottschall-Pass, Katherine; Hurta, Robert

    2012-05-01

    Prostate cancer is one of the most common cancers in the world, and its prevalence is expected to increase appreciably in the coming decades. As such, more research is necessary to understand the etiology, progression and possible preventative measures to delay or to stop the development of this disease. Recently, there has been interest in examining the effects of whole extracts from commonly harvested crops on the behaviour and progression of cancer. Here, we describe the effects of whole cranberry extract (WCE) on the behaviour of DU145 human prostate cancer cells in vitro. Following treatment of DU145 human prostate cancer cells with 10, 25 and 50 μg ml⁻¹ of WCE, respectively for 6 h, WCE significantly decreased the cellular viability of DU145 cells. WCE also decreased the proportion of cells in the G2-M phase of the cell cycle and increased the proportion of cells in the G1 phase of the cell cycle following treatment of cells with 25 and 50 μg ml⁻¹ treatment of WCE for 6 h. These alterations in cell cycle were associated with changes in cell cycle regulatory proteins and other cell cycle associated proteins. WCE decreased the expression of CDK4, cyclin A, cyclin B1, cyclin D1 and cyclin E, and increased the expression of p27. Changes in p16(INK4a) and pRBp107 protein expression levels also were evident, however, the changes noted in p16(INK4a) and pRBp107 protein expression levels were not statistically significant. These findings demonstrate that phytochemical extracts from the American cranberry (Vaccinium macrocarpon) can affect the behaviour of human prostate cancer cells in vitro and further support the potential health benefits associated with cranberries.

  20. Partial sleep deprivation activates the DNA damage response (DDR) and the senescence-associated secretory phenotype (SASP) in aged adult humans.

    PubMed

    Carroll, Judith E; Cole, Steven W; Seeman, Teresa E; Breen, Elizabeth C; Witarama, Tuff; Arevalo, Jesusa M G; Ma, Jeffrey; Irwin, Michael R

    2016-01-01

    Age-related disease risk has been linked to short sleep duration and sleep disturbances; however, the specific molecular pathways linking sleep loss with diseases of aging are poorly defined. Key cellular events seen with aging, which are thought to contribute to disease, may be particularly sensitive to sleep loss. We tested whether one night of partial sleep deprivation (PSD) would increase leukocyte gene expression indicative of DNA damage responses (DDR), the senescence-associated secretory phenotype (SASP), and senescence indicator p16(INK4a) in older adult humans, who are at increased risk for cellular senescence. Community-dwelling older adults aged 61-86years (n=29; 48% male) underwent an experimental partial sleep deprivation (PSD) protocol over 4 nights, including adaptation, an uninterrupted night of sleep, partial sleep deprivation (sleep restricted 3-7AM), and a subsequent full night of sleep. Blood samples were obtained each morning to assess peripheral blood mononuclear cell (PBMC) gene expression using Illumina HT-12 arrays. Analyses of microarray results revealed that SASP (p<.05) and DDR (p=.08) gene expression were elevated from baseline to PSD nights. Gene expression changes were also observed from baseline to PSD in NFKB2, NBS1 and CHK2 (all p's<.05). The senescence marker p16(INK4a) (CDKN2A) was increased 1day after PSD compared to baseline (p<.01), however confirmatory RT-PCR did not replicate this finding. One night of partial sleep deprivation activates PBMC gene expression patterns consistent with biological aging in this older adult sample. PSD enhanced the SASP and increased the accumulation of damage that initiates cell cycle arrest and promotes cellular senescence. These findings causally link sleep deprivation to the molecular processes associated with biological aging.

  1. Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies.

    PubMed

    Prével, Camille; Pellerano, Morgan; González-Vera, Juan A; Henri, Pauline; Meunier, Laurent; Vollaire, Julien; Josserand, Véronique; Morris, May C

    2016-11-15

    Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma.

  2. Partial Sleep Deprivation Activates the DNA Damage Response (DDR) and the Senescence-Associated Secretory Phenotype (SASP) in Aged Adult Humans

    PubMed Central

    Carroll, Judith E.; Cole, Steven W.; Seeman, Teresa E.; Breen, Elizabeth C.; Witarama, Tuff; Arevalo, Jesusa M.G.; Ma, Jeffrey; Irwin, Michael R.

    2015-01-01

    Age-related disease risk has been linked to short sleep duration and sleep disturbances; however, the specific molecular pathways linking sleep loss with diseases of aging are poorly defined. Key cellular events seen with aging, which are thought to contribute to disease, may be particularly sensitive to sleep loss. We tested whether one night of partial sleep deprivation (PSD) would increase leukocyte gene expression indicative of DNA damage responses (DDR), the senescence-associated secretory phenotype (SASP), and senescence indicator p16INK4a in older adult humans, who are at increased risk for cellular senescence. Community-dwelling older adults aged 61-86 years (n=29; 48% male) underwent an experimental partial sleep deprivation (PSD) protocol over 4 nights, including adaptation, an uninterrupted night of sleep, partial sleep deprivation (sleep restricted 3 AM to 7 AM), and a subsequent full night of sleep. Blood samples were obtained each morning to assess peripheral blood mononuclear cell (PBMC) gene expression using Illumina HT-12 arrays. Analyses of microarray results revealed that SASP (p < .05) and DDR (p = .08) gene expression were elevated from baseline to PSD nights. Gene expression changes were also observed from baseline to PSD in NFKB2, NBS1 and CHK2 (all p's < .05). The senescence marker p16INK4a (CDKN2A) was increased one day after PSD compared to baseline (p < .01), however confirmatory RT-PCR did not replicate this finding. One night of partial sleep deprivation activates PBMC gene expression patterns consistent with biological aging in this older adult sample. PSD enhanced the SASP and increased the accumulation of damage that initiates cell cycle arrest and promotes cellular senescence. These findings causally link sleep deprivation to the molecular processes associated with biological aging. PMID:26336034

  3. Efficacy of CDK4 inhibition against sarcomas depends on their levels of CDK4 and p16ink4 mRNA

    PubMed Central

    Perez, Marco; Muñoz-Galván, Sandra; Jiménez-García, Manuel P.; Marín, Juan J.; Carnero, Amancio

    2015-01-01

    Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness and new treatment approaches are needed. Deregulation of the G1 checkpoint is crucial for various oncogenic transformation processes, suggesting that many cancer cell types depend on CDK4/6 activity. Thus, CDK4/6 activity appears to represent a promising therapeutic target for cancer treatment. In the present work, we explore the efficacy of CDK4 inhibition using palbociclib (PD0332991), a highly selective inhibitor of CDK4/6, in a panel of sarcoma cell lines and sarcoma tumor xenografts (PDXs). Palbociclib induces senescence in these cell lines and the responsiveness of these cell lines correlated with their levels of CDK4 mRNA. Palbociclib is also active in vivo against sarcomas displaying high levels of CDK4 but not against sarcomas displaying low levels of CDK4 and high levels of p16ink4a. The analysis of tumors growing after palbociclib showed a clear decrease in the CDK4 levels, indicating that clonal selection occurred in these treated tumors. In summary, our data support the efficacy of CDK4 inhibitors against sarcomas displaying increased CDK4 levels, particularly fibrosarcomas and MPNST. Our results also suggest that high levels of p16ink4a may indicate poor efficacy of CDK4 inhibitors. PMID:26528855

  4. Different Roles of Negative and Positive Components of the Circadian Clock in Oncogene-induced Neoplastic Transformation.

    PubMed

    Katamune, Chiharu; Koyanagi, Satoru; Shiromizu, Shoya; Matsunaga, Naoya; Shimba, Shigeki; Shibata, Shigenobu; Ohdo, Shigehiro

    2016-05-13

    In mammals, circadian rhythms in physiological function are generated by a molecular oscillator driven by transcriptional-translational feedback loop consisting of negative and positive regulators. Disruption of this circadian clock machinery is thought to increase the risk of cancer development, but the potential contributions of each component of circadian clock to oncogenesis have been little explored. Here we reported that negative and positive transcriptional regulators of circadian feedback loop had different roles in oncogene-induced neoplastic transformation. Mouse embryonic fibroblasts prepared from animals deficient in negative circadian clock regulators, Period2 (Per2) or Cryptochrome1/2 (Cry1/2), were prone to transformation induced by co-expression of H-ras(V12) and SV40 large T antigen (SV40LT). In contrast, mouse embryonic fibroblasts prepared from mice deficient in positive circadian clock regulators, Bmal1 or Clock, showed resistance to oncogene-induced transformation. In Per2 mutant and Cry1/2-null cells, the introduction of oncogenes induced expression of ATF4, a potent repressor of cell senescence-associated proteins p16INK4a and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, in Bmal1-null and Clock mutant cells, the expression of ATF4 was not induced by oncogene introduction, which allowed constitutive expression of p16INK4a and p19ARF triggering cellular senescence. Although genetic ablation of either negative or positive transcriptional regulators of the circadian clock leads to disrupted rhythms in physiological functions, our findings define their different contributions to neoplastic cellular transformation.

  5. CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts, “fueling” tumor growth via paracrine interactions, without an increase in neo-angiogenesis

    PubMed Central

    Capparelli, Claudia; Chiavarina, Barbara; Whitaker-Menezes, Diana; Pestell, Timothy G.; Pestell, Richard G.; Hulit, James; Andò, Sebastiano; Howell, Anthony; Martinez-Outschoorn, Ubaldo E.; Sotgia, Federica; Lisanti, Michael P.

    2012-01-01

    Here, we investigated the compartment-specific role of cell cycle arrest and senescence in breast cancer tumor growth. For this purpose, we generated a number of hTERT-immortalized senescent fibroblast cell lines overexpressing CDK inhibitors, such as p16(INK4A), p19(ARF) or p21(WAF1/CIP1). Interestingly, all these senescent fibroblast cell lines showed evidence of increased susceptibility toward the induction of autophagy (either at baseline or after starvation), as well as significant mitochondrial dysfunction. Most importantly, these senescent fibroblasts also dramatically promoted tumor growth (up to ~2-fold), without any comparable increases in tumor angiogenesis. Conversely, we generated human breast cancer cells (MDA-MB-231 cells) overexpressing CDK inhibitors, namely p16(INK4A) or p21(WAF1/CIP1). Senescent MDA-MB-231 cells also showed increased expression of markers of cell cycle arrest and autophagy, including β-galactosidase, as predicted. Senescent MDA-MB-231 cells had retarded tumor growth, with up to a near 2-fold reduction in tumor volume. Thus, the effects of CDK inhibitors are compartment-specific and are related to their metabolic effects, which results in the induction of autophagy and mitochondrial dysfunction. Finally, induction of cell cycle arrest with specific inhibitors (PD0332991) or cellular stressors [hydrogen peroxide (H₂O₂) or starvation] indicated that the onset of autophagy and senescence are inextricably linked biological processes. The compartment-specific induction of senescence (and hence autophagy) may be a new therapeutic target that could be exploited for the successful treatment of human breast cancer patients. PMID:22935696

  6. Functional inactivation of Rb sensitizes cancer cells to TSC2 inactivation induced cell death.

    PubMed

    Danos, Arpad M; Liao, Yang; Li, Xuan; Du, Wei

    2013-01-01

    We showed previously that inactivation of TSC2 induces death in cancer cells lacking the Retinoblastoma (Rb) tumor suppressor under stress conditions, suggesting that inactivation of TSC2 can potentially be used as an approach to specifically kill cancers that have lost WT Rb. As Rb is often inactivated in cancers by overexpression of cyclin D1, loss of p16(ink4a) cdk inhibitor, or expression of viral oncoproteins, it will be interesting to determine if such functional inactivation of Rb would similarly sensitize cancer cells to TSC2 inactivation induced cell death. In addition, many cancers lack functional Pten, resulting in increased PI3K/Akt signaling that has been shown to modulate E2F-induced cell death. Therefore it will be interesting to test whether loss of Pten will affect TSC2 inactivation induced killing of Rb mutant cancer cells. Here, we show that overexpression of Cyclin D1 or the viral oncogene E1a sensitizes cancer cells to TSC2 knockdown induced cell death and growth inhibition. On the other hand, knockdown of p16(ink4a) sensitizes cancer cells to TSC2 knockdown induced cell death in a manner that is likely dependant on serum induction of Cyclin D1 to inactivate the Rb function. Additionally, we demonstrate that loss of Pten does not interfere with TSC2 knockdown induced cell death in Rb mutant cancer cells. Together, these results suggest that TSC2 is potentially a useful target for a large spectrum of cancer types with an inactivated Rb pathway.

  7. eMelanoBase: an online locus-specific variant database for familial melanoma.

    PubMed

    Fung, David C Y; Holland, Elizabeth A; Becker, Therese M; Hayward, Nicholas K; Bressac-de Paillerets, Brigitte; Mann, Graham J

    2003-01-01

    A proportion of melanoma-prone individuals in both familial and non-familial contexts has been shown to carry inactivating mutations in either CDKN2A or, rarely, CDK4. CDKN2A is a complex locus that encodes two unrelated proteins from alternately spliced transcripts that are read in different frames. The alpha transcript (exons 1alpha, 2, and 3) produces the p16INK4A cyclin-dependent kinase inhibitor, while the beta transcript (exons 1beta and 2) is translated as p14ARF, a stabilizing factor of p53 levels through binding to MDM2. Mutations in exon 2 can impair both polypeptides and insertions and deletions in exons 1alpha, 1beta, and 2, which can theoretically generate p16INK4A-p14ARF fusion proteins. No online database currently takes into account all the consequences of these genotypes, a situation compounded by some problematic previous annotations of CDKN2A-related sequences and descriptions of their mutations. As an initiative of the international Melanoma Genetics Consortium, we have therefore established a database of germline variants observed in all loci implicated in familial melanoma susceptibility. Such a comprehensive, publicly accessible database is an essential foundation for research on melanoma susceptibility and its clinical application. Our database serves two types of data as defined by HUGO. The core dataset includes the nucleotide variants on the genomic and transcript levels, amino acid variants, and citation. The ancillary dataset includes keyword description of events at the transcription and translation levels and epidemiological data. The application that handles users' queries was designed in the model-view-controller architecture and was implemented in Java. The object-relational database schema was deduced using functional dependency analysis. We hereby present our first functional prototype of eMelanoBase. The service is accessible via the URL www.wmi.usyd.edu.au:8080/melanoma.html.

  8. A Comparative Analysis of Clinical and Molecular Factors with the Stage of Cervical Cancer in a Brazilian Cohort

    PubMed Central

    Amaro-Filho, Sergio M.; Golub, Jonathan E.; Nuovo, Gerard J.; Cunha, Cynthia B.; Levi, José E.; Villa, Luisa L.; Andrade, Cecília V.; Russomano, Fabio B.; Tristão, Aparecida; Pires, Andrea; Nicol, Alcina F.

    2013-01-01

    Cell cycle protein expression plays an important role in the pathophysiology of cervical cancer. However, few studies have attempted to correlate the use of these biomarkers with the clinical progression of the tumor. Objectives 1) To analyze the expression of Ki-67, p53 and p16INK4a in cervical cancer, 2) to correlate the relative expression of these proteins as well as clinical parameters with the stage of disease, and 3) to determine the HPV DNA prevalence and subtype distribution. Methods Tissue Micro-Arrays (TMA) from patients with invasive cervical cancer (ICC) and controls were analyzed. HPV DNA detection was done by PCR and in situ hybridization. Ki-67, p53 and p16INK4a were analyzed by immunohistochemistry; clinical data was derived from the chart review. Results Advanced tumor stage (III and IV) was strongly associated (p<0.005) with advanced age (>55 years old), with more than four pregnancies and with the lack of formal education. HPV DNA was found in 94.3% of cases with the most prevalent types being HPV16 (67.5%), followed by HPV33 (12.0%) and HPV35 (3.6%). High expression of Ki-67 and p16 was more common in the advanced FIGO stages (p = 0.023). Women with HPV16 tended to be younger (50.9 years; SE 1.9) compared to women with other types (59.9 years; SE 2.8). Conclusion We found that Ki-67 and p16 expression were independently associated with the tumor stage. We also noted that about 1/3 of the cervical cancers in this Brazilian cohort were not associated with HPV types directly targeted by the current HPV vaccines. PMID:23505442

  9. Differential Methylation of the HPV 16 Upstream Regulatory Region during Epithelial Differentiation and Neoplastic Transformation

    PubMed Central

    Vinokurova, Svetlana; von Knebel Doeberitz, Magnus

    2011-01-01

    High risk human papillomaviruses are squamous epitheliotropic viruses that may cause cervical and other cancers. HPV replication depends on squamous epithelial differentiation. Transformation of HPV-infected cells goes along with substantial alteration of the viral gene expression profile and preferentially occurs at transformation zones usually at the uterine cervix. Methylation of the viral genome may affect regulatory features that control transcription and replication of the viral genome. Therefore, we analyzed the methylation pattern of the HPV16 upstream regulatory region (URR) during squamous epithelial differentiation and neoplastic transformation and analyzed how shifts in the HPV URR methylome may affect viral gene expression and replication. HPV 16 positive biopsy sections encompassing all stages of an HPV infection (latent, permissive and transforming) were micro-dissected and DNA was isolated from cell fractions representing the basal, intermediate, and superficial cell layers, each, as well as from transformed p16INK4a-positive cells. We observed fundamental changes in the methylation profile of transcription factor binding sites in the HPV16 upstream regulatory region linked to the squamous epithelial differentiation stage. Squamous epithelial transformation indicated by p16INK4a overexpression was associated with methylation of the distal E2 binding site 1 leading to hyper-activation of the HPV 16 URR. Adjacent normal but HPV 16-infected epithelial areas retained hyper-methylated HPV DNA suggesting that these viral genomes were inactivated. These data suggest that distinct shifts of the HPV 16 methylome are linked to differentiation dependent transcription and replication control and may trigger neoplastic transformation. PMID:21915330

  10. Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor-suppressor activities.

    PubMed

    Kagawa, S; Natsuizaka, M; Whelan, K A; Facompre, N; Naganuma, S; Ohashi, S; Kinugasa, H; Egloff, A M; Basu, D; Gimotty, P A; Klein-Szanto, A J; Bass, A J; Wong, K-K; Diehl, J A; Rustgi, A K; Nakagawa, H

    2015-04-30

    Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor-suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor-suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated β-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16(INK4A)-Rb pathway. Loss of p16(INK4A) or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as transforming growth factor-β-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor-suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.

  11. P19ARF stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2

    PubMed Central

    Tao, Weikang; Levine, Arnold J.

    1999-01-01

    The INK4a-ARF locus encodes two distinct tumor suppressors, p16INK4a and p19ARF. Whereas p16INK4a restrains cell growth through preventing phosphorylation of the retinoblastoma protein, p19ARF acts by attenuating Mdm2-mediated degradation of p53, thereby stabilizing p53. Recent data indicate that Mdm2 shuttles between the nucleus and the cytoplasm and that nucleo-cytoplasmic shuttling of Mdm2 is essential for Mdm2’s ability to promote p53 degradation. Therefore, Mdm2 must export p53 from the nucleus to the cytoplasm where it targets p53 for degradation. We show here that coexpression of p19ARF blocks the nucleo-cytoplasmic shuttling of Mdm2. Moreover, subnuclear localization of Mdm2 changes from the nucleoplasm to the nucleolus in a shuttling time-dependent manner, whereas p19ARF is exclusively located in the nucleolus. In heterokaryons containing Mdm2 and p19ARF, the longer the Mdm2 shuttling is allowed, the more Mdm2 protein colocalizes with p19ARF in the nucleolus, implying that Mdm2 moves from the nucleoplasm to the nucleolus and then associates with p19ARF there. Furthermore, whether or not Mdm2 colocalizes with p19ARF in the nucleolus, p19ARF prevents Mdm2 shuttling. This observation suggests that Mdm2 might be exported through the nucleolus and p19ARF could inhibit the nuclear export of Mdm2 by tethering Mdm2 in the nucleolus. Taken together, p19ARF could stabilize p53 by inhibiting the nuclear export of Mdm2. PMID:10359817

  12. Validation that Metabolic Tumor Volume Predicts Outcome in Head and Neck Cancer

    PubMed Central

    Tang, Chad; Murphy, James D.; Khong, Brian; La, Trang H.; Kong, Christina; Fischbein, Nancy J.; Colevas, A. Dimitrios; Iagaru, Andrei H.; Graves, Edward E.; Loo, Billy W.; Le, Quynh-Thu

    2011-01-01

    Purpose We have previously reported that metabolic tumor volume (MTV) obtained from pre-treatment FDG PET/CT predicted outcome in patients with head-and-neck cancer (HNC). The purpose of this study is to validate these results on an independent dataset, determine if the primary tumor or nodal MTV drives this correlation, and explore the interaction with p16INK4a status as a surrogate marker for HPV. Methods and Materials The validation dataset in this study included 83 patients with squamous cell HNC who had a FDG PET/CT scan prior to definitive radiotherapy. MTV and SUVmax were calculated for the primary tumor, involved nodes, and the combination of both. The primary endpoint was to validate that MTV predicted progression-free survival and overall survival. Secondary analyses included determining the prognostic utility of primary tumor versus nodal MTV. Results Similar to our prior findings, an increase in total MTV of 17 cm3 (difference between 75th and 25th percentile) was associated with a 2.1 fold increase in the risk of disease progression (p=0.0002), and a 2.0 fold increase in the risk of death (p=0.0048). SUVmax was not associated with either outcome. Primary tumor MTV predicted progression-free (HR=1.94; p<0.0001) and overall (HR=1.57; p<0.0001) survival, whereas nodal MTV did not. In addition, MTV predicted progression-free (HR=4.23; p<0.0001) and overall (HR=3.21; p=0.0029) survival in patients with p16INK4a positive oropharyngeal cancer. Conclusions This study validates our previous findings that MTV independently predicts outcomes in HNC. MTV should be considered as a potential risk stratifying biomarker in future studies of HNC. PMID:22270174

  13. Endometrial metaplasias and reactive changes: a spectrum of altered differentiation.

    PubMed

    Nicolae, Alina; Preda, Ovidiu; Nogales, Francisco F

    2011-02-01

    Endometrial metaplasias and changes (EMCs) are conditions frequently overlooked and misdiagnosed. The aim of this review is to update current issues and provide a classification with a practical clinicopathological approach. Hormonal or irritative stimuli are the main inducing factors of EMCs, although some metaplasias have a mutational origin. EMCs vary from reactive, degenerative lesions to those able to associate with malignancy or those having a preneoplastic potential. The most common types of EMCs are ciliated tubal metaplasia (CTM) and mucinous metaplasia (MM), which occur in simple and complex glands, and possibly these architectural changes hold the same prognostic significance as they do in hyperplastic endometrioid lesions. Immunohistochemically, CTM is positive for LhS28, bcl-2, PAX2 and p16(INK4A). Complex CTM is likely to be a precursor of ciliated endometrioid-type carcinomas. MMs should be evaluated architecturally, taking into account that their atypicality is minimal. The differentiation between complex MM and mucinous carcinoma may be extremely difficult. Surface complex, papillary MM in endometrial polyps can be considered as benign. Intestinal-type endometrial MM is rare and its presence should prompt further investigation of associated lesions in the endocervix. Endometrial squamous metaplasia (ESS) is often linked to chronic irritative situations. It should be differentiated from secondary involvement by a human papilomavirus-related cervical lesion. Morular metaplasia is a mutational phenomenon with a distinct phenotype that helps to differentiate it from ESS. Morules are benign, hormonally inert structures that are often markers of complex endometrioid glandular architecture, and they are associated with an attenuated malignancy. Endometrial reactive changes are commonly associated with desquamation or hormonal imbalance. The frequent, p16(INK4A) positive, benign surface papillary syncytial change may be misdiagnosed, in some cases, as

  14. Infection of Lymphoid Cells by Integration-Defective Human Immunodeficiency Virus Type 1 Increases De Novo Methylation

    PubMed Central

    Fang, Jing-Yuan; Mikovits, Judy A.; Bagni, Rachel; Petrow-Sadowski, Cari L.; Ruscetti, Francis W.

    2001-01-01

    DNA methylation, by regulating the transcription of genes, is a major modifier of the eukaryotic genome. DNA methyltransferases (DNMTs) are responsible for both maintenance and de novo methylation. We have reported that human immunodeficiency virus type 1 (HIV-1) infection increases DNMT1 expression and de novo methylation of genes such as the gamma interferon gene in CD4+ cells. Here, we examined the mechanism(s) by which HIV-1 infection increases the cellular capacity to methylate genes. While the RNAs and proteins of all three DNMTs (1, 3a, and 3b) were detected in Hut 78 lymphoid cells, only the expression of DNMT1 was significantly increased 3 to 5 days postinfection. This increase was observed with either wild-type HIV-1 or an integrase (IN) mutant, which renders HIV replication defective, due to the inability of the provirus to integrate into the host genome. Unintegrated viral DNA is a common feature of many retroviral infections and is thought to play a role in pathogenesis. These results indicate another mechanism by which unintegrated viral DNA affects the host. In addition to the increase in overall genomic methylation, hypermethylation and reduced expression of the p16INK4A gene, one of the most commonly altered genes in human cancer, were seen in cells infected with both wild-type and IN-defective HIV-1. Thus, infection of lymphoid cells with integration-defective HIV-1 can increase the methylation of CpG islands in the promoters of genes such as the p16INK4A gene, silencing their expression. PMID:11559808

  15. Arctigenin induces cell cycle arrest by blocking the phosphorylation of Rb via the modulation of cell cycle regulatory proteins in human gastric cancer cells.

    PubMed

    Jeong, Jin Boo; Hong, Se Chul; Jeong, Hyung Jin; Koo, Jin Suk

    2011-10-01

    Gastric cancer is a leading cause of cancer-related deaths, worldwide being second only to lung cancer as a cause of death. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms of arctigenin for anti-tumor effect on gastric cancer have not been examined. This study examined the biological effects of arctigenin on the human gastric cancer cell line SNU-1 and AGS. Cell proliferation was determined by MTT assay. In MTT assay, the proliferation of SNU-1 and AGS cells was significantly inhibited by arctigenin in a time and dose dependent manner, as compared with SNU-1 and AGS cells cultured in the absence of arctigenin. Inhibition of cell proliferation by arctigenin was in part associated with apoptotic cell death, as shown by changes in the expression ratio of Bcl-2 to Bax by arctigenin. Also, arctigenin blocked cell cycle arrest from G(1) to S phase by regulating the expression of cell cycle regulatory proteins such as Rb, cyclin D1, cyclin E, CDK4, CDK2, p21Waf1/Cip1 and p15 INK4b. The antiproliferative effect of arctigenin on SNU-1 and AGS gastric cancer cells revealed in this study suggests that arctigenin has intriguing potential as a chemopreventive or chemotherapeutic agent.

  16. Endothelial Progenitor Cell Dysfunction in Myelodysplastic Syndromes: Possible Contribution of a Defective Vascular Niche to Myelodysplasia12

    PubMed Central

    Teofili, Luciana; Martini, Maurizio; Nuzzolo, Eugenia Rosa; Capodimonti, Sara; Iachininoto, Maria Grazia; Cocomazzi, Alessandra; Fabiani, Emiliano; Voso, Maria Teresa; Larocca, Luigi M.

    2015-01-01

    We set a model to replicate the vascular bone marrow niche by using endothelial colony forming cells (ECFCs), and we used it to explore the vascular niche function in patients with low-risk myelodysplastic syndromes (MDS). Overall, we investigated 56 patients and we observed higher levels of ECFCs in MDS than in healthy controls; moreover, MDS ECFCs were found variably hypermethylated for p15INK4b DAPK1, CDH1, or SOCS1. MDS ECFCs exhibited a marked adhesive capacity to normal mononuclear cells. When normal CD34 + cells were co-cultured with MDS ECFCs, they generated significant lower amounts of CD11b + and CD41 + cells than in co-culture with normal ECFCs. At gene expression profile, several genes involved in cell adhesion were upregulated in MDS ECFCs, while several members of the Wingless and int (Wnt) pathways were underexpressed. Furthermore, at miRNA expression profile, MDS ECFCs hypo-expressed various miRNAs involved in Wnt pathway regulation. The addition of Wnt3A reduced the expression of intercellular cell adhesion molecule-1 on MDS ECFCs and restored the defective expression of markers of differentiation. Overall, our data demonstrate that in low-risk MDS, ECFCs exhibit various primary abnormalities, including putative MDS signatures, and suggest the possible contribution of the vascular niche dysfunction to myelodysplasia. PMID:26025663

  17. Activin A induces growth arrest through a SMAD- dependent pathway in hepatic progenitor cells

    PubMed Central

    2014-01-01

    Background Activin A, an important member of transforming growth factor-β superfamily, is reported to inhibit proliferation of mature hepatocyte. However, the effect of activin A on growth of hepatic progenitor cells is not fully understood. To that end, we attempted to evaluate the potential role of activin A in the regulation of hepatic progenitor cell proliferation. Results Using the 2-acetaminofluorene/partial hepatectomy model, activin A expression decreased immediately after partial hepatectomy and then increased from the 9th to 15th day post surgery, which is associated with the attenuation of oval cell proliferation. Activin A inhibited oval cell line LE6 growth via activating the SMAD signaling pathway, which manifested as the phosphorylation of SMAD2/3, the inhibition of Rb phosphorylation, the suppression of cyclinD1 and cyclinE, and the promotion of p21WAF1/Cip1 and p15INK4B expression. Treatment with activin A antagonist follistatin or blocking SMAD signaling could diminish the anti-proliferative effect of activin A. By contrast, inhibition of the MAPK pathway did not contribute to this effect. Antagonizing activin A activity by follistatin administration enhanced oval cell proliferation in the 2-acetylaminofluorene/partial hepatectomy model. Conclusion Activin A, acting through the SMAD pathway, negatively regulates the proliferation of hepatic progenitor cells. PMID:24628936

  18. Constitutive Smad linker phosphorylation in melanoma: a mechanism of resistance to transforming growth factor-β-mediated growth inhibition.

    PubMed

    Cohen-Solal, Karine A; Merrigan, Kim T; Chan, Joseph L-K; Goydos, James S; Chen, Wenjin; Foran, David J; Liu, Fang; Lasfar, Ahmed; Reiss, Michael

    2011-06-01

    Melanoma cells are resistant to transforming growth factor-β (TGFβ)-induced cell-cycle arrest. In this study, we investigated a mechanism of resistance involving a regulatory domain, called linker region, in Smad2 and Smad3, main downstream effectors of TGFβ. Melanoma cells in culture and tumor samples exhibited constitutive Smad2 and Smad3 linker phosphorylation. Treatment of melanoma cells with the MEK1/2 inhibitor, U0126, or the two pan-CDK and GSK3 inhibitors, Flavopiridol and R547, resulted in decreased linker phosphorylation of Smad2 and Smad3. Overexpression of the linker phosphorylation-resistant Smad3 EPSM mutant in melanoma cells resulted in an increase in expression of p15(INK4B) and p21(WAF1) , as compared with cells transfected with wild-type (WT) Smad3. In addition, the cell numbers of EPSM Smad3-expressing melanoma cells were significantly reduced compared with WT Smad3-expressing cells. These results suggest that the linker phosphorylation of Smad3 contributes to the resistance of melanoma cells to TGFβ-mediated growth inhibition.

  19. TGF-{beta}{sub 1}-induced cardiac myofibroblasts are nonproliferating functional cells carrying DNA damages

    SciTech Connect

    Petrov, Victor V. Pelt, Jos F. van; Vermeesch, Joris R.; Van Duppen, Viktor J.; Vekemans, Katrien; Fagard, Robert H.; Lijnen, Paul J.

    2008-04-15

    TGF-{beta}{sub 1} induces differentiation and total inhibition of cardiac MyoFb cell division and DNA synthesis. These effects of TGF-{beta}{sub 1} are irreversible. Inhibition of MyoFb proliferation is accompanied with the expression of Smad1, Mad1, p15Ink4B and total inhibition of telomerase activity. Surprisingly, TGF-{beta}{sub 1}-activated MyoFbs are growth-arrested not only at G1-phase but also at S-phase of the cell cycle. Staining with TUNEL indicates that these cells carry DNA damages. However, the absolute majority of MyoFbs are non-apoptotic cells as established with two apoptosis-specific methods, flow cytometry and caspase-dependent cleavage of cytokeratin 18. Expression in MyoFbs of proliferative cell nuclear antigen even in the absence of serum confirms that these MyoFbs perform repair of DNA damages. These results suggest that TGF-{beta}{sub 1}-activated MyoFbs can be growth-arrested by two checkpoints, the G1/S checkpoint, which prevents cells from entering S-phase and the intra-S checkpoint, which is activated by encountering DNA damage during the S phase or by unrepaired damage that escapes the G1/S checkpoint. Despite carrying of the DNA damages TGF-{beta}{sub 1}-activated MyoFbs are highly functional cells producing lysyl oxidase and contracting the collagen matrix.

  20. Maintenance treatment with azacytidine for patients with high-risk myelodysplastic syndromes (MDS) or acute myeloid leukaemia following MDS in complete remission after induction chemotherapy.

    PubMed

    Grövdal, Michael; Karimi, Mohsen; Khan, Rasheed; Aggerholm, Anni; Antunovic, Petar; Astermark, Jan; Bernell, Per; Engström, Lena-Maria; Kjeldsen, Lars; Linder, Olle; Nilsson, Lars; Olsson, Anna; Holm, Mette S; Tangen, Jon M; Wallvik, Jonas; Oberg, Gunnar; Hokland, Peter; Jacobsen, Sten E; Porwit, Anna; Hellström-Lindberg, Eva

    2010-08-01

    This prospective Phase II study is the first to assess the feasibility and efficacy of maintenance 5-azacytidine for older patients with high-risk myelodysplastic syndrome (MDS), chronic myelomonocytic leukaemia and MDS-acute myeloid leukaemia syndromes in complete remission (CR) after induction chemotherapy. Sixty patients were enrolled and treated by standard induction chemotherapy. Patients that reached CR started maintenance therapy with subcutaneous azacytidine, 5/28 d until relapse. Promoter-methylation status of CDKN2B (P15 ink4b), CDH1 and HIC1 was examined pre-induction, in CR and 6, 12 and 24 months post CR. Twenty-four (40%) patients achieved CR after induction chemotherapy and 23 started maintenance treatment with azacytidine. Median CR duration was 13.5 months, >24 months in 17% of the patients, and 18-30.5 months in the four patients with trisomy 8. CR duration was not associated with CDKN2B methylation status or karyotype. Median overall survival was 20 months. Hypermethylation of CDH1 was significantly associated with low CR rate, early relapse, and short overall survival (P = 0.003). 5-azacytidine treatment, at a dose of 60 mg/m(2) was well tolerated. Grade III-IV thrombocytopenia and neutropenia occurred after 9.5 and 30% of the cycles, respectively, while haemoglobin levels increased during treatment. 5-azacytidine treatment is safe, feasible and may be of benefit in a subset of patients.

  1. The long non-coding RNA ANRIL promotes proliferation and cell cycle progression and inhibits apoptosis and senescence in epithelial ovarian cancer

    PubMed Central

    Qiu, Jun-jun; Wang, Yan; Liu, Ying-lei; Zhang, Ying; Ding, Jing-xin; Hua, Ke-qin

    2016-01-01

    Antisense non-coding RNA in the INK4 locus (ANRIL) has been implicated in a variety of cancers. In the present study, we evaluated ANRIL expression in epithelial ovarian cancer (EOC) and defined its clinical implications and biological functions. ANRIL was overexpressed in EOC tissues relative to normal controls. Overexpression correlated with advanced International Federation of Gynecologists and Obstetricians stage and high histological grade. Multivariate analysis indicated that ANRIL is an independent prognostic factor for overall survival in EOC. Gain- and loss-of-function experiments demonstrated that ANRIL promotes EOC cell proliferation both in vitro and in vivo. The proliferative effect was linked to the promotion of cell cycle progression and inhibition of apoptosis and senescence. Down-regulation of P15INK4B and up-regulation of Bcl-2 by ANRIL may partially explain ANRIL-induced EOC cell proliferation. This study is the first to establish that ANRIL promotes EOC progression and is a potential prognostic biomarker. PMID:27095571

  2. Part II-mechanism of adaptation: A549 cells adapt to high concentration of nitric oxide through bypass of cell cycle checkpoints.

    PubMed

    Aqil, Madeeha; Deliu, Zane; Elseth, Kim M; Shen, Grace; Xue, Jiaping; Radosevich, James A

    2014-03-01

    Previous work has shown enhanced survival capacity in high nitric oxide (HNO)-adapted tumor cells. In Part I of this series of manuscripts, we have shown that A549-HNO cells demonstrate an improved growth profile under UV and X-ray radiation treatment. These cells exhibit increased expression of proteins involved in DNA damage recognition and repair pathway, both the non-homologous end joining pathway and homologous recombination. These include Ku80, DNA-PK, XLF ligase and MRN complex proteins. Further, the A549-HNO cells show high levels of ATM, ATR, Chk1 and Chk2, and phospho-p53. Activation of these molecules may lead to cell cycle arrest and apoptosis due to DNA damage. This is observed in parent A549 cells in response to NO donor treatment; however, the A549-HNO cells proliferate and inhibit apoptosis. Cell cycle analysis showed slowed progression through S phase which will allow time for DNA repair. Thus, to better understand the increased growth rate in A549-HNO when compared to the parent cell line A549, we studied molecular mechanisms involved in cell cycle regulation in A549-HNO cells. During the initial time period of NO donor treatment, we observe high levels of cyclin/Cdk complexes involved in regulating various stages of the cell cycle. This would lead to bypass of G1-S and G2-M checkpoints. The HNO cells also show much higher expression of Cdc25A. Cdc25A activates Cdk molecules involved in different phases of the cell cycle. In addition, there is enhanced phosphorylation of the Rb protein in HNO cells. This leads to inactivation of Rb/E2F checkpoint regulating G1-S transition. This may lead to faster progression in S phase. Thus, all of these perturbations in HNO cells lead to accelerated cell cycle progression and a higher growth rate. We also assessed expression of cell cycle inhibitors in HNO cells. Interestingly, the HNO cells show a significant decline in p21CIP1 at initial time points, but with prolonged exposure, the levels were much higher

  3. Synergistic effects of acyclic retinoid and OSI-461 on growth inhibition and gene expression in human hepatoma cells.

    PubMed

    Shimizu, Masahito; Suzui, Masumi; Deguchi, Atsuko; Lim, Jin T E; Xiao, Danhua; Hayes, Julia H; Papadopoulos, Kyriakos P; Weinstein, I Bernard

    2004-10-01

    Hepatoma is one of the most frequently occurring cancers worldwide. However, effective chemotherapeutic agents for this disease have not been developed. Acyclic retinoid, a novel synthetic retinoid, can reduce the incidence of postsurgical recurrence of hepatoma and improve the survival rate. OSI-461, a potent derivative of exisulind, can increase intracellular levels of cyclic GMP, which leads to activation of protein kinase G and induction of apoptosis in cancer cells. In the present study, we examined the combined effects of acyclic retinoid plus OSI-461 in the HepG2 human hepatoma cell line. We found that the combination of as little as 1.0 micromol/L acyclic retinoid and 0.01 micromol/L OSI-461 exerted synergistic inhibition of the growth of HepG2 cells. Combined treatment with low concentrations of these two agents also acted synergistically to induce apoptosis in HepG2 cells through induction of Bax and Apaf-1, reduction of Bcl-2 and Bcl-xL, and activation of caspase-3, -8, and -9. OSI-461 enhanced the G0-G1 arrest caused by acyclic retinoid, and the combination of these agents caused a synergistic decrease in the levels of expression of cyclin D1 protein and mRNA, inhibited cyclin D1 promoter activity, decreased the level of hyperphosphorylated forms of the Rb protein, induced increased cellular levels of the p21(CIP1) protein and mRNA, and stimulated p21(CIP1) promoter activity. Moreover, OSI-461 enhanced the ability of acyclic retinoid to induce increased cellular levels of retinoic acid receptor beta and to stimulate retinoic acid response element-chloramphenicol acetyltransferase activity. A hypothetical model involving concerted effects on p21(CIP1) and retinoic acid receptor beta expression is proposed to explain these synergistic effects. Our results suggest that the combination of acyclic retinoid plus OSI-461 might be an effective regimen for the chemoprevention and chemotherapy of human hepatoma and possibly other malignancies.

  4. The RNA binding proteins RBM38 and DND1 are repressed in AML and have a novel function in APL differentiation.

    PubMed

    Wampfler, Julian; Federzoni, Elena A; Torbett, Bruce E; Fey, Martin F; Tschan, Mario P

    2016-02-01

    The RNA binding proteins RBM binding motif protein 38 (RBM38) and DEAD END 1 (DND1) selectively stabilize mRNAs by attenuating RNAse activity or protecting them from micro(mi)RNA-mediated cleavage. Furthermore, both proteins can efficiently stabilize the mRNA of the cell cycle inhibitor p21(CIP1). Since acute myeloid leukemia (AML) differentiation requires cell cycle arrest and RBM38 as well as DND1 have antiproliferative functions, we hypothesized that decreased RBM38 and DND1 expression may contribute to the differentiation block seen in this disease. We first quantified RBM38 and DND1 mRNA expression in clinical AML patient samples and CD34(+) progenitor cells and mature granulocytes from healthy donors. We found significantly lower RBM38 and DND1 mRNA levels in AML blasts and CD34(+) progenitor cells as compared to mature neutrophils from healthy donors. Furthermore, the lowest expression of both RBM38 and DND1 mRNA correlated with t(8;21). In addition, neutrophil differentiation of CD34(+) cells in vitro with G-CSF (granulocyte colony stimulating factor) resulted in a significant increase of RBM38 and DND1 mRNA levels. Similarly, neutrophil differentiation of NB4 acute promyelocytic leukemia (APL) cells was associated with a significant induction of RBM38 and DND1 expression. To address the function of RBM38 and DND1 in neutrophil differentiation, we generated two independent NB4RBM38 as well as DND1 knockdown cell lines. Inhibition of both RBM38 and DND1 mRNA significantly attenuated NB4 differentiation and resulted in decreased p21(CIP1) mRNA expression. Our results clearly indicate that expression of the RNA binding proteins RBM38 and DND1 is repressed in primary AML patients, that neutrophil differentiation is dependent on increased expression of both proteins, and that these proteins have a critical role in regulating p21(CIP1) expression during APL differentiation.

  5. Cardenolide glycosides from the seeds of Digitalis purpurea exhibit carcinoma-specific cytotoxicity toward renal adenocarcinoma and hepatocellular carcinoma cells.

    PubMed

    Fujino, Tomofumi; Kuroda, Minpei; Matsuo, Yukiko; Kubo, Satoshi; Tamura, Chikako; Sakamoto, Nami; Mimaki, Yoshihiro; Hayakawa, Makio

    2015-01-01

    Four cardenolide glycosides, glucodigifucoside (2), 3'-O-acetylglucoevatromonoside (9), digitoxigenin 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-3-O-acetyl-β-D-digitoxopyranoside (11), and purpureaglycoside A (12), isolated from the seeds of Digitalis purpurea, exhibited potent cytotoxicity against human renal adenocarcinoma cell line ACHN. These compounds exhibited significantly lower IC50 values against ACHN than that against normal human renal proximal tubule-derived cell line HK-2. In particular, 2 exhibited the most potent and carcinoma-specific cytotoxicity, with a sixfold lower IC50 value against ACHN than that against HK-2. Measurement of cyclin-dependent kinase inhibitor levels revealed that upregulation of p21/Cip1 expression was involved in the carcinoma-specific cytotoxicity of 2. Further, compound 2 also exhibited the carcinoma-specific cytotoxicity toward hepatocellular carcinoma cell line.

  6. Flavonoid-induced autophagy in hormone sensitive breast cancer cells.

    PubMed

    Brunelli, Elisa; Pinton, Giulia; Bellini, Paolo; Minassi, Alberto; Appendino, Giovanni; Moro, Laura

    2009-09-01

    The activity of 8-prenylapigenin (8-PA) and its 3'-methoxylated analogue isocannflavin B (IsoB) was investigated in estrogen-dependent T47-D and estrogen-independent MDA-MB-231 human breast cancer cell lines. 8-PA showed a biphasic effect on T47-D cell proliferation, while no significant effect was observed on MDA-MB-231 cells. Conversely, IsoB exhibited only an inhibitory effect on T47-D cell proliferation, accompanied by the appearance of an intense intracytoplasmic vacuolization of autophagic origin. Moreover, biochemical analysis showed that IsoB reduced Akt phosphorylation and p21(Cip1) expression in T47-D cells. These data show that the prenylflavone moiety is a versatile platform for the induction and modulation of bioactivity.

  7. New perspectives on iron: an introduction.

    PubMed

    Boldt, D H

    1999-10-01

    Iron is an essential nutritional element for all life forms. Iron plays critical roles in electron transport and cellular respiration, cell proliferation and differentiation, and regulation of gene expression. Two emerging new functions for iron are its necessary role in supporting transcription of certain key genes required for cell growth and function [eg, nitric oxide synthase, protein kinase C-beta, p21 (CIP1/WAF1)] and its complex role in hematopoietic cell differentiation. However, iron is also potentially deleterious. Reactive oxygen species generated by Fenton chemistry may contribute to major pathological processes such as cancer, atherosclerosis, and neurodegenerative diseases. Iron-generated reactive oxygen species may also function in normal intracellular signaling. Therefore, roles of iron are both essential and extraordinarily diverse. This symposium explores this diversity by covering topics of iron absorption and transport, the regulation of gene expression by iron responsive proteins, the cellular biology of heme, hereditary hemochromatosis, and clinical use of serum transferrin receptor measurements.

  8. Potent anti-proliferative actions of a non-diuretic glucosamine derivative of ethacrynic acid.

    PubMed

    Punganuru, Surendra R; Mostofa, A G M; Madala, Hanumantha Rao; Basak, Debasish; Srivenugopal, Kalkunte S

    2016-06-15

    Ethacrynic acid (EA), a known inhibitor of the neoplastic marker glutathione S-transferase P1 and other GSTs, exerts a weak antiproliferative activity against human cancer cells. The clinical use of EA (Edecrin) as an anticancer drug is limited by its potent loop diuretic activity. In this study, we developed a non-diuretic 2-amino-2-deoxy-d-glucose conjugated EA (EAG) to target tumors cells via the highly expressed glucose transporter 1 (GLUT1). Cell survival assays revealed that EAG had little effect on normal cells, but was cytotoxic 3 to 4.5-fold greater than EA. Mechanistically, the EAG induced selective cell death in cancer cells by inhibiting GSTP1 and generating abundant reactive oxygen species. Furthermore, EAG induced p21(cip1) expression and a G2/M cell cycle block irrespective of the p53 gene status in tumor cells. These data encourage the development of new EA analogs.

  9. A Pleiotropic RNA-Binding Protein Controls Distinct Cell Cycle Checkpoints to Drive Resistance of p53-Defective Tumors to Chemotherapy.

    PubMed

    Cannell, Ian G; Merrick, Karl A; Morandell, Sandra; Zhu, Chang-Qi; Braun, Christian J; Grant, Robert A; Cameron, Eleanor R; Tsao, Ming-Sound; Hemann, Michael T; Yaffe, Michael B

    2015-11-09

    In normal cells, p53 is activated by DNA damage checkpoint kinases to simultaneously control the G1/S and G2/M cell cycle checkpoints through transcriptional induction of p21(cip1) and Gadd45α. In p53-mutant tumors, cell cycle checkpoints are rewired, leading to dependency on the p38/MK2 pathway to survive DNA-damaging chemotherapy. Here we show that the RNA binding protein hnRNPA0 is the "successor" to p53 for checkpoint control. Like p53, hnRNPA0 is activated by a checkpoint kinase (MK2) and simultaneously controls both cell cycle checkpoints through distinct target mRNAs, but unlike p53, this is through the post-transcriptional stabilization of p27(Kip1) and Gadd45α mRNAs. This pathway drives cisplatin resistance in lung cancer, demonstrating the importance of post-transcriptional RNA control to chemotherapy response.

  10. PKCη promotes senescence induced by oxidative stress and chemotherapy

    PubMed Central

    Zurgil, U; Ben-Ari, A; Atias, K; Isakov, N; Apte, R; Livneh, E

    2014-01-01

    Senescence is characterized by permanent cell-cycle arrest despite continued viability and metabolic activity, in conjunction with the secretion of a complex mixture of extracellular proteins and soluble factors known as the senescence-associated secretory phenotype (SASP). Cellular senescence has been shown to prevent the proliferation of potentially tumorigenic cells, and is thus generally considered a tumor suppressive process. However, some SASP components may act as pro-tumorigenic mediators on premalignant cells in the microenvironment. A limited number of studies indicated that protein kinase C (PKC) has a role in senescence, with different isoforms having opposing effects. It is therefore important to elucidate the functional role of specific PKCs in senescence. Here we show that PKCη, an epithelial specific and anti-apoptotic kinase, promotes senescence induced by oxidative stress and DNA damage. We further demonstrate that PKCη promotes senescence through its ability to upregulate the expression of the cell cycle inhibitors p21Cip1 and p27Kip1 and enhance transcription and secretion of interleukin-6 (IL-6). Moreover, we demonstrate that PKCη creates a positive loop for reinforcing senescence by increasing the transcription of both IL-6 and IL-6 receptor, whereas the expression of IL-8 is specifically suppressed by PKCη. Thus, the presence/absence of PKCη modulates major components of SASP. Furthermore, we show that the human polymorphic variant of PKCη, 374I, that exhibits higher kinase activity in comparison to WT-374V, is also more effective in IL-6 secretion, p21Cip1 expression and the promotion of senescence, further supporting a role for PKCη in senescence. As there is now considerable interest in senescence activation/elimination to control tumor progression, it is first crucial to reveal the molecular regulators of senescence. This will improve our ability to develop new strategies to harness senescence as a potential cancer therapy in the

  11. MGMT Inhibition Restores ERα Functional Sensitivity to Antiestrogen Therapy

    PubMed Central

    Bobustuc, George C; Smith, Joshua S; Maddipatla, Sreeram; Jeudy, Sheila; Limaye, Arati; Isley, Beth; Caparas, Maria-Lourdes M; Constantino, Susan M; Shah, Nikita; Baker, Cheryl H; Srivenugopal, Kalkunte S; Baidas, Said; Konduri, Santhi D

    2012-01-01

    Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O6 methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O6-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21cip1/waf1. We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21cip1/waf1 and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance. PMID:22549111

  12. Complex cell cycle abnormalities caused by human T-lymphotropic virus type 1 Tax.

    PubMed

    Yang, Liangpeng; Kotomura, Naoe; Ho, Yik-Khuan; Zhi, Huijun; Bixler, Sandra; Schell, Michael J; Giam, Chou-Zen

    2011-03-01

    Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL), a malignancy of CD4(+) T cells whose etiology is thought to be associated with the viral trans-activator Tax. We have shown recently that Tax can drastically upregulate the expression of p27(Kip1) and p21(CIP1/WAF1) through protein stabilization and mRNA trans-activation and stabilization, respectively. The Tax-induced surge in p21(CIP1/WAF1) and p27(Kip1) begins in S phase and results in cellular senescence. Importantly, HeLa and SupT1 T cells infected by HTLV-1 also arrest in senescence, thus challenging the notion that HTLV-1 infection causes cell proliferation. Here we use time-lapse microscopy to investigate the effect of Tax on cell cycle progression in two reporter cell lines, HeLa/18x21-EGFP and HeLa-FUCCI, that express enhanced green fluorescent protein (EGFP) under the control of 18 copies of the Tax-responsive 21-bp repeat element and fluorescent ubiquitin cell cycle indicators, respectively. Tax-expressing HeLa cells exhibit elongated or stalled cell cycle phases. Many of them bypass mitosis and become single senescent cells as evidenced by the expression of senescence-associated β-galactosidase. Such cells have twice the normal equivalent of cellular contents and hence are enlarged, with exaggerated nuclei. Interestingly, nocodazole treatment revealed a small variant population of HeLa/18x21-EGFP cells that could progress into mitosis normally with high levels of Tax expression, suggesting that genetic or epigenetic changes that prevent Tax-induced senescence can occur spontaneously at a detectable frequency.

  13. Inhibition of Cell Proliferation and Growth of Pancreatic Cancer by Silencing of Carbohydrate Sulfotransferase 15 In Vitro and in a Xenograft Model

    PubMed Central

    Shibazaki, Yuichiro; Yoneyama, Hiroyuki; Fujii, Masato; Hashiguchi, Taishi; Ito, Zensho; Kajihara, Mikio; Misawa, Takeyuki; Homma, Sadamu; Ohkusa, Toshifumi

    2015-01-01

    Chondroitin sulfate E (CS-E), a highly sulfated glycosaminoglycan, is known to promote tumor invasion and metastasis. Because the presence of CS-E is detected in both tumor and stromal cells in pancreatic ductal adenocarcinoma (PDAC), multistage involvement of CS-E in the development of PDAC has been considered. However, its involvement in the early stage of PDAC progression is still not fully understood. In this study, to clarify the direct role of CS-E in tumor, but not stromal, cells of PDAC, we focused on carbohydrate sulfotransferase 15 (CHST15), a specific enzyme that biosynthesizes CS-E, and investigated the effects of the CHST15 siRNA on tumor cell proliferation in vitro and growth in vivo. CHST15 mRNA is highly expressed in the human pancreatic cancer cell lines PANC-1, MIA PaCa-2, Capan-1 and Capan-2. CHST15 siRNA significantly inhibited the expression of CHST15 mRNA in these four cells in vitro. Silencing of the CHST15 gene in the cells was associated with significant reduction of proliferation and up-regulation of the cell cycle inhibitor-related gene p21CIP1/WAF1. In a subcutaneous xenograft tumor model of PANC-1 in nude mice, a single intratumoral injection of CHST15 siRNA almost completely suppressed tumor growth. Reduced CHST15 protein signals associated with tumor necrosis were observed with the treatment with CHST15 siRNA. These results provide evidence of the direct action of CHST15 on the proliferation of pancreatic tumor cells partly through the p21CIP1/WAF1 pathway. Thus, CHST15-CS-E axis-mediated tumor cell proliferation could be a novel therapeutic target in the early stage of PDAC progression. PMID:26642349

  14. Ablation of human telomerase reverse transcriptase (hTERT) induces cellular senescence in gastric cancer through a galectin-3 dependent mechanism

    PubMed Central

    La, Sun-Hyuk; Kim, Seok-Jun; Kang, Hyeok-Gu; Lee, Han-Woong; Chun, Kyung-Hee

    2016-01-01

    The human Telomerase Reverse Transcriptase (hTERT) gene encodes a rate-limiting catalytic subunit of telomerase that maintains genomic integrity. Suppression of hTERT expression could induce cellular senescence and is considered a potent approach for gastric cancer therapy. However, control of hTERT expression and function remains poorly understood in gastric cancer. In this study, we demonstrated that high expression levels of hTERT in malignant tissues are correlated with poor survival probability in gastric cancer patients. Knockdown of hTERT expression retarded cell proliferation and cellular senescence, which was confirmed by increased protein expression levels of p21cip1 and p27kip1, and decreased phosphorylation of Rb. In contrast, overexpression of hTERT increased cell proliferation and decreased cellular senescence. Remarkably, the down-regulation of hTERT expression was detected in lgals3−/− mouse embryo fibroblasts (MEFs). Knockdown of galectin-3 decreased the expression of hTERT in gastric cancer cells. Galectin-3 ablation-induced cellular senescence was rescued by concomitant overexpression of hTERT. hTERT ablation-induced cellular senescence and p21cip1 and p27kip1 expression was rescued by concomitant overexpression of galectin-3. The size of tumor burdens was increased in hTERT-overexpressed gastric cancer cells xenografted mice, whereas it was repressed by concomitant depletion of galectin-3. Additionally, we determined that the N-terminal domain of galectin-3 directly interacted with hTERT. The telomeric activity of hTERT was also decreased by galectin-3 ablation. Taken together, ablation of hTERT induces cellular senescence and inhibits the growth of gastric cancer cells, suggesting that it could be a potent target in gastric cancer therapy. We also propose that galectin-3 is an important regulator of hTERT expression and telomeric activity in gastric tumorigenesis. PMID:27494887

  15. Distinct Glucocorticoid Receptor Transcriptional Regulatory Surfaces Mediate the Cytotoxic and Cytostatic Effects of Glucocorticoids

    PubMed Central

    Rogatsky, Inez; Hittelman, Adam B.; Pearce, David; Garabedian, Michael J.

    1999-01-01

    Glucocorticoids act through the glucocorticoid receptor (GR), which can function as a transcriptional activator or repressor, to elicit cytostatic and cytotoxic effects in a variety of cells. The molecular mechanisms regulating these events and the target genes affected by the activated receptor remain largely undefined. Using cultured human osteosarcoma cells as a model for the GR antiproliferative effect, we demonstrate that in U20S cells, GR activation leads to irreversible growth inhibition, apoptosis, and repression of Bcl2. This cytotoxic effect is mediated by GR’s transcriptional repression function, since transactivation-deficient mutants and ligands still bring about apoptosis and Bcl2 down-regulation. In contrast, the antiproliferative effect of GR in SAOS2 cells is reversible, does not result in apoptosis or repression of Bcl2, and is a function of the receptor’s ability to stimulate transcription. Thus, the cytotoxic versus cytostatic outcome of glucocorticoid treatment is cell context dependent. Interestingly, the cytostatic effect of glucocorticoids in SAOS2 cells involves multiple GR activation surfaces. GR mutants and ligands that disrupt individual transcriptional activation functions (activation function 1 [AF-1] and AF-2) or receptor dimerization fail to fully inhibit cellular proliferation and, remarkably, discriminate between the targets of GR’s cytostatic action, the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. Induction of p21Cip1 is agonist dependent and requires AF-2 but not AF-1 or GR dimerization. In contrast, induction of p27Kip1 is agonist independent, does not require AF-2 or AF-1, but depends on GR dimerization. Our findings indicate that multiple GR transcriptional regulatory mechanisms that employ distinct receptor surfaces are used to evoke either the cytostatic or cytotoxic response to glucocorticoids. PMID:10373553

  16. Low-dose etoposide-treatment induces endoreplication and cell death accompanied by cytoskeletal alterations in A549 cells: Does the response involve senescence? The possible role of vimentin

    PubMed Central

    2013-01-01

    Background Senescence in the population of cells is often described as a program of restricted proliferative capacity, which is manifested by broad morphological and biochemical changes including a metabolic shift towards an autophagic-like response and a genotoxic-stress related induction of polyploidy. Concomitantly, the cell cycle progression of a senescent cell is believed to be irreversibly arrested. Recent reports suggest that this phenomenon may have an influence on the therapeutic outcome of anticancer treatment. The aim of this study was to verify the possible involvement of this program in the response to the treatment of the A549 cell population with low doses of etoposide, as well as to describe accompanying cytoskeletal alterations. Methods After treatment with etoposide, selected biochemical and morphological parameters were examined, including: the activity of senescence-associated ß-galactosidase, SAHF formation, cell cycle progression, the induction of p21Cip1/Waf1/Sdi1 and cyclin D1, DNA strand breaks, the disruption of cell membrane asymmetry/integrity and ultrastructural alterations. Vimentin and G-actin cytoskeleton was evaluated both cytometrically and microscopically. Results and conclusions Etoposide induced a senescence-like phenotype in the population of A549 cells. Morphological alterations were nevertheless not directly coupled with other senescence markers including a stable cell cycle arrest, SAHF formation or p21Cip1/Waf1/Sdi1 induction. Instead, a polyploid, TUNEL-positive fraction of cells visibly grew in number. Also upregulation of cyclin D1 was observed. Here we present preliminary evidence, based on microscopic analyses, that suggest a possible role of vimentin in nuclear alterations accompanying polyploidization-depolyploidization events following genotoxic insults. PMID:23383739

  17. Metformin inhibits growth and enhances radiation response of non-small cell lung cancer (NSCLC) through ATM and AMPK

    PubMed Central

    Storozhuk, Y; Hopmans, S N; Sanli, T; Barron, C; Tsiani, E; Cutz, J-C; Pond, G; Wright, J; Singh, G; Tsakiridis, T

    2013-01-01

    Background: We examined the potential of metformin (MET) to enhance non-small cell lung cancer (NSCLC) responses to ionising radiation (IR). Methods: Human NSCLC cells, mouse embryonic fibroblasts from wild-type and AMP-activated kinase (AMPK) α1/2-subunit−/− embryos (AMPKα1/2−/−-MEFs) and NSCLC tumours grafted into Balb/c-nude mice were treated with IR and MET and subjected to proliferation, clonogenic, immunoblotting, cell cycle and apoptosis assays and immunohistochemistry (IHC). Results: Metformin (2.5 μℳ–5 mℳ) inhibited proliferation and radio-sensitised NSCLC cells. Metformin (i) activated the ataxia telengiectasia-mutated (ATM)–AMPK–p53/p21cip1 and inhibited the Akt–mammalian target of rapamycin (mTOR)–eIF4E-binding protein 1 (4EBP1) pathways, (ii) induced G1 cycle arrest and (iii) enhanced apoptosis. ATM inhibition blocked MET and IR activation of AMPK. Non-small cell lung cancer cells with inhibited AMPK and AMPKα1/2−/−-MEFs were resistant to the antiproliferative effects of MET and IR. Metformin or IR inhibited xenograft growth and combined treatment enhanced it further than each treatment alone. Ionising radiation and MET induced (i) sustained activation of ATM–AMPK–p53/p21cip1 and inhibition of Akt–mTOR–4EBP1 pathways in tumours, (ii) reduced expression of angiogenesis and (iii) enhanced expression of apoptosis markers. Conclusion: Clinically achievable MET doses inhibit NSCLC cell and tumour growth and sensitise them to IR. Metformin and IR mediate their action through an ATM–AMPK-dependent pathway. Our results suggest that MET can be a clinically useful adjunct to radiotherapy in NSCLC. PMID:23632475

  18. In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency

    PubMed Central

    Tourlakis, Marina E.; Zhang, Siyi; Ball, Heather L.; Gandhi, Rikesh; Liu, Hongrui; Zhong, Jian; Yuan, Julie S.; Guidos, Cynthia J.; Durie, Peter R.; Rommens, Johanna M.

    2015-01-01

    Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15Ink4b and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to

  19. In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency.

    PubMed

    Tourlakis, Marina E; Zhang, Siyi; Ball, Heather L; Gandhi, Rikesh; Liu, Hongrui; Zhong, Jian; Yuan, Julie S; Guidos, Cynthia J; Durie, Peter R; Rommens, Johanna M

    2015-06-01

    Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15(Ink4b) and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to

  20. 2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

    SciTech Connect

    Jeong, Jin Boo; Jeong, Hyung Jin

    2010-10-01

    Research highlights: {yields} 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. {yields} 2M4VP inhibited hyper-phosphorylation of Rb protein. {yields} 2M4VP induced cell cycle arrest from G1 to S. {yields} 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. {yields} 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

  1. The proinflammatory LTB4/BLT1 signal axis confers resistance to TGF-β1-induced growth inhibition by targeting Smad3 linker region

    PubMed Central

    Park, Seong Ji; Jo, Eun Ji; Lee, Young K.; Lim, Seunghwan; Kim, Jae-Hong; Letterio, John J.; Liu, Fang; Kim, Seong-Jin; Kim, Byung-Chul

    2015-01-01

    Leukotriene B4 (LTB4) is a potent pro-inflammatory eicosanoid that is derived from arachidonic acid, and its signaling is known to have a tumor-promoting role in several cancer types. In this study, we investigated whether enhanced LTB4 signaling confers resistance to the cytostatic transforming growth factor-β1 (TGF-β1) response. We found that LTB4 pretreatment or ectopic expression of BLT1, a high affinity LTB4 receptor, fully abrogated TGF-β1-induced cell cycle arrest and expression of p15INK4B and p27KIP1. Mechanism study revealed that LTB4-mediated suppression of TGF-β1-induced Smad3 activation and growth inhibition was due to enhanced phosphorylation of Smad3 linker region (pSmad3L) through activation of BLT1-NAD(P)H oxidase (NOX)-reactive oxygen species (ROS)-epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3-K)-extracellular signal-activated kinase1/2 (ERK1/2)-linked signaling cascade. Furthermore, the LTB4/BLT1 signaling pathway leading to pSmad3L was constitutively activated in breast cancer cells and was correlated with TGF-β1-resistant growth of the cells in vitro and in vivo. In human breast cancer tissues, the expression level of pSmad3L (Thr179) had a positive correlation with BLT1 expression. Collectively, our data demonstrate for the first time that the induction of pSmad3L through BLT1-NOX-ROS-EGFR-PI3K-ERK1/2 signaling pathway is a key mechanism by which LTB4 blocks the anti-proliferative responses of TGF-β1, providing a novel mechanistic insight into the connection between enhanced inflammatory signal and cancer cell growth. PMID:26497676

  2. TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions.

    PubMed

    Mori, Shigeo; Matsuzaki, Koichi; Yoshida, Katsunori; Furukawa, Fukiko; Tahashi, Yoshiya; Yamagata, Hideo; Sekimoto, Go; Seki, Toshihito; Matsui, Hirofumi; Nishizawa, Mikio; Fujisawa, Jun-ichi; Okazaki, Kazuichi

    2004-09-23

    Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-beta (TGF-beta) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-beta treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-beta or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-beta signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-beta allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-beta-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-beta signals. Moreover, a combined treatment with HGF and TGF-beta led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-beta-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-beta transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

  3. The proinflammatory LTB4/BLT1 signal axis confers resistance to TGF-β1-induced growth inhibition by targeting Smad3 linker region.

    PubMed

    Jeon, Woo-Kwang; Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji; Lee, Young K; Lim, Seunghwan; Kim, Jae-Hong; Letterio, John J; Liu, Fang; Kim, Seong-Jin; Kim, Byung-Chul

    2015-12-08

    Leukotriene B4 (LTB4) is a potent pro-inflammatory eicosanoid that is derived from arachidonic acid, and its signaling is known to have a tumor-promoting role in several cancer types. In this study, we investigated whether enhanced LTB4 signaling confers resistance to the cytostatic transforming growth factor-β1 (TGF-β1) response. We found that LTB4 pretreatment or ectopic expression of BLT1, a high affinity LTB4 receptor, fully abrogated TGF-β1-induced cell cycle arrest and expression of p15INK4B and p27KIP1. Mechanism study revealed that LTB4-mediated suppression of TGF-β1-induced Smad3 activation and growth inhibition was due to enhanced phosphorylation of Smad3 linker region (pSmad3L) through activation of BLT1-NAD(P)H oxidase (NOX)-reactive oxygen species (ROS)-epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3-K)-extracellular signal-activated kinase1/2 (ERK1/2)-linked signaling cascade. Furthermore, the LTB4/BLT1 signaling pathway leading to pSmad3L was constitutively activated in breast cancer cells and was correlated with TGF-β1-resistant growth of the cells in vitro and in vivo. In human breast cancer tissues, the expression level of pSmad3L (Thr179) had a positive correlation with BLT1 expression. Collectively, our data demonstrate for the first time that the induction of pSmad3L through BLT1-NOX-ROS-EGFR-PI3K-ERK1/2 signaling pathway is a key mechanism by which LTB4 blocks the anti-proliferative responses of TGF-β1, providing a novel mechanistic insight into the connection between enhanced inflammatory signal and cancer cell growth.

  4. Epstein-Barr virus nuclear antigen 3A promotes cellular proliferation by repression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1.

    PubMed

    Tursiella, Melissa L; Bowman, Emily R; Wanzeck, Keith C; Throm, Robert E; Liao, Jason; Zhu, Junjia; Sample, Clare E

    2014-10-01

    Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14(ARF) and p16(INK4a) expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14(ARF) and p16I(NK4a). By contrast, p16(INK4a) was not detectably expressed in Wp-R BL and the low-level expression of p14(ARF) was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21(WAF1/CIP1), a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21(WAF1/CIP1) expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the

  5. Polo-like kinase 3 and phosphoT273 caspase-8 are associated with improved local tumor control and survival in patients with anal carcinoma treated with concomitant chemoradiotherapy

    PubMed Central

    Helmke, Christina; Balermpas, Panagiotis; Fokas, Emmanouil; Wieland, Ulrike; Rave-Fränk, Margret; Kitz, Julia; Matthess, Yves; Raab, Monika; Strebhardt, Klaus; Rödel, Claus

    2016-01-01

    We have recently shown that caspase-8 is a new substrate of Polo-like kinase 3 (Plk3) that phosphorylates the protein on residue T273 thereby promoting its pro-apoptotic function. In the present study we aimed to investigate the clinical relevance of Plk3 expression and phosphorylation of caspase-8 at T273 in patients with anal squamous cell carcinoma (SSC) treated with 5-fluorouracil and mitomycin C-based chemoradiotherapy (CRT). Immunohistochemical detection of the markers was performed in pretreatment biopsy specimens of 95 patients and was correlated with clinical/histopathologic characteristics including HPV-16 virus load/p16INK4a expression and cumulative incidence of local and distant failure, cancer specific survival (CSS), and overall survival (OS). We observed significant positive correlations between Plk3 expression, pT273 caspase-8 signal, and levels of HPV-16 virus DNA load/p16INK4a detection. Patients with high scores of Plk3 and pT273 caspase-8 showed increased local control (p = 0.011; p = 0.001), increased CSS (p = 0.011; p = 0.013) and OS (p = 0.024; p = 0.001), while the levels of pT273 caspase-8 were significantly associated (p = 0.033) with distant metastases. In multivariate analyses Plk3 expression remained significant for local failure (p = 0.018), CSS (p = 0.016) and OS (p = 0.023). Moreover, a combined HPV16 DNA load and Plk3 or pT273 caspase-8 variable revealed a significant correlation to decreased local failure (p = 0.001; p = 0.009), increased CSS (p = 0.016; p = 0.023) and OS (p = 0.003; p = 0.003). In conclusion these data indicate that elevated levels of Plk3 and pT273 caspase-8 are correlated with favorable clinical outcome in patients with anal SCC treated with concomitant CRT. PMID:27462786

  6. Identification of Senescent Cells in the Bone Microenvironment

    PubMed Central

    Farr, Joshua N; Fraser, Daniel G; Wang, Haitao; Jaehn, Katharina; Ogrodnik, Mikolaj B; Weivoda, Megan M; Drake, Matthew T; Tchkonia, Tamara; LeBrasseur, Nathan K; Kirkland, James L; Bonewald, Lynda F; Pignolo, Robert J; Monroe, David G; Khosla, Sundeep

    2017-01-01

    Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16Ink4a, profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence-associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age-related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16Ink4a expression by real-time quantitative polymerase chain reaction (rt-qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence-associated distension of satellites (SADS), ie, large-scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction-induced foci (TIFs) than osteocytes from young mice. Corresponding with the age-associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age-associated upregulation in myeloid cells. These data show that with aging, a subset of cells

  7. Breast primary epithelial cells that escape p16-dependent stasis enter a telomere-driven crisis state.

    PubMed

    Feijoo, Purificación; Terradas, Mariona; Soler, David; Domínguez, Daniel; Tusell, Laura; Genescà, Anna

    2016-01-13

    Breast cancer is the most common malignant disease in women, but some basic questions remain in breast cancer biology. To answer these, several cell models were developed. Recently, the use of improved cell-culture conditions has enabled the development of a new primary cell model with certain luminal characteristics. This model is relevant because, after the introduction of a specific set of genetic elements, the transformed cells yielded tumors resembling human adenocarcinomas in mice. The use of improved cell-culture conditions supporting the growth of these breast primary epithelial cells was expected to delay or eliminate stress-induced senescence and lead to the propagation of normal cells. However, no studies have been carried out to investigate these points. Propagation of breast primary epithelial cells was performed in WIT medium on Primaria plates. Immunofluorescence, western blot and qRT-PCR were used to detect molecular markers, and to determine the integrity of DNA damage-response pathways. Promoter methylation of p16 (INK4a) was assessed by pyrosequencing. In order to obtain a dynamic picture of chromosome instability over time in culture, we applied FISH methodologies. To better link chromosome instability with excessive telomere attrition, we introduced the telomerase reverse transcriptase human gene using a lentiviral vector. We report here that breast primary epithelial cells propagated in vitro with WIT medium on Primaria plates express some luminal characteristics, but not a complete luminal lineage phenotype. They undergo a p16-dependent stress-induced senescence (stasis), and the cells that escape stasis finally enter a crisis state with rampant chromosome instability. Chromosome instability in these cells is driven by excessive telomere attrition, as distributions of chromosomes involved in aberrations correlate with the profiles of telomere signal-free ends. Importantly, ectopic expression of the human TERT gene rescued their chromosomal

  8. Branchiogenic carcinoma with high-risk-type human papillomavirus infection: A case report

    PubMed Central

    Maeda, Hiroyuki; Deng, Zeyi; Ikegami, Taro; Matayoshi, Sen; Agena, Shinya; Kiyuna, Asanori; Yamashita, Yukashi; Uehara, Takayuki; Ganaha, Akira; Suzuki, Mikio

    2016-01-01

    Branchiogenic carcinoma (BC) usually appears as a mass lesion with a predominant cystic component. Since lymph node metastasis from oropharyngeal carcinoma (OPC) has a cystic appearance, it is occasionally difficult to distinguish between BC and nodal metastases from clinically silent OPC. Factors associated with the malignant transformation process in BC remain obscure. The present study reports the case of a 56-year-old man with a right cystic cervical mass that was diagnosed as squamous cell carcinoma based on examination by fine-needle aspiration biopsy. The primary tumor could not be detected despite several imaging examinations, a pan-endoscopy of the head and neck, esophagus and stomach, biopsies of the head and neck regions, and bilateral tonsillectomies. The pathological findings of the surgical specimens from a radical neck dissection were consistent with the histological characteristics of BC, with evidence of transition from dysplasia through intraepithelial carcinoma to invasive carcinoma. Normal squamous epithelium and dysplastic and cancerous portions in the BC showed strong p16INK4a immunoreactivity. The expression of p16INK4a was also observed in all 9 nodal metastases in the neck dissection specimens. The cystic formation observed in the BC was not observed in the nodal metastases. As the presence of human papillomavirus-16 in the tumor was confirmed by polymerase chain reaction, quantitative polymerase chain reaction was employed for the measurement of human papillomavirus-16 viral load and integration. The results showed that the viral load of human papillomavirus-16 was 3.01×107/50 ng genomic DNA, and the E2/E6 ratio was 0.13, so the integration state was judged to be the mixed type. To the best of our knowledge, this is the first report of BC associated with high-risk-type human papillomavirus infection. The study indicates that a human papillomavirus-positive neck mass may not necessarily be OPC, but that it could be BC with a poor prognosis

  9. Germ line transmission of the Cdk4(R24C) mutation facilitates tumorigenesis and escape from cellular senescence.

    PubMed

    Rane, Sushil G; Cosenza, Stephen C; Mettus, Richard V; Reddy, E Premkumar

    2002-01-01

    Mutations in CDK4 and its key kinase inhibitor p16(INK4a) have been implicated in the genesis and progression of familial human melanoma. The importance of the CDK4 locus in human cancer first became evident following the identification of a germ line CDK4-Arg24Cys (R24C) mutation, which abolishes the ability of CDK4 to bind to p16(INK4a). To determine the role of the Cdk4(R24C) germ line mutation in the genesis of other cancer types, we introduced the R24C mutation in the Cdk4 locus of mice by using Cre-loxP-mediated "knock-in" technology. Cdk4(R24C/R24C) mouse embryo fibroblasts (MEFs) displayed increased Cdk4 kinase activity resulting in hyperphosphorylation of all three members of the Rb family, pRb, p107, and p130. MEFs derived from Cdk4(R24C/R24C) mice displayed decreased doubling times, escape from replicative senescence, and escape sensitivity to contact-induced growth arrest. These MEFs also exhibited a high degree of susceptibility to oncogene-induced transformation, suggesting that the Cdk4(R24C) mutation can serve as a primary event in the progression towards a fully transformed phenotype. In agreement with the in vitro data, homozygous Cdk4(R24C/R24C) mice developed tumors of various etiology within 8 to 10 months of their life span. The majority of these tumors were found in the pancreas, pituitary, brain, mammary tissue, and skin. In addition, Cdk4(R24C/R24C) mice showed extraordinary susceptibility to carcinogens and developed papillomas within the first 8 to 10 weeks following cutaneous application of the carcinogens 9,10-di-methyl-1,2-benz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). This report formally establishes that the activation of Cdk4 is sufficient to promote cancer in many tissues. The observation that a wide variety of tumors develop in mice harboring the Cdk4(R24C) mutation offers a genetic proof that Cdk4 activation may constitute a central event in the genesis of many types of cancers in addition to melanoma.

  10. Ionizing radiation and aging: rejuvenating an old idea

    PubMed Central

    Richardson, Richard B.

    2009-01-01

    This paper reviews the contemporary evidence that radiation can accelerate aging, degenerative health effects and mortality. Around the 1960s, the idea that ionizing radiation caused premature aging was dismissed as the radiation-induced health effects appeared to be virtually confined to neoplasms. More recently, radiation has become associated with a much wider spectrum of age-related diseases, including cardiovascular disease; although some diseases of old age, such as diabetes, are notably absent as a radiation risk. On the basis of recent research, is there a stronger case today to be made linking radiation and aging? Comparison is made between the now-known biological mechanisms of aging and those of radiation, including oxidative stress, chromosomal damage, apoptosis, stem cell exhaustion and inflammation. The association between radiation effects and the free-radical theory of aging as the causative hypothesis seems to be more compelling than that between radiation and the nutrient-sensing TOR pathway. Premature aging has been assessed by biomarkers in calorie restriction studies; yet, biomarkers such as telomere erosion and p16INK4a are ambiguous for radiation-induced aging. Some animal studies suggest low dose radiation may even demonstrate hormesis health benefits. Regardless, there is virtually no support for a life span extending hypothesis for A-bomb survivors and other exposed subjects. PMID:20157573

  11. Thymomegaly, Microsplenia, and Defective Homeostatic Proliferation of Peripheral Lymphocytes in p51-Ets1 Isoform-Specific Null Mice▿

    PubMed Central

    Higuchi, Tsukasa; Bartel, Frank O.; Masuya, Masahiro; Deguchi, Takao; Henderson, Kelly W.; Li, Runzhao; Muise-Helmericks, Robin C.; Kern, Michael J.; Watson, Dennis K.; Spyropoulos, Demetri D.

    2007-01-01

    Ets1 is a member of the Ets transcription factor family. Alternative splicing of exon VII results in two naturally occurring protein isoforms: full-length Ets1 (p51-Ets1) and Ets1ΔVII (p42-Ets1). These isoforms bear key distinctions regarding protein-protein interactions, DNA binding kinetics, and transcriptional target specificity. Disruption of both Ets1 isoforms in mice results in the loss of detectable NK and NKT cell activity and defects in B and T lymphocytes. We generated mice that express only the Ets1ΔVII isoform. Ets1ΔVII homozygous mice express no p51-Ets1 and elevated levels of the p42-Ets1 protein relative to the wild type and display increased perinatal lethality, thymomegaly, and peripheral lymphopenia. Proliferation was increased in both the thymus and the spleen, while apoptosis was decreased in the thymus and increased in the spleen of homozygotes. Significant elevations of CD8+ and CD8+CD4+ thymocytes were observed. Lymphoid cell (CD19+, CD4+, and CD8+) reductions were predominantly responsible for diminished spleen cellularity, with fewer memory cells and a failure of homeostatic proliferation to maintain peripheral lymphocytes. Collectively, the Ets1ΔVII mutants demonstrate lymphocyte maturation defects associated with misregulation of p16Ink4a, p27Kip1, and CD44. Thus, a balance in the differential regulation of Ets1 isoforms represents a potential mechanism in the control of lymphoid maturation and homeostasis. PMID:17339335

  12. Molecular and Genomic Alterations in Glioblastoma Multiforme.

    PubMed

    Crespo, Ines; Vital, Ana Louisa; Gonzalez-Tablas, María; Patino, María del Carmen; Otero, Alvaro; Lopes, María Celeste; de Oliveira, Catarina; Domingues, Patricia; Orfao, Alberto; Tabernero, Maria Dolores

    2015-07-01

    In recent years, important advances have been achieved in the understanding of the molecular biology of glioblastoma multiforme (GBM); thus, complex genetic alterations and genomic profiles, which recurrently involve multiple signaling pathways, have been defined, leading to the first molecular/genetic classification of the disease. In this regard, different genetic alterations and genetic pathways appear to distinguish primary (eg, EGFR amplification) versus secondary (eg, IDH1/2 or TP53 mutation) GBM. Such genetic alterations target distinct combinations of the growth factor receptor-ras signaling pathways, as well as the phosphatidylinositol 3-kinase/phosphatase and tensin homolog/AKT, retinoblastoma/cyclin-dependent kinase (CDK) N2A-p16(INK4A), and TP53/mouse double minute (MDM) 2/MDM4/CDKN2A-p14(ARF) pathways, in cells that present features associated with key stages of normal neurogenesis and (normal) central nervous system cell types. This translates into well-defined genomic profiles that have been recently classified by The Cancer Genome Atlas Consortium into four subtypes: classic, mesenchymal, proneural, and neural GBM. Herein, we review the most relevant genetic alterations of primary versus secondary GBM, the specific signaling pathways involved, and the overall genomic profile of this genetically heterogeneous group of malignant tumors.

  13. Deregulation of DNMT1, DNMT3B and miR-29s in Burkitt lymphoma suggests novel contribution for disease pathogenesis.

    PubMed

    Robaina, Marcela C; Mazzoccoli, Luciano; Arruda, Viviane Oliveira; Reis, Flaviana Ruade de Souza; Apa, Alexandre Gustavo; de Rezende, Lidia Maria Magalhães; Klumb, Claudete Esteves

    2015-04-01

    Methylation of CpG islands in promoter gene regions is frequently observed in lymphomas. DNA methylation is established by DNA methyltransferases (DNMTs). DNMT1 maintains methylation patterns, while DNMT3A and DNMT3B are critical for de novo DNA methylation. Little is known about the expression of DNMTs in lymphomas. DNMT3A and 3B genes can be regulated post-transcriptionally by miR-29 family. Here, we demonstrated for the first time the overexpression of DNMT1 and DNMT3B in Burkitt lymphoma (BL) tumor samples (69% and 86%, respectively). Specifically, the treatment of two BL cell lines with the DNMT inhibitor 5-aza-dC decreased DNMT1 and DNMT3B protein levels and inhibited cell growth. Additionally, miR-29a, miR-29b and miR-29c levels were significantly decreased in the BL tumor samples. Besides, the ectopic expression of miR-29a, miR-29b and miR-29c reduced the DNMT3B expression and miR-29a and miR-29b lead to increase of p16(INK4a) mRNA expression. Altogether, our data suggest that deregulation of DNMT1, DNMT3B and miR29 may be involved in BL pathogenesis.

  14. The interplay between p16 serine phosphorylation and arginine methylation determines its function in modulating cellular apoptosis and senescence

    PubMed Central

    Lu, Yang; Ma, Wenlong; Li, Zhongwei; Lu, Jun; Wang, Xiuli

    2017-01-01

    Cyclin-dependent kinase inhibitor p16INK4a (p16) primarily functions as a negative regulator of the retinoblastoma protein (Rb) -E2F pathway, thus plays critical role in cell cycle progression, cellular senescence and apoptosis. In this study, we showed that the methylation of Arg 138 and the phosphorylation of Ser 140 on p16 were critical for the control of cell proliferation and apoptosis. Compared to wild type p16, mutant p16R138K possessed improved function in preventing cell proliferation and inducing apoptosis, while the Ser 140 mutation (p16S140A) exhibited the opposite alteration. We also demonstrated that H2O2 was able to induce the phosphorylation of p16, which facilitated the interaction between CDK4 (Cyclin-dependent protein kinase) and p16, in 293T (human emborynic kidney) cells. Furthermore, the elevated arginine methylation in p16S140A mutant and increased serine phosphorylation in p16R138K mutant suggest that a antagonizing mechanism coordinating Arg 138 methylation and Ser 140 phosphorylation to regulates p16 function as well as cellular apoptosis and senescence. These findings will therefore contribute to therapeutic treatment for p16-related gene therapy by providing theoretical and experimental evidence. PMID:28120917

  15. p16 loss rescues functional decline of Brca1-deficient mammary stem cells.

    PubMed

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

    2017-02-22

    Recent evidence indicates that the accumulation of endogenous DNA damage can induce senescence and limit the function of adult stem cells. It remains elusive whether deficiency in DNA damage repair is associated with the functional alteration of mammary stem cells. In this article, we reported that senescence was induced in mammary epithelial cells during aging along with increased expression of p16Ink4a (p16), an inhibitor of CDK4 and CKD6. Loss of p16 abrogated the age-induced senescence in mammary epithelial cells and significantly increased mammary stem cell function. We showed that loss of Brca1, a tumor suppressor that functions in DNA damage repair, in the mammary epithelium induced senescence with induction of p16 and a decline of stem cell function, which was rescued by p16 loss. These data not only answer the question as to whether deficiency in DNA damage repair is associated with the functional decline of mammary stem cells, but also identify the role of p16 in suppressing Brca1-deficient mammary stem cell function.

  16. Attenuation of p38α MAPK stress response signaling delays the in vivo aging of skeletal muscle myofibers and progenitor cells

    PubMed Central

    Papaconstantinou, John; Wang, Chen Z.; Zhang, Min; Yang, San; Deford, James; Bulavin, Dmitry V.; Ansari, Naseem H.

    2015-01-01

    Functional competence and self-renewal of mammalian skeletal muscle myofibers and progenitor cells declines with age. Progression of the muscle aging phenotype involves the decline of juvenile protective factors i.e., proteins whose beneficial functions translate directly to the quality of life, and self-renewal of progenitor cells. These characteristics occur simultaneously with the age-associated increase of p38α stress response signaling. This suggests that the maintenance of low levels of p38α activity of juvenile tissues may delay or attenuate aging. We used the dominant negative haploinsufficient p38α mouse (DN-p38αAF/+) to demonstrate that in vivo attenuation of p38α activity in the gastrocnemius of the aged mutant delays age-associated processes that include: a) the decline of the juvenile protective factors, BubR1, aldehyde dehydrogenase 1A (ALDH1A1), and aldehyde dehydrogenase 2 (ALDH2); b) attenuated expression of p16Ink4a and p19Arf tumor suppressor genes of the Cdkn2a locus; c) decreased levels of hydroxynonenal protein adducts, expression of COX2 and iNOS; d) decline of the senescent progenitor cell pool level and d) the loss of gastrocnemius muscle mass. We propose that elevated P-p38α activity promotes skeletal muscle aging and that the homeostasis of p38α impacts the maintenance of a beneficial healthspan. PMID:26423835

  17. Diffuse large B-cell lymphomas with CDKN2A deletion have a distinct gene expression signature and a poor prognosis under R-CHOP treatment: a GELA study.

    PubMed

    Jardin, Fabrice; Jais, Jean-Philippe; Molina, Thierry-Jo; Parmentier, Françoise; Picquenot, Jean-Michel; Ruminy, Philippe; Tilly, Hervé; Bastard, Christian; Salles, Gilles-André; Feugier, Pierre; Thieblemont, Catherine; Gisselbrecht, Christian; de Reynies, Aurelien; Coiffier, Bertrand; Haioun, Corinne; Leroy, Karen

    2010-08-19

    Genomic alterations play a crucial role in the development and progression of diffuse large B-cell lymphomas (DLBCLs). We determined gene copy number alterations (GCNAs) of TP53, CDKN2A, CDKN1B, BCL2, MYC, REL, and RB1 with a single polymerase chain reaction (PCR) assay (quantitative multiplex PCR of short fragments [QMPSF]) in a cohort of 114 patients with DLBCL to assess their prognostic value and relationship with the gene expression profile. Losses of TP53 and CDKN2A, observed in 8% and 35% of patients, respectively, were significantly associated with a shorter survival after rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) treatment, independently of the International Prognostic Index and of the cell of origin. Analysis of the 9p21 genomic region indicated that transcripts encoding p14ARF and p16INK4A were both disrupted in most patients with CDKN2A deletion. These patients predominantly had an activated B-cell profile and showed a specific gene expression signature, characterized by dysregulation of the RB/E2F pathway, activation of cellular metabolism, and decreased immune and inflammatory responses. These features may constitute the molecular basis sustaining the unfavorable outcome and chemoresistance of this DLBCL subgroup. Detection of TP53 and CDKN2A loss by QMPSF is a powerful tool that could be used for patient stratification in future clinical trials.

  18. Senescence induced by RECQL4 dysfunction contributes to Rothmund–Thomson syndrome features in mice

    PubMed Central

    Lu, H; Fang, E F; Sykora, P; Kulikowicz, T; Zhang, Y; Becker, K G; Croteau, D L; Bohr, V A

    2014-01-01

    Cellular senescence refers to irreversible growth arrest of primary eukaryotic cells, a process thought to contribute to aging-related degeneration and disease. Deficiency of RecQ helicase RECQL4 leads to Rothmund–Thomson syndrome (RTS), and we have investigated whether senescence is involved using cellular approaches and a mouse model. We first systematically investigated whether depletion of RECQL4 and the other four human RecQ helicases, BLM, WRN, RECQL1 and RECQL5, impacts the proliferative potential of human primary fibroblasts. BLM-, WRN- and RECQL4-depleted cells display increased staining of senescence-associated β-galactosidase (SA-β-gal), higher expression of p16INK4a or/and p21WAF1 and accumulated persistent DNA damage foci. These features were less frequent in RECQL1- and RECQL5-depleted cells. We have mapped the region in RECQL4 that prevents cellular senescence to its N-terminal region and helicase domain. We further investigated senescence features in an RTS mouse model, Recql4-deficient mice (Recql4HD). Tail fibroblasts from Recql4HD showed increased SA-β-gal staining and increased DNA damage foci. We also identified sparser tail hair and fewer blood cells in Recql4HD mice accompanied with increased senescence in tail hair follicles and in bone marrow cells. In conclusion, dysfunction of RECQL4 increases DNA damage and triggers premature senescence in both human and mouse cells, which may contribute to symptoms in RTS patients. PMID:24832598

  19. Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

    SciTech Connect

    Novak, Petr; Jensen, Taylor J.; Garbe, James C.; Stampfer, Martha R.; Futscher, Bernard W.

    2009-04-20

    The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16(INK4A) expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that mayprove useful in breast cancer risk assessment.

  20. Tissue depletion of taurine accelerates skeletal muscle senescence and leads to early death in mice.

    PubMed

    Ito, Takashi; Yoshikawa, Natsumi; Inui, Takaaki; Miyazaki, Natsuko; Schaffer, Stephen W; Azuma, Junichi

    2014-01-01

    Taurine (2-aminoethanesulfonic acid) is found in milimolar concentrations in mammalian tissues. One of its main functions is osmoregulation; however, it also exhibits cytoprotective activity by diminishing injury caused by stress and disease. Taurine depletion is associated with several defects, many of which are found in the aging animal, suggesting that taurine might exert anti-aging actions. Therefore, in the present study, we examined the hypothesis that taurine depletion accelerates aging by reducing longevity and accelerating aging-associated tissue damage. Tissue taurine depletion in taurine transporter knockout (TauTKO) mouse was found to shorten lifespan and accelerate skeletal muscle histological and functional defects, including an increase in central nuclei containing myotubes, a reduction in mitochondrial complex 1 activity and an induction in an aging biomarker, Cyclin-dependent kinase 4 inhibitor A (p16INK4a). Tissue taurine depletion also enhances unfolded protein response (UPR), which may be associated with an improvement in protein folding by taurine. Our data reveal that tissue taurine depletion affects longevity and cellular senescence; an effect possibly linked to a disturbance in protein folding.

  1. p16 — EDRN Public Portal

    Cancer.gov

    The CDKN2A locus generates several transcript variants which differ in their first exons. Two distinct transcripts are produced from different promoters: p16 (INK4A) and p14 (ARF). At least three alternatively spliced variants encoding distinct proteins have been reported, two of which (p16 and p14) encode structurally related isoforms known to function as inhibitors of CDK4 kinase. The remaining transcript includes an alternate first exon located 20 Kb upstream of the remainder of the gene; this transcript contains an alternate open reading frame (ARF) that specifies a protein which is structurally unrelated to the products of the other variants. This ARF product functions as a stabilizer of the tumor suppressor protein p53 as it can interact with, and sequester, MDM1, a protein responsible for the degradation of p53. In spite of the structural and functional differences, the CDK inhibitor isoforms and the ARF product encoded by this gene, through the regulatory roles of CDK4 and p53 in cell cycle G1 progression, share a common functionality in cell cycle G1 control. This gene is frequently mutated or deleted in a wide variety of tumors, and is known to be an important tumor suppressor gene.

  2. Post-ART epidermodysplasia verruciformis in a patient with AIDS.

    PubMed

    da Silva, Leila Cristina Ferreira; Miranda, Angelica Espinosa; Ferreira, Luiz Carlos de Lima; da Silva, Roberto Moreira; Mira, Marcelo Távora; Talhari, Carolina; Talhari, Sinésio

    2010-01-01

    Epidermodysplasia verruciformis (EV) is a rare disorder characterized by persistent human papillomavirus (HPV) infection. Here, we describe a 48-year-old, black, married male with AIDS, presenting a 1-year history of asymptomatic hypopigmented lesions that appeared 3 years after antiretroviral therapy (ART) initiation. Pre-ART, the initial CD4 count was 32 cells/mm(3) and the skin lesions appeared when the CD4 count reached 122 cells/mm(3). Dermatological examination demonstrated thin, scaly, slightly verrucous hypopigmented macules and papules, isolated or presenting with a linear aspect (Köbner phenomenon) in some areas, distributed on the neck, trunk, and superior and inferior members. Skin biopsy of a macular lesion revealed epidermal acanthosis with vacuolated keratinocytes presenting blue-gray pallor, arranged in clusters at the granular and upper spinous layer. Immunohistochemistry revealed expression of p16( INK4a) with diffuse positivity in the upper third of the epithelium, corresponding to the vacuolated keratinocytes. Polymerase chain reaction (PCR) was positive for type 12 HPV, and a diagnosis of EV-like associated to AIDS was made. EV-like is a rare disease and in this patent might be a manifestation of immune reconstitution inflammatory syndrome.

  3. Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation.

    PubMed

    Pickering, M T; Kowalik, T F

    2006-02-02

    Although it is unclear which cellular factor(s) is responsible for the genetic instability associated with initiating and sustaining cell transformation, it is known that many cancers have mutations that inactivate the Rb-mediated proliferation pathway. We show here that pRb inactivation and the resultant deregulation of one E2F family member, E2F1, leads to DNA double-strand break (DSB) accumulation in normal diploid human cells. These DSBs occur independent of Atm, p53, caspases, reactive oxygen species, and apoptosis. Moreover, E2F1 does not contribute to c-Myc-associated DSBs, indicating that the DSBs associated with these oncoproteins arise through distinct pathways. We also find E2F1-associated DSBs in an Rb mutated cancer cell line in the absence of an exogenous DSB stimulus. These basal, E2F1-associated DSBs are not observed in a p16(ink4a) inactivated cancer cell line that retains functional pRb, unless pRb is depleted. Thus, Rb status is key to regulating both the proliferation promoting functions associated with E2F and for preventing DNA damage accumulation if E2F1 becomes deregulated. Taken together, these data suggest that loss of Rb creates strong selective pressure, via DSB accumulation, for inactivating p53 mutations and that E2F1 contributes to the genetic instability associated with transformation and tumorigenesis.

  4. Epidermal Nbn deletion causes premature hair loss and a phenotype resembling psoriasiform dermatitis

    PubMed Central

    Seidel, Philipp; Remus, Martina; Delacher, Michael; Grigaravicius, Paulius; Reuss, David E.; Frappart, Lucien; von Deimling, Andreas; Feuerer, Markus; Abdollahi, Amir; Frappart, Pierre-Olivier

    2016-01-01

    Nijmegen Breakage Syndrome is a disease caused by NBN mutations. Here, we report a novel function of Nbn in skin homeostasis. We found that Nbn deficiency in hair follicle (HF) progenitors promoted increased DNA damage signaling, stimulating p16Ink4a up-regulation, Trp53 stabilization and cytokines secretion leading to HF-growth arrest and hair loss. At later stages, the basal keratinocytes layer exhibited also enhanced DNA damage response but in contrast to the one in HF progenitor was not associated with pro-inflammatory cytokines expression, but rather increased proliferation, lack of differentiation and immune response resembling psoriasiform dermatitis. Simultaneous Nbn and Trp53 inactivation significantly exacerbated this phenotype, due to the lack of inhibition of pro-inflammatory cytokines secretion by Trp53. Altogether, we demonstrated novel functions of Nbn in HF maintenance and prevention of skin inflammation and we provide a mechanistic explanation that links cell intrinsic DNA maintenance with large scale morphological tissue alterations. PMID:27050272

  5. In vitro aging of rat lung cells. Downregulation of telomerase activity and continuous decrease of telomere length are not incompatible with malignant transformation.

    PubMed

    Petitot, Fabrice; Lebeau, Jérôme; Dano, Laurent; Lectard, Bruno; Altmeyer, Sandrine; Levalois, Céline; Chevillard, Sylvie

    2003-05-15

    Most normal mammalian somatic cells cultivated in vitro enter replicative senescence after a finite number of divisions, as a consequence of the progressive shortening of telomeres during proliferation that reflects one aspect of organism/cellular aging. The situation appears more complex in rodent cells due to physiological telomerase expression in most somatic normal tissues, great telomere length, and the difficulties of finding suitable in vitro culture conditions. To study in vitro aging of rat lung epithelial cells, we have developed primary culture conditions adapted to rat fresh lung explants and have studied for 1 year (50 passages) the changes in cellular proliferation and mortality, genetic instability, telomerase activity, telomere length, and tumorigenic potential. We have observed an absence of senescence and/or crisis, a transient genetic instability, the persistence of a differentiated Clara cell phenotype, a steady decrease in telomerase activity followed by a low residual activity together with a continuous decrease in telomere length, a constant rate of proliferation, and the acquisition of tumorigenic potential. The bypass of the growth arrest and the acquisition of long-term growth properties could be explained by the loss of p16(INK4a) expression, the ARF/p53 pathway not being altered. In conclusion, these results clearly indicate that, in rat lung epithelial cells, in vitro transformation and acquisition of tumorigenic properties can occur even if the telomere length is still decreasing and telomerase activity remains downregulated.

  6. Attenuation of p38α MAPK stress response signaling delays the in vivo aging of skeletal muscle myofibers and progenitor cells.

    PubMed

    Papaconstantinou, John; Wang, Chen Z; Zhang, Min; Yang, San; Deford, James; Bulavin, Dmitry V; Ansari, Naseem H

    2015-09-01

    Functional competence and self-renewal of mammalian skeletal muscle myofibers and progenitor cells declines with age. Progression of the muscle aging phenotype involves the decline of juvenile protective factorsi.e., proteins whose beneficial functions translate directly to the quality of life, and self-renewal of progenitor cells. These characteristics occur simultaneously with the age-associated increase of p38α stress response signaling. This suggests that the maintenance of low levels of p38α activity of juvenile tissues may delay or attenuate aging. We used the dominant negative haploinsufficient p38α mouse (DN-p38α(AF/+)) to demonstrate that in vivo attenuation of p38α activity in the gastrocnemius of the aged mutant delays age-associated processes that include: a) the decline of the juvenile protective factors, BubR1, aldehyde dehydrogenase 1A (ALDH1A1), and aldehyde dehydrogenase 2 (ALDH2); b) attenuated expression of p16(Ink4a) and p19(Arf) tumor suppressor genes of the Cdkn2a locus; c) decreased levels of hydroxynonenal protein adducts, expression of COX2 and iNOS; d) decline of the senescent progenitor cell pool level and d) the loss of gastrocnemius muscle mass. We propose that elevated P-p38α activity promotes skeletal muscle aging and that the homeostasis of p38α impacts the maintenance of a beneficial healthspan.

  7. Ionizing radiation and aging: rejuvenating an old idea.

    PubMed

    Richardson, Richard B

    2009-11-17

    This paper reviews the contemporary evidence that radiation can accelerate aging, degenerative health effects and mortality. Around the 1960s, the idea that ionizing radiation caused premature aging was dismissed as the radiation-induced health effects appeared to be virtually confined to neoplasms. More recently, radiation has become associated with a much wider spectrum of age-related diseases, including cardiovascular disease; although some diseases of old age, such as diabetes, are notably absent as a radiation risk. On the basis of recent research, is there a stronger case today to be made linking radiation and aging? Comparison is made between the now-known biological mechanisms of aging and those of radiation, including oxidative stress, chromosomal damage, apoptosis, stem cell exhaustion and inflammation. The association between radiation effects and the free-radical theory of aging as the causative hypothesis seems to be more compelling than that between radiation and the nutrient-sensing TOR pathway. Premature aging has been assessed by biomarkers in calorie restriction studies; yet, biomarkers such as telomere erosion and p16(INK4a) are ambiguous for radiation-induced aging. Some animal studies suggest low dose radiation may even demonstrate hormesis health benefits. Regardless, there is virtually no support for a life span extending hypothesis for A-bomb survivors and other exposed subjects.

  8. Cervical histopathology variability among laboratories: a population-based statewide investigation.

    PubMed

    Gage, Julia C; Schiffman, Mark; Hunt, William C; Joste, Nancy; Ghosh, Arpita; Wentzensen, Nicolas; Wheeler, Cosette M

    2013-03-01

    To inform the proposed systematic adjudicative staining of cervical intraepithelial neoplasia grade 2 (CIN2) and equivocal diagnoses, we characterized diagnostic heterogeneity across 15 laboratories. Laboratory-specific distributions of 37,486 biopsy specimen diagnoses were compared after adjusting for preceding cytology. In a subset of preceding cytology specimens, HPV16 genotyping was considered an indicator of lesion severity. Distributions of normal and CIN1 diagnoses varied widely, with laboratories favoring either normal (5.5%-57.7%) or CIN1 diagnoses (23.3%-86.7%; P < .001 for normal:CIN1 variability). Excluding extreme values, 6.2% to 14.4% of diagnoses were CIN2 (P < .001). For CIN2 diagnoses, HPV16 positivity in the preceding cytology varied between 39.0% in the largest laboratory and 57.4% in others (P < .001), suggesting differential interpretation, not population differences, as a cause of variability. In conclusion, the frequency of diagnoses requiring special staining (p16(INK4a) immunostaining) to adjudicate equivocal CIN2 will be sizable and vary between laboratories, especially if extended to a fraction of CIN1 lesions.

  9. Renal tubular Notch signaling triggers a prosenescent state after acute kidney injury.

    PubMed

    Sörensen-Zender, Inga; Rong, Song; Susnik, Nathan; Zender, Steffen; Pennekamp, Petra; Melk, Anette; Haller, Hermann; Schmitt, Roland

    2014-04-15

    The aging kidney has a diminished regenerative potential and an increased tendency to develop tubular atrophy and fibrosis after acute injury. In this study, we found that activation of tubular epithelial Notch1 signaling was prolonged in the aging kidney after ischemia/reperfusion (IR) damage. To analyze the consequences of sustained Notch activation, we generated mice with conditional inducible expression of Notch1 intracellular domain (NICD) in proximal tubules. NICD kidneys were analyzed 1 and 4 wk after renal IR. Conditional NICD expression was associated with aggravated tubular damage, a fibrotic phenotype, and the expression of cellular senescence markers p21 and p16(INK4a). In wild-type mice pharmacological inhibition of Notch using the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) improved tubulo-interstitial damage and antagonized the prosenescent pathway activation after IR. In vitro, activation of Notch signaling with delta-like-ligand-4 caused prosenescent changes in tubular cells while inhibition with DAPT attenuated these changes. In conclusion, our data suggest that sustained epithelial Notch activation after IR might contribute to the inferior outcome of old kidneys after injury. Sustained epithelial activation of Notch is associated with a prosenescent phenotype and maladaptive repair.

  10. Recurrent chromosomal aberrations in INK4a/ARF defective primary lymphomas predict drug responses in vivo.

    PubMed

    Helmrich, Anne; Lee, Soyoung; O'Brien, Patricia; Dörken, Bernd; Lowe, Scott W; Schröck, Evelin; Schmitt, Clemens A

    2005-06-16

    Predicting responsiveness to anticancer therapy based on molecular findings at diagnosis is important to optimize treatment decisions. Although clinical outcome correlates with distinct mutations in some cancer entities, treatment responses within these lesion-stratified subgroups still remain heterogeneous, underscoring the need for additional prognosticators. We previously demonstrated that defined genetic defects at the INK4a/ARF locus, which encodes the tumor suppressors p16INK4a and ARF, not only accelerated lymphomagenesis in the Emu-myc transgenic mouse but also interfered with treatment sensitivity. In this study, we take a nonbiased genome-wide approach to examine whether the responsiveness of these lymphomas can be further stratified based on cytogenetic information at diagnosis. Indeed, using spectral karyotyping, comparative genomic hybridization, and fluorescence in situ hybridization in 38 primary lymphomas, we find recurrent cytogenetic alterations that refine the predictive value of INK4a/ARF lesions on drug responses in vivo: gain of chromosome 14, which was never detected in INK4a/ARFnull lymphomas, defined an ARFnull subgroup with superior treatment outcome. Gain of chromosome 6 was identified as a recurrent chromosomal aberration that predisposed ARFnull tumors to their subsequent INK4a loss during therapy. These data illustrate how cytogenetic information from cancer specimens might complement established prognostic markers and may improve anticancer treatment strategies.

  11. Epigenetics and pancreatic cancer: pathophysiology and novel treatment aspects.

    PubMed

    Neureiter, Daniel; Jäger, Tarkan; Ocker, Matthias; Kiesslich, Tobias

    2014-06-28

    An improvement in pancreatic cancer treatment represents an urgent medical goal. Late diagnosis and high intrinsic resistance to conventional chemotherapy has led to a dismal overall prognosis that has remained unchanged during the past decades. Increasing knowledge about the molecular pathogenesis of the disease has shown that genetic alterations, such as mutations of K-ras, and especially epigenetic dysregulation of tumor-associated genes, such as silencing of the tumor suppressor p16(ink4a), are hallmarks of pancreatic cancer. Here, we describe genes that are commonly affected by epigenetic dysregulation in pancreatic cancer via DNA methylation, histone acetylation or miRNA (microRNA) expression, and review the implications on pancreatic cancer biology such as epithelial-mesenchymal transition, morphological pattern formation, or cancer stem cell regulation during carcinogenesis from PanIN (pancreatic intraepithelial lesions) to invasive cancer and resistance development. Epigenetic drugs, such as DNA methyltransferases or histone deactylase inhibitors, have shown promising preclinical results in pancreatic cancer and are currently in early phases of clinical development. Combinations of epigenetic drugs with established cytotoxic drugs or targeted therapies are promising approaches to improve the poor response and survival rate of pancreatic cancer patients.

  12. Autocrine IL-6 mediates pituitary tumor senescence.

    PubMed

    Sapochnik, Melanie; Haedo, Mariana R; Fuertes, Mariana; Ajler, Pablo; Carrizo, Guillermo; Cervio, Andrés; Sevlever, Gustavo; Stalla, Günter K; Arzt, Eduardo

    2017-01-17

    Cellular senescence is a stable proliferative arrest state. Pituitary adenomas are frequent and mostly benign, but the mechanism for this remains unknown. IL-6 is involved in pituitary tumor progression and is produced by the tumoral cells. In a cell autonomous fashion, IL-6 participates in oncogene-induced senescence in transduced human melanocytes. Here we prove that autocrine IL-6 participates in pituitary tumor senescence. Endogenous IL-6 inhibition in somatotroph MtT/S shRNA stable clones results in decreased SA-β-gal activity and p16INK4a but increased pRb, proliferation and invasion. Nude mice injected with IL-6 silenced clones develop tumors contrary to MtT/S wild type that do not, demonstrating that clones that escape senescence are capable of becoming tumorigenic. When endogenous IL-6 is silenced, cell cultures derived from positive SA-β-gal human tumor samples decrease the expression of the senescence marker. Our results establish that IL-6 contributes to maintain senescence by its autocrine action, providing a natural model of IL-6 mediated benign adenoma senescence.

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

    PubMed Central

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

    2016-01-01

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

  14. Mice lacking JunB are osteopenic due to cell-autonomous osteoblast and osteoclast defects.

    PubMed

    Kenner, Lukas; Hoebertz, Astrid; Beil, F Timo; Beil, Timo; Keon, Niamh; Karreth, Florian; Eferl, Robert; Scheuch, Harald; Szremska, Agnieszka; Amling, Michael; Schorpp-Kistner, Marina; Angel, Peter; Wagner, Erwin F

    2004-02-16

    Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. JunBDelta/Delta mice are born viable, but develop severe low turnover osteopenia caused by apparent cell-autonomous osteoblast and osteoclast defects before a chronic myeloid leukemia-like disease. Although JunB was reported to be a negative regulator of cell proliferation, junBDelta/Delta osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16(INK4a) levels, but exhibit decreased cyclin D1 and cyclin A expression. Runx2 is transiently increased during osteoblast differentiation in vitro, whereas mature osteoblast markers such as osteocalcin and bone sialoprotein are strongly reduced. To support a cell-autonomous function of JunB in osteoclasts, junB was inactivated specifically in the macrophage-osteoclast lineage. Mutant mice develop an osteopetrosis-like phenotype with increased bone mass and reduced numbers of osteoclasts. Thus, these data reveal a novel function of JunB as a positive regulator controlling primarily osteoblast as well as osteoclast activity.

  15. Ionizing radiation promotes the acquisition of a senescence-associated secretory phenotype and impairs angiogenic capacity in cerebromicrovascular endothelial cells: role of increased DNA damage and decreased DNA repair capacity in microvascular radiosensitivity.

    PubMed

    Ungvari, Zoltan; Podlutsky, Andrej; Sosnowska, Danuta; Tucsek, Zsuzsanna; Toth, Peter; Deak, Ferenc; Gautam, Tripti; Csiszar, Anna; Sonntag, William E

    2013-12-01

    Cerebromicrovascular rarefaction is believed to play a central role in cognitive impairment in patients receiving whole-brain irradiation therapy. To elucidate the mechanism underlying the deleterious effects of γ-irradiation on the cerebral microcirculation, rat primary cerebromicrovascular endothelial cells (CMVECs) were irradiated in vitro. We found that in CMVECs, γ-irradiation (2-8 Gy) elicited increased DNA damage, which was repaired less efficiently in CMVECs compared with neurons, microglia, and astrocytes. Increased genomic injury in CMVECs associated with increased apoptotic cell death. In the surviving cells, γ-irradiation promotes premature senescence (indicated by SA-β-galactosidase positivity and upregulation of p16 (INK4a) ), which was associated with impaired angiogenic capacity (decreased proliferation and tube-forming capacity). γ-Irradiated CMVECs acquired a senescence-associated secretory phenotype, characterized by upregulation of proinflammatory cytokines and chemokines (including IL-6, IL-1α, and MCP-1). Collectively, increased vulnerability of γ-irradiated CMVECs and their impaired angiogenic capacity likely contribute to cerebromicrovascular rarefaction and prevent regeneration of the microvasculature postirradiation. The acquisition of a senescence-associated secretory phenotype in irradiated CMVECs is biologically highly significant as changes in the cytokine microenvironment in the hippocampus may affect diverse biological processes relevant for normal neuronal function (including regulation of neurogenesis and the maintenance of the blood brain barrier).

  16. A Novel Aspect of Tumorigenesis-BMI1 Functions in Regulating DNA Damage Response.

    PubMed

    Lin, Xiaozeng; Ojo, Diane; Wei, Fengxiang; Wong, Nicholas; Gu, Yan; Tang, Damu

    2015-12-01

    BMI1 plays critical roles in maintaining the self-renewal of hematopoietic, neural, intestinal stem cells, and cancer stem cells (CSCs) for a variety of cancer types. BMI1 promotes cell proliferative life span and epithelial to mesenchymal transition (EMT). Upregulation of BMI1 occurs in multiple cancer types and is associated with poor prognosis. Mechanistically, BMI1 is a subunit of the Polycomb repressive complex 1 (PRC1), and binds the catalytic RING2/RING1b subunit to form a functional E3 ubiquitin ligase. Through mono-ubiquitination of histone H2A at lysine 119 (H2A-K119Ub), BMI1 represses multiple gene loci; among these, the INK4A/ARF locus has been most thoroughly investigated. The locus encodes the p16INK4A and p14/p19ARF tumor suppressors that function in the pRb and p53 pathways, respectively. Its repression contributes to BMI1-derived tumorigenesis. BMI1 also possesses other oncogenic functions, specifically its regulative role in DNA damage response (DDR). In this process, BMI1 ubiquitinates histone H2A and γH2AX, thereby facilitating the repair of double-stranded DNA breaks (DSBs) through stimulating homologous recombination and non-homologous end joining. Additionally, BMI1 compromises DSB-induced checkpoint activation independent of its-associated E3 ubiquitin ligase activity. We review the emerging role of BMI1 in DDR regulation and discuss its impact on BMI1-derived tumorigenesis.

  17. Cancer-targeted IL-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation

    PubMed Central

    Schilbach, Karin; Alkhaled, Mohammed; Welker, Christian; Eckert, Franziska; Blank, Gregor; Ziegler, Hendrik; Sterk, Marco; Müller, Friederike; Sonntag, Katja; Wieder, Thomas; Braumüller, Heidi; Schmitt, Julia; Eyrich, Matthias; Schleicher, Sabine; Seitz, Christian; Erbacher, Annika; Pichler, Bernd J; Müller, Hartmut; Tighe, Robert; Lim, Annick; Gillies, Stephen D; Strittmatter, Wolfgang; Röcken, Martin; Handgretinger, Rupert

    2015-01-01

    Stimulating the immune system to attack cancer is a promising approach, even for the control of advanced cancers. Several cytokines that promote interferon-γ-dominated immune responses show antitumor activity, with interleukin 12 (IL-12) being of major importance. Here, we used an antibody-IL-12 fusion protein (NHS-IL12) that binds histones of necrotic cells to treat human sarcoma in humanized mice. Following sarcoma engraftment, NHS-IL12 therapy was combined with either engineered IL-7 (FcIL-7) or IL-2 (IL-2MAB602) for continuous cytokine bioavailability. NHS-IL12 strongly induced innate and adaptive antitumor immunity when combined with IL-7 or IL-2. NHS-IL12 therapy significantly improved survival of sarcoma-bearing mice and caused long-term remissions when combined with IL-2. NHS-IL12 induced pronounced cancer cell senescence, as documented by strong expression of senescence-associated p16INK4a and nuclear translocation of p-HP1γ, and permanent arrest of cancer cell proliferation. In addition, this cancer immunotherapy initiated the induction of myogenic differentiation, further promoting the hypothesis that efficient antitumor immunity includes mechanisms different from cytotoxicity for efficient cancer control in vivo. PMID:26140238

  18. Acute myeloid leukemia requires Hhex to enable PRC2-mediated epigenetic repression of Cdkn2a

    PubMed Central

    Shields, Benjamin J.; Jackson, Jacob T.; Metcalf, Donald; Shi, Wei; Huang, Qiutong; Garnham, Alexandra L.; Glaser, Stefan P.; Beck, Dominik; Pimanda, John E.; Bogue, Clifford W.; Smyth, Gordon K.; Alexander, Warren S.; McCormack, Matthew P.

    2016-01-01

    Unlike clustered HOX genes, the role of nonclustered homeobox gene family members in hematopoiesis and leukemogenesis has not been extensively studied. Here we found that the hematopoietically expressed homeobox gene Hhex is overexpressed in acute myeloid leukemia (AML) and is essential for the initiation and propagation of MLL-ENL-induced AML but dispensable for normal myelopoiesis, indicating a specific requirement for Hhex for leukemic growth. Loss of Hhex leads to expression of the Cdkn2a-encoded tumor suppressors p16INK4a and p19ARF, which are required for growth arrest and myeloid differentiation following Hhex deletion. Mechanistically, we show that Hhex binds to the Cdkn2a locus and directly interacts with the Polycomb-repressive complex 2 (PRC2) to enable H3K27me3-mediated epigenetic repression. Thus, Hhex is a potential therapeutic target that is specifically required for AML stem cells to repress tumor suppressor pathways and enable continued self-renewal. PMID:26728554

  19. c-myc copy number gain is a powerful prognosticator of disease outcome in cervical dysplasia.

    PubMed

    Kübler, Kirsten; Heinenberg, Sally; Rudlowski, Christian; Keyver-Paik, Mignon-Denise; Abramian, Alina; Merkelbach-Bruse, Sabine; Büttner, Reinhard; Kuhn, Walther; Schildhaus, Hans-Ulrich

    2015-01-20

    Cervical carcinoma develops from preneoplasia by a multistep process. Although most low-grade dysplastic lesions will regress without intervention and even high-grade changes exhibit a substantial rate of regression, a small percentage of dysplasia will progress over time. Thus, indicators are needed to estimate the biological risk and to help avoid overtreatment in women who desire to preserve fertility. In addition to the classical biomarkers, PCR-ELISA-determined HPV genotype and immunohistochemically assessed p16INK4a and Ki-67 expression, cells with integrated HPV and copy number gain of TERC and c-myc were quantified in a panel of 104 benign, intraepithelial neoplastic (CIN I, II, III) and cancerous lesions using fluorescence in situ hybridization. Optimal cut-off values were calculated; Kaplan-Meier curves and a Cox proportional hazard regression model were used to evaluate prognostic signatures. The assay reliably identified HPV integration, TERC and c-myc copy number gain as determined by comparisons with established biomarkers. All biomarker levels increased with the progression of the disease. However, only c-myc copy number gain independently prognosticated a low probability of dysplastic regression. Our results suggest that c-myc plays a key role in the process of dysplastic transformation and might thus be exploited for treatment and follow-up decision-making of cervical dysplasia.

  20. Senescence-like Phenotypes in Human Nevi

    PubMed Central

    Joselow, Andrew; Lynn, Darren; Terzian, Tamara; Box, Neil F.

    2016-01-01

    Summary Cellular senescence is an irreversible arrest of cell proliferation at the G1 stage of the cell cycle in which cells become refractory to growth stimuli. Senescence is a critical and potent defense mechanism that mammalian cells have to suppress tumors. While there are many ways to induce a senescence response, oncogene-induced senescence (OIS) remains key to inhibiting progression of cells that have acquired oncogenic mutations. In primary cells in culture, OIS induces a set of measurable phenotypic and behavioral changes, in addition to cell cycle exit. Senescence-associated β-Galactosidase (SA-β-Gal) activity is a main hallmark of senescent cells, along with morphological changes that may depend on the oncogene that is activated, or on the primary cell type. Characteristic cellular changes of senescence include increased size, flattening, multi-nucleation, and extensive vacuolation. At the molecular level, tumor suppressor genes such as p53 and p16INK4a may play a role in initiation or maintenance of OIS. Activation of a DNA damage response and a senescence-associated secretory phenotype could delineate the onset of senescence. Despite advances in our understanding of how OIS suppresses some tumor types, the in vivo role of OIS in melanocytic nevi and melanoma remains poorly understood and not validated. In an effort to stimulate research in this field, we review in this chapter the known markers of senescence and provide experimental protocols for their identification by immunofluorescent staining in melanocytic nevi and malignant melanoma. PMID:27812879

  1. Rb-dependent cellular senescence, multinucleation and susceptibility to oncogenic transformation through PKC scaffolding by SSeCKS/AKAP12

    PubMed Central

    Akakura, Shin; Nochajski, Peter; Gao, Lingqiu; Sotomayor, Paul; Matsui, Sei-ichi

    2010-01-01

    A subset of AKAPs (A Kinase Anchoring Proteins) regulate signaling and cytoskeletal pathways through the spaciotemporal scaffolding of multiple protein kinases (PK), such as PKC and PKA, and associations with the plasma membrane and the actin-based cytoskeleton. SSeCKS/Gravin/Akap12 expression is severely downregulated in many advanced cancers and exhibits tumor- and metastasis-suppressing activity. akap12-null (KO) mice develop prostatic hyperplasia with focal dysplasia, but the precise mechanism how Akap12 prevents oncogenic progression remains unclear. Here, we show that KO mouse embryonic fibroblasts (MEF) exhibit premature senescence marked by polyploidy and multinucleation, and by increased susceptibility to oncogenic transformation. Although p53 and Rb pathways are activated in the absence of Akap12, senescence is dependent on Rb. Senescence is driven by the activation of PKCα, which induces p16Ink4a/Rb through a MEK-dependent downregulation of Id1, and PKCδ, which downregulates Lats1/Warts, a mitotic exit network kinase required for cytokinesis. Our data strongly suggest that Akap12 controls Rb-mediated cell aging and oncogenic progression by directly scaffolding and attenuating PKCα/δ. PMID:21099353

  2. MicroRNAs in cancer: lessons from melanoma.

    PubMed

    Greenberg, Eyal; Nemlich, Yael; Markel, Gal

    2014-01-01

    Melanoma is a high-grade, poorly differentiated malignant tumor of pigment-producing cells (melanocytes), accounting for more than 70% of the skin cancer related deaths. Although new lines of targeted therapy and immunotherapy were introduced lately, durable responses are not common as it is hard to target the elusive metastatic phenotype. microRNAs (miRNAs) are short non-coding RNA molecules that function as specific epigenetic regulators of the transcriptome. miRNAs are involved in a broad spectrum of physiological and pathological processes, including cancer-related functions such as proliferation, cell cycle, migration, invasion, immune evasion and drug resistance. These functions are mostly regulated in melanoma through four molecular deregulated pathways, including the RAS/MAPK pathway, the MITF pathway, the p16INK4A-CDK4-RB pathway and the PI3K-AKT pathway. miRNAs provide a strong platform for delineation of cancer mechanisms. Here we review the diverse roles of miRNAs in melanoma cell biology. Studying miRNA-mediated regulation of aggressive and tumor related features is expected to provide novel mechanistic insights that may pave the way for new diagnostic, prognostic and predictive tools as well as new molecular targets for future therapy.

  3. Normal repair of ultraviolet radiation-induced DNA damage in familial melanoma without CDKN2A or CDK4 gene mutation.

    PubMed

    Shannon, J A; Matias, C; Luxford, C; Kefford, R F; Mann, G J

    1999-04-01

    Excessive sun exposure and family history are strong risk factors for the development of cutaneous melanoma. Inherited susceptibility to this type of skin cancer could therefore result from constitutively impaired capacity to repair ultraviolet (UV)-induced DNA lesions. While a proportion of familial melanoma kindreds exhibit germline mutations in the cell cycle regulatory gene CDKN2A (p16INK4a) or its protein target, cyclin-dependent kinase 4 (CDK4), the biochemical basis of most familial melanoma is unknown. We have examined lymphoblastoid cell lines from melanoma-affected and unaffected individuals from large hereditary melanoma kindreds which are not attributable to CDKN2A or CDK4 gene mutation. These lines were tested for sensitivity of clonogenic growth to UV radiation and for their ability to repair transfected UV-damaged plasmid templates (host cell reactivation). Two of seven affected-unaffected pairs differed in colony survival after exposure to UVB radiation; however, no significant differences were observed in the host-cell reactivation assays. These results indicate that melanoma susceptibility genes other than CDKN2A and CDK4 do not impair net capacity to repair UV-induced DNA damage.

  4. Downregulation of peroxiredoxin-3 by hydrophobic bile acid induces mitochondrial dysfunction and cellular senescence in human trophoblasts

    PubMed Central

    Wu, Wei-Bin; Menon, Ramkumar; Xu, Yue-Ying; Zhao, Jiu-Ru; Wang, Yan-Lin; Liu, Yuan; Zhang, Hui-Juan

    2016-01-01

    Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific disorder characterised by raised bile acids in foetal-maternal circulation, which threatens perinatal health. During the progression of ICP, the effect of oxidative stress is underscored. Peroxiredoxin-3 (PRDX3) is a mitochondrial antioxidant enzyme that is crucial to balance intracellular oxidative stress. However, the role of PRDX3 in placental trophoblast cells under ICP is not fully understood. We demonstrated that the level of PRDX3 was downregulated in ICP placentas as well as bile acids–treated trophoblast cells and villous explant in vitro. Toxic levels of bile acids and PRDX3 knockdown induced oxidative stress and mitochondrial dysfunction in trophoblast cells. Moreover, silencing of PRDX3 in trophoblast cell line HTR8/SVneo induced growth arrest and cellular senescence via activation of p38-mitogen-activated protein kinase (MAPK) and induction of p21WAF1/CIP and p16INK4A. Additionally, enhanced cellular senescence, determined by senescence-associated beta-galactosidase staining, was obviously attenuated by p38-MAPK inhibitor SB203580. Our data determined that exposure to bile acid decreased PRDX3 level in human trophoblasts. PRDX3 protected trophoblast cells against mitochondrial dysfunction and cellular senescence induced by oxidative stress. Our results suggest that decreased PRDX3 by excessive bile acids in trophoblasts plays a critical role in the pathogenesis and progression of ICP. PMID:27958341

  5. Human skin neural crest progenitor cells are susceptible to BRAF(V600E)-induced transformation.

    PubMed

    Kumar, S M; Dai, J; Li, S; Yang, R; Yu, H; Nathanson, K L; Liu, S; Zhou, H; Guo, J; Xu, X

    2014-02-13

    Adult stem cells are multipotent and persist in small numbers in adult tissues throughout the lifespan of an organism. Unlike differentiated cells, adult stem cells are intrinsically resistant to senescence. It is unclear how adult stem cells in solid organs respond to oncogenic stimulation and whether these cells have a role in tumor initiation. We report here that expression of BRAF(V600E) in human neural crest progenitor cells (hNCPCs) did not induce growth arrest as seen in human melanocytes, but instead, increased their cell proliferation capacity. These cells (hNCPCs(V600E)) acquired anchorage-independent growth ability and were weakly tumorigenic in vivo. Unlike in human melanocytes, BRAF(V600E) expression in hNCPCs did not induce p16(INK4a) expression. BRAF(V600E) induced elevated expression of CDK2, CDK4, MITF and EST1/2 protein in hNCPCs, and also induced melanocytic differentiation of these cells. Furthermore, overexpression of MITF in hNCPCs(V600E) dramatically increased their tumorigenicity and resulted in fully transformed tumor cells. These findings indicate that hNCPCs are susceptible to BRAF(V600E)-induced transformation, and MITF potentiates the oncogenic effect of BRAF(V600E) in these progenitor cells. These results suggest that the hNCPCs are potential targets for BRAF(V600E)-induced melanocytic tumor formation.

  6. Recent insights into the molecular mechanisms involved in aging and the malignant transformation of adult stem/progenitor cells and their therapeutic implications

    PubMed Central

    Mimeault, Murielle; Batra, Surinder K.

    2013-01-01

    Recent advancements in tissue-resident adult stem/progenitor cell research have revealed that enhanced telomere attrition, oxidative stress, ultraviolet radiation exposure and oncogenic events leading to severe DNA damages and genomic instability may occur in these immature and regenerative cells during chronological aging. Particularly, the alterations in key signaling components controlling their self-renewal capacity and an up-regulation of tumor suppressor gene products such as p16INK4A, p19ARF, ataxia-telangiectasia mutated (ATM) kinase, p53and/or theforkhead box O (FOXOs) family of transcription factors may result in their dysfunctions, growth arrest and senescence or apoptotic death during the aging process. These molecular events may culminate in a progressive decline in the regenerative functions and the number of tissue-resident adult stem/progenitor cells, and age-related disease development. Conversely, the telomerase re-activation and accumulation of numerous genetic and/or epigenetic alterations in adult stem/progenitor cells with advancing age may result in their immortalization and malignant transformation into highly leukemic or tumorigenic cancer-initiating cells and cancer initiation. Therefore, the cell-replacement and gene therapies and molecular targeting of aged and dysfunctional adult stem/progenitor cells including their malignant counterpart, cancer-initiating cells, hold great promise for treating and even curing diverse devastating human diseases. These diseases include premature aging diseases, hematopoietic, cardiovascular, musculoskeletal, pulmonary, ocular, urogenital, neurodegenerative and skin disorders and aggressive and recurrent cancers. PMID:19114129

  7. The human papillomavirus type 11 E1/\\E4 protein is not essential for viral genome amplification.

    PubMed

    Fang, L; Budgeon, L R; Doorbar, J; Briggs, E R; Howett, M K

    2006-08-01

    An abundant human papillomavirus (HPV) protein E1/\\E4 is expressed late in the virus life cycle in the terminally differentiated layers of epithelia. The expression of E1/\\E4 usually coincides with the onset of viral DNA amplification. However, the function of E1/\\E4 in viral life cycle is not completely understood. To examine the role of E1/\\E4 in the virus life cycle, we introduced a single nucleotide change in the HPV-11 genome to result in a truncation of E1/\\E4 protein without affecting the E2 amino acid sequence. This mutated HPV-11 genome was introduced into a human foreskin keratinocyte cell line immortalized by the catalytic subunit of human telomerase, deficient in p16(INK4a) expression, and previously shown to support the HPV-11 life cycle when grown in organotypic raft culture. We have demonstrated that E1/\\E4 is dispensable for HPV-11 viral DNA amplification in the late stages of the viral life cycle.

  8. A novel role for high-mobility group a proteins in cellular senescence and heterochromatin formation.

    PubMed

    Narita, Masashi; Narita, Masako; Krizhanovsky, Valery; Nuñez, Sabrina; Chicas, Agustin; Hearn, Stephen A; Myers, Michael P; Lowe, Scott W

    2006-08-11

    Cellular senescence is a stable state of proliferative arrest that provides a barrier to malignant transformation and contributes to the antitumor activity of certain chemotherapies. Senescent cells can accumulate senescence-associated heterochromatic foci (SAHFs), which may provide a chromatin buffer that prevents activation of proliferation-associated genes by mitogenic transcription factors. Surprisingly, we show that the High-Mobility Group A (HMGA) proteins, which can promote tumorigenesis, accumulate on the chromatin of senescent fibroblasts and are essential structural components of SAHFs. HMGA proteins cooperate with the p16(INK4a) tumor suppressor to promote SAHF formation and proliferative arrest and stabilize senescence by contributing to the repression of proliferation-associated genes. These antiproliferative activities are canceled by coexpression of the HDM2 and CDK4 oncogenes, which are often coamplified with HMGA2 in human cancers. Our results identify a component of the senescence machinery that contributes to heterochromatin formation and imply that HMGA proteins also act in tumor suppressor networks.

  9. The Challenge of Cancer Genomics in Rare Nervous System Neoplasms: Malignant Peripheral Nerve Sheath Tumors as a Paradigm for Cross-Species Comparative Oncogenomics.

    PubMed

    Carroll, Steven L

    2016-03-01

    Comprehensive genomic analyses of common nervous system cancers provide new insights into their pathogenesis, diagnosis, and treatment. Although analogous studies of rare nervous system tumors are needed, there are major barriers to performing such studies. Cross-species comparative oncogenomics, identifying driver mutations in mouse cancer models and validating them in human tumors, is a promising alternative. Although still in its infancy, this approach is being applied to malignant peripheral nerve sheath tumors (MPNSTs), rare Schwann cell-derived malignancies that occur sporadically, after radiotherapy, and in neurofibromatosis type 1. Studies of human neurofibromatosis type 1-associated tumors suggest that NF1 tumor suppressor loss in Schwann cells triggers cell-autonomous and intercellular changes, resulting in development of benign neurofibromas; subsequent neurofibroma-MPNST progression is caused by aberrant growth factor signaling and mutations affecting the p16(INK4A)-cyclin D1-CDK4-Rb and p19(ARF)-Mdm2-p53 cell cycle pathways. Mice with Nf1, Trp53, and/or Cdkn2a mutations that overexpress the Schwann cell mitogen neuregulin-1 or overexpress the epidermal growth factor receptor validate observations in human tumors and, to various degrees, model human tumorigenesis. Genomic analyses of MPNSTs arising in neuregulin-1 and epidermal growth factor receptor-overexpressing mice and forward genetic screens with Sleeping Beauty transposons implicate additional signaling cascades in MPNST pathogenesis. These studies confirm the utility of mouse models for MPNST driver gene discovery and provide new insights into the complexity of MPNST pathogenesis.

  10. Practical issues in the application of p16 immunohistochemistry in diagnostic pathology.

    PubMed

    Mahajan, Aparna

    2016-05-01

    The p16 tumor suppressor gene (CDKN2A) is a member of the INK4 class of cell cycle inhibitors and is located on chromosome 9p21. The p16 protein binds to cyclin-dependent kinases 4 and 6 and maintains the retinoblastoma gene product in its hypophosphorylated state, which in turn binds to E2F transcription factor and prevents cell cycle progression. Expression of p16 protein is increased in aging cells. Immunohistochemistry for p16ink4a is most widely used as a surrogate maker for high-risk human papilloma virus infection in formalin-fixed, paraffin-embedded tissues. The most widely researched, accepted, and practiced use of p16 immunostain is in the lower anogenital tract. In addition, p16 immunostain is widely used for oropharyngeal squamous cell carcinoma. Its applications have also been extended to gynecologic tumors, which are unrelated to human papillomavirus. This article aims to review the literature on the diagnostic utility of p16 immunohistochemistry and highlight the practical issues in the application and interpretation of this stain.

  11. The interplay between p16 serine phosphorylation and arginine methylation determines its function in modulating cellular apoptosis and senescence.

    PubMed

    Lu, Yang; Ma, Wenlong; Li, Zhongwei; Lu, Jun; Wang, Xiuli

    2017-01-25

    Cyclin-dependent kinase inhibitor p16(INK4a) (p16) primarily functions as a negative regulator of the retinoblastoma protein (Rb) -E2F pathway, thus plays critical role in cell cycle progression, cellular senescence and apoptosis. In this study, we showed that the methylation of Arg 138 and the phosphorylation of Ser 140 on p16 were critical for the control of cell proliferation and apoptosis. Compared to wild type p16, mutant p16R138K possessed improved function in preventing cell proliferation and inducing apoptosis, while the Ser 140 mutation (p16S140A) exhibited the opposite alteration. We also demonstrated that H2O2 was able to induce the phosphorylation of p16, which facilitated the interaction between CDK4 (Cyclin-dependent protein kinase) and p16, in 293T (human emborynic kidney) cells. Furthermore, the elevated arginine methylation in p16S140A mutant and increased serine phosphorylation in p16R138K mutant suggest that a antagonizing mechanism coordinating Arg 138 methylation and Ser 140 phosphorylation to regulates p16 function as well as cellular apoptosis and senescence. These findings will therefore contribute to therapeutic treatment for p16-related gene therapy by providing theoretical and experimental evidence.

  12. Magnolol causes alterations in the cell cycle in androgen insensitive human prostate cancer cells in vitro by affecting expression of key cell cycle regulatory proteins.

    PubMed

    McKeown, Brendan T; McDougall, Luke; Catalli, Adriana; Hurta, Robert A R

    2014-01-01

    Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 μM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16(INK4a), p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.

  13. A hyaluronic acid-based compound inhibits fibroblast senescence induced by oxidative stress in vitro and prevents oral mucositis in vivo.

    PubMed

    Cirillo, Nicola; Vicidomini, Antonio; McCullough, Michael; Gambardella, Antonio; Hassona, Yazan; Prime, Stephen S; Colella, Giuseppe

    2015-07-01

    Virtually all patients receiving radio- and chemotherapy for cancer develop oral mucositis, a severe and highly debilitating condition. The onset of mucositis is thought to involve the production of reactive oxygen species (ROS) in the submucosa. Here we investigated a possible protective effect of a commercial formulation of hyaluronic acid (HA) enriched with amino acids (Mucosamin(®)) against the damage induced by oxidative stress both in vitro and in vivo. Transient exposure of normal human oral fibroblasts to hydrogen peroxide (H(2)O(2)) led to irreversible senescence, as demonstrated by sustained increase in the levels of p16(INK4A) and SA-βGal. Conditioned media from senescent fibroblasts induced detrimental effects on keratinocytes, as shown by reduced metabolic activity and migration capability. Pre-treatment with Mucosamin(®) prevented H(2)O(2) -induced, but not TGF-β-induced, fibroblast senescence with a concomitant reduction of fibroblast-induced loss of keratinocyte vitality and functional activity. Finally, data from a case-series of patients undergoing radio/chemotherapy strongly suggested that prophylactic use of the hyaluronic acid-based compound in the form of a spray may be effective in preventing the onset of oral mucositis.

  14. Loss-of-function screening to identify miRNAs involved in senescence: tumor suppressor activity of miRNA-335 and its new target CARF

    PubMed Central

    Yu, Yue; Gao, Ran; Kaul, Zeenia; Li, Ling; Kato, Yoshio; Zhang, Zhenya; Groden, Joanna; Kaul, Sunil C; Wadhwa, Renu

    2016-01-01

    Significance of microRNAs (miRs), small non-coding molecules, has been implicated in a variety of biological processes. Here, we recruited retroviral insertional mutagenesis to obtain induction of an arbitrary noncoding RNAs, and coupled it with a cell based loss-of-function (5-Aza-2′-deoxycytidine (5Aza-dC)-induced senescence bypass) screening system. Cells that escaped 5-Aza-dC-induced senescence were subjected to miR-microarray analysis with respect to the untreated control. We identified miR-335 as one of the upregulated miRs. In order to characterize the functional significance, we overexpressed miR-335 in human cancer cells and found that it caused growth suppression. We demonstrate that the latter accounted for inhibition of 5-Aza-dC incorporation into the cell genome, enabling them to escape from induction of senescence. We also report that CARF (Collaborator of ARF) is a new target of miR-335 that regulates its growth suppressor function by complex crosstalk with other proteins including p16INK4A, pRB, HDM2 and p21WAF1. PMID:27457128

  15. Bmi1 regulates murine intestinal stem cell proliferation and self-renewal downstream of Notch.

    PubMed

    López-Arribillaga, Erika; Rodilla, Verónica; Pellegrinet, Luca; Guiu, Jordi; Iglesias, Mar; Roman, Angel Carlos; Gutarra, Susana; González, Susana; Muñoz-Cánoves, Pura; Fernández-Salguero, Pedro; Radtke, Freddy; Bigas, Anna; Espinosa, Lluís

    2015-01-01

    Genetic data indicate that abrogation of Notch-Rbpj or Wnt-β-catenin pathways results in the loss of the intestinal stem cells (ISCs). However, whether the effect of Notch is direct or due to the aberrant differentiation of the transit-amplifying cells into post-mitotic goblet cells is unknown. To address this issue, we have generated composite tamoxifen-inducible intestine-specific genetic mouse models and analyzed the expression of intestinal differentiation markers. Importantly, we found that activation of β-catenin partially rescues the differentiation phenotype of Rbpj deletion mutants, but not the loss of the ISC compartment. Moreover, we identified Bmi1, which is expressed in the ISC and progenitor compartments, as a gene that is co-regulated by Notch and β-catenin. Loss of Bmi1 resulted in reduced proliferation in the ISC compartment accompanied by p16(INK4a) and p19(ARF) (splice variants of Cdkn2a) accumulation, and increased differentiation to the post-mitotic goblet cell lineage that partially mimics Notch loss-of-function defects. Finally, we provide evidence that Bmi1 contributes to ISC self-renewal.

  16. Ionizing radiation-induced long-term expression of senescence markers in mice is independent of p53 and immune status

    PubMed Central

    Le, Oanh; Rodier, Francis; Fontaine, Francois; Coppe, Jean-Philippe; Campisi, Judith; DeGregori, James; Laverdiére, Caroline; Kokta, Victor; Haddad, Elie; Beauséjour, Christian M.

    2010-01-01

    Summary Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with life-long delayed repair and regeneration of irradiated tissues. We hypothesised that IR-induced senescence markers could persist long-term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45 weeks, the maximum time analysed) DNA damage foci and increased p16INK4a expression, two hallmarks of cellular senescence and aging. BrdU labelling experiments revealed that IR-induced damaged cells are preferentially eliminated, at least partially, in a tissue dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild-type and Rag2−/−γC−/− mice, the latter being deficient in T, B and NK cells. Together, our results provide compelling evidence that exposure to IR induces long-term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors. PMID:20331441

  17. Sweating the small stuff: microRNAs and genetic changes define pancreatic cancer.

    PubMed

    Tang, Siuwah; Bonaroti, Jillian; Unlu, Sebnem; Liang, Xiaoyan; Tang, Daolin; Zeh, Herbert J; Lotze, Michael T

    2013-07-01

    MicroRNAs (miRNAs) are 18- to 22-nucleotide-long, single-stranded, noncoding RNAs that regulate important biological processes including differentiation, proliferation, and response to cellular stressors such as hypoxia, nutrient depletion, and traversion of the cell cycle by controlling protein expression within the cell. Many investigators have profiled cancer tissue and serum miRNAs to identify potential therapeutic targets, understand the pathways involved in tumorigenesis, and identify diagnostic tumor signatures. In the setting of pancreatic cancer, obtaining pancreatic tissue is invasive and impractical for early diagnosis. Several groups have profiled miRNAs that are present in the blood as a means to diagnose tumor progression and predict prognosis/survival or drug resistance. Several miRNA signatures found in pancreatic tissue and the peripheral blood, as well as the pathways that are associated with pancreatic cancer, are reviewed here in detail. Three miRNA biomarkers (miR-21, miR-155, and miR-200) have been repetitively identified in both pancreatic cancer tissue and patients' blood. Those miRNAs regulate and are regulated by the central genetic and epigenetic changes observed in pancreatic cancer including p53, transforming growth factor β, p16(INK4A), BRCA1/2, and Kras. These miRNAs are involved in DNA repair, cell cycle, and cell invasion and also play important roles in promoting metastases.

  18. Geographic heterogeneity in the prevalence of human papillomavirus in head and neck cancer.

    PubMed

    Anantharaman, Devasena; Abedi-Ardekani, Behnoush; Beachler, Daniel C; Gheit, Tarik; Olshan, Andrew F; Wisniewski, Kathy; Wunsch-Filho, Victor; Toporcov, Tatiana N; Tajara, Eloiza H; Levi, José Eduardo; Moyses, Raquel A; Boccia, Stefania; Cadoni, Gabriella; Rindi, Guido; Ahrens, Wolfgang; Merletti, Franco; Conway, David I; Wright, Sylvia; Carreira, Christine; Renard, Helene; Chopard, Priscilia; McKay-Chopin, Sandrine; Scelo, Ghislaine; Tommasino, Massimo; Brennan, Paul; D'Souza, Gypsyamber

    2017-05-01

    Human papillomavirus (HPV) causes oropharyngeal squamous cell carcinoma (OPSCC), although strongly divergent results have been reported regarding the prevalence of HPV16 in different countries, whether this represents important differences in etiology remains unclear. Applying rigorous protocols for sample processing, we centrally evaluated 1,420 head and neck tumors (533 oropharynx, 395 oral cavity and 482 larynx) from studies conducted in the US, Europe and Brazil for mucosal HPV DNA and p16(INK4a) expression to evaluate regional heterogeneity in the proportion of HPV16-associated OPSCC and other head and neck cancer, and to assess covariates associated with the risk of HPV16-positive OPSCC. While majority of OPSCC in the US (60%) were HPV16-positive, this proportion was 31% in Europe and only 4% in Brazil (p < 0.01). Similar differences were observed for other head and neck tumors, ranging from 7% in the US and 5% in Europe, to 0% in South America. The odds of HPV16-positive OPSCC declined with increasing pack years of smoking (OR: 0.75; 95% CI: 0.64-0.87) and drink years of alcohol use (OR: 0.64; 95% CI: 0.54-0.76). These results suggest that while the contribution of HPV16 is substantial for the oropharynx, it remains limited for oral cavity and laryngeal cancers.

  19. Interaction with CCNH/CDK7 facilitates CtBP2 promoting esophageal squamous cell carcinoma (ESCC) metastasis via upregulating epithelial-mesenchymal transition (EMT) progression.

    PubMed

    Zhang, Jianguo; Zhu, Junya; Yang, Lei; Guan, Chengqi; Ni, Runzhou; Wang, Yuchan; Ji, Lili; Tian, Ye

    2015-09-01

    CtBP2, as a transcriptional corepressor of epithelial-specific genes, has been reported to promote tumor due to upregulating epithelial-mesenchymal transition (EMT) in cancer cells. CtBP2 was also demonstrated to contribute to the proliferation of esophageal squamous cell carcinoma (ESCC) cells through a negative transcriptional regulation of p16(INK4A). In this study, for the first time, we reported that CtBP2 expression, along with CCNH/CDK7, was higher in ESCC tissues with lymph node metastases than in those without lymph node metastases. Moreover, both CtBP2 and CCNH/CDK7 were positively correlated with E-cadherin, tumor grade, and tumor metastasis. However, the concrete mechanism of CtBP2's role in enhancing ESCC migration remains incompletely understood. We confirmed that CCNH/CDK7 could directly interact with CtBP2 in ESCC cells in vivo and in vitro. Furthermore, our data demonstrate for the first time that CtBP2 enhanced the migration of ESCC cells in a CCNH/CDK7-dependent manner. Our results indicated that CCNH/CDK7-CtBP2 axis may augment ESCC cell migration, and targeting the interaction of both may provide a novel therapeutic target of ESCC.

  20. The association between EGFR variant III, HPV, p16, c-MET, EGFR gene copy number and response to EGFR inhibitors in patients with recurrent or metastatic squamous cell carcinoma of the head and neck

    PubMed Central

    2011-01-01

    Background We examine the potential prognostic and predictive roles of EGFR variant III mutation, EGFR gene copy number (GCN), human papillomavirus (HPV) infection, c-MET and p16INK4A protein expression in recurrent or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN). Methods We analyzed the archival tumor specimens of 53 patients who were treated in 4 phase II trials for R/M SCCHN. Two trials involved the EGFR inhibitor erlotinib, and 2 trials involved non-EGFR targeted agents. EGFRvIII mutation was determined by quantitative RT-PCR, HPV DNA by Linear Array Genotyping, p16 and c-MET protein expression by immunohistochemistry, and EGFR GCN by FISH. Results EGFRvIII mutation, detected in 22 patients (42%), was associated with better disease control, but no difference was seen between erlotinib-treated versus non-erlotinib treated patients. EGFRvIII was not associated with TTP or OS. The presence of HPV DNA (38%), p16 immunostaining (32%), c-MET high expression (58%) and EGFR amplification (27%), were not associated with response, TTP or OS. Conclusion EGFRvIII mutation, present in about 40% of SCCHN, appears to be an unexpected prognostic biomarker associated with better disease control in R/M SCCHN regardless of treatment with erlotinib. Larger prospective studies are required to validate its significance. PMID:21352589

  1. Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines.

    PubMed

    Young, Richard J; Waldeck, Kelly; Martin, Claire; Foo, Jung H; Cameron, Donald P; Kirby, Laura; Do, Hongdo; Mitchell, Catherine; Cullinane, Carleen; Liu, Wendy; Fox, Stephen B; Dutton-Regester, Ken; Hayward, Nicholas K; Jene, Nicholas; Dobrovic, Alexander; Pearson, Richard B; Christensen, James G; Randolph, Sophia; McArthur, Grant A; Sheppard, Karen E

    2014-07-01

    We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.

  2. Establishing a panel of chemo-resistant mesothelioma models for investigating chemo-resistance and identifying new treatments for mesothelioma

    PubMed Central

    Hudson, Amanda L.; Weir, Chris; Moon, Elizabeth; Harvie, Rozelle; Klebe, Sonja; Clarke, Stephen J.; Pavlakis, Nick; Howell, Viive M.

    2014-01-01

    Mesothelioma is inherently chemo-resistant with only 50% of patients responding to the standard of care treatments, and consequently it has a very grim prognosis. The aim of this study was to establish a panel of chemo-resistant mesothelioma models with clinically relevant levels of resistance as tools for investigating chemo-resistance and identifying new treatments for mesothelioma. Chemo-resistant cell lines were established in vitro and characterized in vivo using syngeneic Fischer rats. Tumors derived from all chemo-resistant cell lines were immunohistochemically classified as mesothelioma. Homozygous deletion of p16INK4A/p14ARF and increased expression of several ATP-binding cassette transporters were demonstrated, consistent with findings in human mesothelioma. Further, the acquisition of chemo-resistance in vitro resulted in changes to tumor morphology and overall survival. In conclusion, these models display many features corresponding with the human disease, and provide the first series of matched parental and chemo-resistant models for in vitro and in vivo mesothelioma studies. PMID:25141917

  3. Inherited coding variants at the CDKN2A locus influence susceptibility to acute lymphoblastic leukaemia in children

    PubMed Central

    Xu, Heng; Zhang, Hui; Yang, Wenjian; Yadav, Rachita; Morrison, Alanna C.; Qian, Maoxiang; Devidas, Meenakshi; Liu, Yu; Perez-Andreu, Virginia; Zhao, Xujie; Gastier-Foster, Julie M.; Lupo, Philip J.; Neale, Geoff; Raetz, Elizabeth; Larsen, Eric; Bowman, W. Paul; Carroll, William L.; Winick, Naomi; Williams, Richard; Hansen, Torben; Holm, Jens-Christian; Mardis, Elaine; Fulton, Robert; Pui, Ching-Hon; Zhang, Jinghui; Mullighan, Charles G.; Evans, William E.; Hunger, Stephen P.; Gupta, Ramneek; Schmiegelow, Kjeld; Loh, Mignon L.; Relling, Mary V.; Yang, Jun J.

    2015-01-01

    There is increasing evidence from genome-wide association studies for a strong inherited genetic basis of susceptibility to acute lymphoblastic leukaemia (ALL) in children, yet the effects of protein-coding variants on ALL risk have not been systematically evaluated. Here we show a missense variant in CDKN2A associated with the development of ALL at genome-wide significance (rs3731249, P=9.4 × 10−23, odds ratio=2.23). Functional studies indicate that this hypomorphic variant results in reduced tumour suppressor function of p16INK4A, increases the susceptibility to leukaemic transformation of haematopoietic progenitor cells, and is preferentially retained in ALL tumour cells. Resequencing the CDKN2A–CDKN2B locus in 2,407 childhood ALL cases reveals 19 additional putative functional germline variants. These results provide direct functional evidence for the influence of inherited genetic variation on ALL risk, highlighting the important and complex roles of CDKN2A–CDKN2B tumour suppressors in leukaemogenesis. PMID:26104880

  4. Adiponectin Suppresses UVB-Induced Premature Senescence and hBD2 Overexpression in Human Keratinocytes

    PubMed Central

    Kim, MinJeong; Park, Kui Young; Lee, Mi-Kyung; Jin, Taewon; Seo, Seong Jun

    2016-01-01

    Recent studies have revealed that adiponectin can suppress cellular inflammatory signaling pathways. This study aimed to elucidate the effect of adiponectin on the unregulated production of hBD2 in UVB-induced premature senescent keratinocytes. We constructed an in vitro model of premature senescent keratinocytes through repeated exposure to low energy UVB. After repeated low energy UVB exposure, there was significant generation of reactive oxygen species (ROS) and induction of senescence-associated markers, including senescence associated beta-galactosidase activity and expression of p16INK4a and histone H2AX. In addition, the present clinical study showed higher expression of hBD2 in sun-exposed skin of elderly group, and the overexpression of hBD2 was observed by c-Fos activation in vitro. Adiponectin has the ability to scavenge ROS and consequently inhibit MAPKs and SA-markers in UVB-exposed keratinocytes. An inhibitor study demonstrated that adiponectin downregulated hBD2 mRNA expression through suppression of the AP-1 transcription factor components c-Fos via inactivation of p38 MAPK. Collectively, the dysregulated production of hBD2 by the induction of oxidative stress was attenuated by adiponectin through the suppression of p38 and JNK/SAPK MAPK signaling in UVB-mediated premature senescent inducible conditions. These results suggest the feasibility of adiponectin as an anti-photoaging and anti-inflammatory agent in the skin. PMID:27526049

  5. ATR maintains select progenitors during nervous system development

    PubMed Central

    Lee, Youngsoo; Shull, Erin RP; Frappart, Pierre-Olivier; Katyal, Sachin; Enriquez-Rios, Vanessa; Zhao, Jingfeng; Russell, Helen R; Brown, Eric J; McKinnon, Peter J

    2012-01-01

    The ATR (ATM (ataxia telangiectasia mutated) and rad3-related) checkpoint kinase is considered critical for signalling DNA replication stress and its dysfunction can lead to the neurodevelopmental disorder, ATR-Seckel syndrome. To understand how ATR functions during neurogenesis, we conditionally deleted Atr broadly throughout the murine nervous system, or in a restricted manner in the dorsal telencephalon. Unexpectedly, in both scenarios, Atr loss impacted neurogenesis relatively late during neural development involving only certain progenitor populations. Whereas the Atr-deficient embryonic cerebellar external germinal layer underwent p53- (and p16Ink4a/Arf)-independent proliferation arrest, other brain regions suffered apoptosis that was partially p53 dependent. In contrast to other organs, in the nervous system, p53 loss did not worsen the outcome of Atr inactivation. Coincident inactivation of Atm also did not affect the phenotype after Atr deletion, supporting non-overlapping physiological roles for these related DNA damage-response kinases in the brain. Rather than an essential general role in preventing replication stress, our data indicate that ATR functions to monitor genomic integrity in a selective spatiotemporal manner during neurogenesis. PMID:22266795

  6. Synchronous high-grade squamous intraepithelial lesion and adenocarcinoma in situ of cervix in a young woman presenting with hyperchromatic crowded groups in the cervical cytology specimen: report of a case.

    PubMed

    Zafar, Nadeem; Balazs, Louisa; Benstein, Barbara D

    2008-11-01

    We report a 29-year-old woman who underwent routine gynecologic evaluation at a community clinic and had a cervical sample drawn for liquid-based cytologic evaluation. At cytology, many hyperchromatic crowded groups (HCG) were present, but a consensus could not be established whether the abnormal cells were primarily glandular or squamous with secondary endocervical glandular involvement. An interpretation of atypical endocervical cells, favor neoplastic, was rendered and biopsy advised if clinically appropriate. At biopsy, the cervix contained synchronous squamous cell carcinoma in situ, secondarily involving endocervical glands, and neighboring adenocarcinoma in situ. Immunohistochemistry for Ki-67 and p16(INK4A) crisply and precisely stained both the lesions, clearly separating them from the adjacent uninvolved mucosa. This case re-emphasizes the challenge associated with accurate evaluation of HCG at cytology, the significance of ancillary testing for surrogate markers of high-risk HPV (HR-HPV) infection, the need for adjunct testing for HPV-DNA in the setting of HCG at cervical cytology, and a recommendation to set up studies to evaluate the role of surrogate markers of HR-HPV infection in cytologic samples with HCG.

  7. Mn(III) meso-tetrakis-(N-ethylpyridinium-2-yl) porphyrin mitigates total body irradiation-induced long-term bone marrow suppression.

    PubMed

    Li, Hongliang; Wang, Yong; Pazhanisamy, Senthil K; Shao, Lijian; Batinic-Haberle, Ines; Meng, Aimin; Zhou, Daohong

    2011-07-01

    Our recent studies showed that total body irradiation (TBI) induces long-term bone marrow (BM) suppression in part by induction of hematopoietic stem cell (HSC) senescence through reactive oxygen species (ROS). In this study, we examined if Mn(III) meso-tetrakis-(N-ethylpyridinium-2-yl) porphyrin (MnTE), a superoxide dismutase mimetic and potent antioxidant, can mitigate TBI-induced long-term BM injury in a mouse model. Our results showed that post-TBI treatment with MnTE significantly inhibited the increases in ROS production and DNA damage in HSCs and the reduction in HSC frequency and clonogenic function induced by TBI. In fact, the clonogenic function of HSCs from irradiated mice after MnTE treatment was comparable to that of HSCs from normal controls on a per-HSC basis, suggesting that MnTE treatment inhibited the induction of HSC senescence by TBI. This suggestion is supported by the finding that MnTE treatment also reduced the expression of p16(Ink4a) (p16) mRNA in HSCs induced by TBI and improved the long-term and multilineage engraftment of irradiated HSCs after transplantation. Therefore, the results from this study demonstrate that MnTE has the potential to be used as a therapeutic agent to mitigate TBI-induced long-term BM suppression by inhibiting ionizing radiation-induced HSC senescence through the ROS-p16 pathway.

  8. Total body irradiation causes long-term mouse BM injury via induction of HSC premature senescence in an Ink4a- and Arf-independent manner.

    PubMed

    Shao, Lijian; Feng, Wei; Li, Hongliang; Gardner, David; Luo, Yi; Wang, Yong; Liu, Lingbo; Meng, Aimin; Sharpless, Norman E; Zhou, Daohong

    2014-05-15

    Exposure to total body irradiation (TBI) induces not only acute hematopoietic radiation syndrome but also long-term or residual bone marrow (BM) injury. This residual BM injury is mainly attributed to permanent damage to hematopoietic stem cells (HSCs), including impaired self-renewal, decreased long-term repopulating capacity, and myeloid skewing. These HSC defects were associated with significant increases in production of reactive oxygen species (ROS), expression of p16(Ink4a) (p16) and Arf mRNA, and senescence-associated β-galacotosidase (SA-β-gal) activity, but not with telomere shortening or increased apoptosis, suggesting that TBI induces residual BM injury via induction of HSC premature senescence. This suggestion is supported by the finding that SA-β-gal(+) HSC-enriched LSK cells showed more pronounced defects in clonogenic activity in vitro and long-term engraftment after transplantation than SA-β-gal(-) LSK cells isolated from irradiated mice. However, genetic deletion of p16 and/or Arf had no effect on TBI-induced residual BM suppression and HSC senescence, because HSCs from irradiated p16 and/or Arf knockout (KO) mice exhibited changes similar to those seen in HSCs from wild-type mice after exposure to TBI. These findings provide important new insights into the mechanism by which TBI causes long-term BM suppression (eg, via induction of premature senescence of HSCs in a p16-Arf-independent manner).

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

    PubMed Central

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

    2014-01-01

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

  10. Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells

    PubMed Central

    Takayama, Naoya; Nishimura, Satoshi; Nakamura, Sou; Shimizu, Takafumi; Ohnishi, Ryoko; Endo, Hiroshi; Yamaguchi, Tomoyuki; Otsu, Makoto; Nishimura, Ken; Nakanishi, Mahito; Sawaguchi, Akira; Nagai, Ryozo; Takahashi, Kazutoshi; Yamanaka, Shinya; Nakauchi, Hiromitsu

    2010-01-01

    Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells, but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors, we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation, reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a+CD42b+ platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b+ platelets were present in thrombi after laser-induced vessel wall injury. In contrast, sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression, decreased GATA1 expression, and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression, particularly its later decline, is key to producing functional platelets from selected iPSC clones. PMID:21098095

  11. Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 tax oncoproteins modulate cell cycle progression and apoptosis.

    PubMed

    Sieburg, Michelle; Tripp, Adam; Ma, Jung-Woo; Feuer, Gerold

    2004-10-01

    Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia and lymphoma, an aggressive clonal malignancy of human CD4-bearing T lymphocytes. HTLV-2, although highly related to HTLV-1 at the molecular level, has not been conclusively linked to development of lymphoproliferative disorders. Differences between the biological activities of the respective tax gene products (Tax1 and Tax2) may be one factor which accounts for the differential pathogenicities associated with infection. To develop an in vitro model to investigate and compare the effects of constitutive expression of Tax1 and Tax2, Jurkat T-cell lines were infected with lentivirus vectors encoding Tax1 and Tax2 in conjunction with green fluorescent protein, and stably transduced clonal cell lines were generated by serial dilution in the absence of drug selection. Jurkat cells that constitutively express Tax1 and Tax2 (Tax1/Jurkat and Tax2/Jurkat, respectively) showed notably reduced kinetics of cellular replication, and Tax1 inhibited cellular replication to a higher degree in comparison to Tax2. Tax1 markedly activated transcription from the cdk inhibitor p21(cip1/waf1) promoter in comparison to Tax2, suggesting that upregulation of p21(cip1/waf1) may account for the differential inhibition of cellular replication kinetics displayed by Tax1/Jurkat and Tax2/Jurkat cells. The presence of binucleated and multinucleated cells, reminiscent of large lymphocytes with cleaved or cerebriform nuclei often seen in HTLV-1- and -2-seropositive patients, was noted in cultures expressing Tax1 and Tax2. Although Tax1 and Tax2 expression mediated elevated resistance to apoptosis in Jurkat cells after serum deprivation, Tax1 was unique in protection from apoptosis after exposure to camptothecin and etoposide, inhibitors of topoisomerase I and II, respectively. Characterization of the unique phenotypes displayed by Tax1 and Tax2 in vitro will provide information as to the relative roles of

  12. Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation

    PubMed Central

    2013-01-01

    Background Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. In the frog, Xenopus laevis, three types of CDK inhibitors have been described: p27Xic1 (Xic1) which shares sequence homology with both p21Cip1 and p27Kip1 from mammals, p16Xic2 (Xic2) which shares sequence homology with p21Cip1, and p17Xic3 (Xic3) which shares sequence homology with p27Kip1. While past studies have demonstrated that during DNA polymerase switching, Xic1 is targeted for protein turnover dependent upon DNA, Proliferating Cell Nuclear Antigen (PCNA), and the ubiquitin ligase CRL4Cdt2, little is known about the processes that regulate Xic2 or Xic3. Methods We used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation. Results Our studies indicated that following primer synthesis during the initiation of DNA replication, Xic2 is targeted for DNA- and PCNA-dependent ubiquitin-mediated proteolysis and that Cdt2 can promote Xic2 turnover. Additionally, during interphase, Xic2 is phosphorylated by CDK2 at Ser-98 and Ser-131 in a DNA-independent manner, inhibiting Xic2 turnover. In the presence of double-stranded DNA ends, Xic2 is also phosphorylated at Ser-78 and Ser-81 by a caffeine-sensitive kinase, but this phosphorylation does not alter Xic2 turnover. Conversely, in the presence or absence of DNA, Xic3 was stable in the Xenopus interphase egg extract and did not exhibit a shift indicative of phosphorylation. Conclusions During interphase, Xic2 is targeted for DNA- and PCNA-dependent proteolysis that is negatively regulated by CDK2 phosphorylation. During a response to DNA damage, Xic2 may be alternatively regulated by phosphorylation by a caffeine-sensitive kinase. Our studies suggest that the three types of Xenopus CDK inhibitors, Xic1, Xic2, and Xic3 appear to be uniquely regulated which may reflect their specialized roles during cell

  13. Multiple effects of Xihuang pill aqueous extract on the Hs578T triple-negative breast cancer cell line

    PubMed Central

    Zheng, Wenxian; Han, Shuyan; Jiang, Shantong; Pang, Lina; Li, Xiaohong; Liu, Xijuan; Cao, Minhua; Li, Pingping

    2016-01-01

    The management of triple-negative breast cancer (TNBC) is challenging due to the aggressive behavior, lack of therapeutic options and relatively poor prognosis. Xihuang pill (XHP) is a well-known Traditional Chinese Medicine with anticancer activity. The aim of the present study was to investigate whether the aqueous extract of XHP (AEXHP) has anti-proliferative activity against the Hs578T TNBC cell line, and to elucidate its molecular mechanisms of action. First, an MTT assay was used to evaluate the anti-proliferative activity of AEXHP on the Hs578T cell line; furthermore, the cell cycle distribution, mitochondrial membrane potential and apoptotic rate were determined by flow cytometry, and western blot analysis was used to assess the expression of apoptosis and cell cycle regulatory proteins to investigate the mechanisms of action. The results revealed that the cell viability was significantly inhibited by AEXHP in a dose- and time-dependent manner. Apoptosis and mitochondrial membrane potential loss were detected, and after treatment with 4, 8 and 12 mg/ml AEXHP for 24 h, cleaved caspase-3 was 1.70-, 1.81- and 1.84-fold of that of the control, while procaspase-3, procaspase-8, cleaved caspase-8, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and the Bcl-2/Bax ratio were not significantly affected. Cell cycle analysis revealed that treatment with AEXHP led to S-phase arrest of Hs578T cells. Furthermore, AEXHP treatment resulted in decreased expression of cyclin A and cyclin dependent kinase 2 (CDK2), and increased expression of cyclin E and p21Cip1, as compared to the control group. In conclusion, the viability of Hs578T cells was significantly inhibited by AEXHP in a dose- and time-dependent manner, the likely mechanisms of which being induction of apoptosis, probably via the intrinsic, Bcl-2-independent pathway, and cell cycle arrest in S phase due to decreased expression of cyclin A and CDK2, and increased expression of cyclin E and p21Cip1. PMID

  14. Structural basis for recruitment of human flap endonuclease 1 to PCNA

    PubMed Central

    Sakurai, Shigeru; Kitano, Ken; Yamaguchi, Hiroto; Hamada, Keisuke; Okada, Kengo; Fukuda, Kotaro; Uchida, Makiyo; Ohtsuka, Eiko; Morioka, Hiroshi; Hakoshima, Toshio

    2005-01-01

    Flap endonuclease-1 (FEN1) is a key enzyme for maintaining genomic stability and replication. Proliferating cell nuclear antigen (PCNA) binds FEN1 and stimulates its endonuclease activity. The structural basis of the FEN1–PCNA interaction was revealed by the crystal structure of the complex between human FEN1 and PCNA. The main interface involves the C-terminal tail of FEN1, which forms two β-strands connected by a short helix, the βA–αA–βB motif, participating in β–β and hydrophobic interactions with PCNA. These interactions are similar to those previously observed for the p21CIP1/WAF1 peptide. However, this structure involving the full-length enzyme has revealed additional interfaces that are involved in the core domain. The interactions at the interfaces maintain the enzyme in an inactive ‘locked-down' orientation and might be utilized in rapid DNA-tracking by preserving the central hole of PCNA for sliding along the DNA. A hinge region present between the core domain and the C-terminal tail of FEN1 would play a role in switching the FEN1 orientation from an inactive to an active orientation. PMID:15616578

  15. The Drosophila F-box protein dSkp2 regulates cell proliferation by targeting Dacapo for degradation.

    PubMed

    Dui, Wen; Wei, Bin; He, Feng; Lu, Wei; Li, Changqing; Liang, Xuehong; Ma, Jun; Jiao, Renjie

    2013-06-01

    Cell cycle progression is controlled by a complex regulatory network consisting of interacting positive and negative factors. In humans, the positive regulator Skp2, an F-box protein, has been a subject of intense investigation in part because of its oncogenic activity. By contrast, the molecular and developmental functions of its Drosophila homologue, dSkp2, are poorly understood. Here we investigate the role of dSkp2 by focusing on its functional relationship with Dacapo (Dap), the Drosophila homologue of the cyclin-dependent kinase inhibitors p21(cip1)/p27(kip1)/p57(kip2). We show that dSkp2 interacts physically with Dap and has a role in targeting Dap for ubiquitination and proteasome-mediated degradation. We present evidence that dSkp2 regulates cell cycle progression by antagonizing Dap in vivo. dSkp2 knockdown reduces cell density in the wing by prolonging the cell doubling time. In addition, the wing phenotype caused by dSkp2 knockdown resembles that caused by dap overexpression and can be partially suppressed by reducing the gene dose of dap. Our study thus documents a conserved functional relationship between dSkp2 and Dap in their control of cell cycle progression, suggesting the possibility of using Drosophila as a model system to study Skp2-mediated tumorigenesis.

  16. GCS-100, a novel galectin-3 antagonist, modulates MCL-1, NOXA, and cell cycle to induce myeloma cell death

    PubMed Central

    Streetly, Matthew J.; Maharaj, Lenushka; Joel, Simon; Schey, Steve A.; Gribben, John G.

    2010-01-01

    GCS-100 is a galectin-3 antagonist with an acceptable human safety profile that has been demonstrated to have an antimyeloma effect in the context of bortezomib resistance. In the present study, the mechanisms of action of GCS-100 are elucidated in myeloma cell lines and primary tumor cells. GCS-100 induced inhibition of proliferation, accumulation of cells in sub-G1 and G1 phases, and apoptosis with activation of both caspase-8 and -9 pathways. Dose- and time-dependent decreases in MCL-1 and BCL-XL levels also occurred, accompanied by a rapid induction of NOXA protein, whereas BCL-2, BAX, BAK, BIM, BAD, BID, and PUMA remained unchanged. The cell-cycle inhibitor p21Cip1 was up-regulated by GCS-100, whereas the procycling proteins CYCLIN E2, CYCLIN D2, and CDK6 were all reduced. Reduction in signal transduction was associated with lower levels of activated IκBα, IκB kinase, and AKT as well as lack of IκBα and AKT activation after appropriate cytokine stimulation (insulin-like growth factor-1, tumor necrosis factor-α). Primary myeloma cells showed a direct reduction in proliferation and viability. These data demonstrate that the novel therapeutic molecule, GCS-100, is a potent modifier of myeloma cell biology targeting apoptosis, cell cycle, and intracellular signaling and has potential for myeloma therapy. PMID:20190189

  17. From Immunity and Vaccines to Mammalian Regeneration

    PubMed Central

    Heber-Katz, Ellen

    2015-01-01

    Our current understanding of major histocompatibility complex (MHC)-mediated antigen presentation in self and nonself immune recognition was derived from immunological studies of autoimmunity and virus-host interactions, respectively. The trimolecular complex of the MHC molecule, antigen, and T-cell receptor accounts for the phenomena of immunodominance and MHC degeneracy in both types of responses and constrains vaccine development. Out of such considerations, we developed a simple peptide vaccine construct that obviates immunodominance, resulting in a broadly protective T-cell response in the absence of antibody. In the course of autoimmunity studies, we identified the MRL mouse strain as a mammalian model of amphibian-like regeneration. A significant level of DNA damage in the cells from this mouse pointed to the role of the cell cycle checkpoint gene CDKN1a, or p21cip1/waf1. The MRL mouse has highly reduced levels of this molecule, and a genetic knockout of this single gene in otherwise nonregenerating strains led to an MRL-type regenerative response, indicating that the ability to regenerate has not been lost during evolution. PMID:26116734

  18. Induced cancer stem-like cells as a model for biological screening and discovery of agents targeting phenotypic traits of cancer stem cell.

    PubMed

    Nishi, Mayuko; Akutsu, Hidenori; Kudoh, Ayumi; Kimura, Hirokazu; Yamamoto, Naoki; Umezawa, Akihiro; Lee, Sam W; Ryo, Akihide

    2014-09-30

    Cancer stem cells (CSCs) retain the capacity to propagate themselves through self-renewal and to produce heterogeneous lineages of cancer cells constituting the tumor. Novel drugs that target CSCs can potentially eliminate the tumor initiating cell population therefore resulting in complete cure of the cancer. We recently established a CSC-like model using induced pluripotent stem cell (iPSC) technology to reprogram and partially differentiate human mammary epithelial MCF-10A cells. Using the induced CSC-like (iCSCL) model, we developed a phenotypic drug assay system to identify agents that inhibit the stemness and self-renewal properties of CSCs. The selectivity of the agents was assessed using three distinct assays characterized by cell viability, cellular stemness and tumor sphere formation. Using this approach, we found that withaferin A (WA), an Ayurvedic medicine constituent, was a potent inhibitor of CSC stemness leading to cellular senescence primarily via the induction of p21Cip1 expression. Moreover, WA exhibited strong anti-tumorigenic activity against the iCSCL. These results indicate that our iCSCL model provides an innovative high throughput platform for a simple, easy, and cost-effective method to search for novel CSC-targeting drugs. Furthermore, our current study identified WA as a putative drug candidate for abrogating the stemness and tumor initiating ability of CSCs.

  19. A Disease-Causing Variant in PCNA Disrupts a Promiscuous Protein Binding Site.

    PubMed

    Duffy, Caroline M; Hilbert, Brendan J; Kelch, Brian A

    2016-03-27

    The eukaryotic DNA polymerase sliding clamp, proliferating cell nuclear antigen or PCNA, is a ring-shaped protein complex that surrounds DNA to act as a sliding platform for increasing processivity of cellular replicases and for coordinating various cellular pathways with DNA replication. A single point mutation, Ser228Ile, in the human PCNA gene was recently identified to cause a disease whose symptoms resemble those of DNA damage and repair disorders. The mutation lies near the binding site for most PCNA-interacting proteins. However, the structural consequences of the S228I mutation are unknown. Here, we describe the structure of the disease-causing variant, which reveals a large conformational change that dramatically transforms the binding pocket for PCNA client proteins. We show that the mutation markedly alters the binding energetics for some client proteins, while another, p21(CIP1), is only mildly affected. Structures of the disease variant bound to peptides derived from two PCNA partner proteins reveal that the binding pocket can adjust conformation to accommodate some ligands, indicating that the binding site is dynamic and pliable. Our work has implications for the plasticity of the binding site in PCNA and reveals how a disease mutation selectively alters interactions to a promiscuous binding site that is critical for DNA metabolism.

  20. High fat diet triggers cell cycle arrest and excessive apoptosis of granulosa cells during the follicular development.

    PubMed

    Wu, Yanqing; Zhang, Zhenghong; Liao, Xinghui; Wang, Zhengchao

    2015-10-23

    The regulatory mechanism of granulosa cells (GCs) proliferation during the follicular development is complicated and multifactorial, which is essential for the oocyte growth and normal ovarian functions. To investigate the role of high fat diet (HFD) on the proliferation of GCs, 4-week old female mice were fed with HFD or normal control diet (NC) for 15 weeks or 20 weeks and then detected the expression level of some regulatory molecules of cell cycle and apoptosis. The abnormal ovarian morphology was observed at 20 weeks. Further mechanistic studies indicated that HFD induced-obesity caused elevated apoptotic levels in GCs of the ovaries in a time-dependent manner. Moreover, cell cycle progress was also impacted after HFD fed. The cell cycle inhibitors, p27(Kip1) and p21(Cip1), were significantly induced in the ovaries from the mice in HFD group when compared with that in the ovaries from the mice in NC group. Subsequently, the expression levels of Cyclin D1, D3 and CDK4 were also significantly influenced in the ovaries from the mice fed with HFD in a time-dependent manner. The present results suggested that HFD induced-obesity may trigger cell cycle arrest and excessive apoptosis of GCs, causing the abnormal follicular development and ovarian function failure.

  1. MicroRNA-106a suppresses proliferation, migration, and invasion of bladder cancer cells by modulating MAPK signaling, cell cycle regulators, and Ets-1-mediated MMP-2 expression.

    PubMed

    Shin, Seung-Shick; Park, Sung-Soo; Hwang, Byungdoo; Kim, Won Tae; Choi, Yung Hyun; Kim, Wun-Jae; Moon, Sung-Kwon

    2016-10-01

    Despite the clinical significance of tumorigenesis, little is known about the cellular signaling networks of microRNAs (miRs). Here we report a new finding that mir‑106a regulates the proliferation, migration, and invasion of bladder cancer cells. Basal expression levels of mir‑106a were significantly lower in bladder cancer cells than in normal urothelial cells. Overexpression of mir‑106a suppressed the proliferation of bladder cancer cell line EJ. Transient transfection of mir‑106a into EJ cells led to downregulation of ERK phosphorylation and upregulation of p38 and JNK phosphorylation over their levels in the control. Flow cytometry analysis revealed that mir‑106a-transfected cells accumulated in the G1-phase of the cell cycle, and cyclin D1 and CDK6 were significantly downregulated. This G1-phase cell cycle arrest was due in part to the upregulation of p21CIP1/WAF1. In addition, mir‑106a overexpression blocked the wound-healing migration and invasion of EJ cells. Furthermore, mir‑106a transfection resulted in decreased expression of MMP-2 and diminished binding activity of transcription factor Ets-1 in EJ cells. Collectively, we report the novel mir‑106a-mediated molecular signaling networks that regulate the proliferation, migration, and invasion of bladder cancer cells, suggesting that mir‑106a may be a therapeutic target for treating advanced bladder tumors.

  2. Aspirin acetylates wild type and mutant p53 in colon cancer cells: identification of aspirin acetylated sites on recombinant p53.

    PubMed

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D Ramesh; Marimuthu, Srinivasan; Alfonso, Lloyd F; Bhat, G Jayarama

    2016-05-01

    Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anti-cancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. We demonstrate that aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21(CIP1). Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anti-cancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin.

  3. The all-trans retinoic acid (atRA)-regulated gene Calmin (Clmn) regulates cell cycle exit and neurite outgrowth in murine neuroblastoma (Neuro2a) cells.

    PubMed

    Marzinke, Mark A; Clagett-Dame, Margaret

    2012-01-01

    The vitamin A metabolite all-trans retinoic acid (atRA) functions in nervous system development and regulates cell proliferation and differentiation. Neuroblastoma cells (SH-SY5Y and Neuro2a or N2A) exposed to atRA undergo growth inhibition and neuronal differentiation, both of which are preceded by an increase in Clmn mRNA. Treatment of N2A cells with atRA produces a reduction in phosphohistone 3 immunostaining and BrdU incorporation, both indicators of a reduction in cell proliferation. These effects are nearly eliminated in atRA-treated shClmn knockdown cells. Loss of Clmn in the mouse N2A cell line also results in a significant reduction of atRA-mediated neurite outgrowth, a response that can be rescued by reintroduction of the Clmn sequence. In contrast, ectopic overexpression of Clmn produces an increase in the cyclin dependent kinase inhibitor, p21(Cip1), a decrease in cyclin D1 protein and an increase in hypophosphorylated Rb, showing that Clmn participates in G(1)/S arrest. Clmn overexpression alone is sufficient to inhibit N2A cell proliferation, whereas both Clmn and atRA must be present to induce neurite outgrowth. This study shows that the atRA-responsive gene Clmn promotes exit from the cell cycle, a requisite event for neuronal differentiation.

  4. Cell cycle-dependent control of polarised development by a cyclin-dependent kinase-like protein in the Fucus zygote.

    PubMed

    Corellou, F; Brownlee, C; Kloareg, B; Bouget, F Y

    2001-11-01

    Although iterative development can be uncoupled from morphogenesis in plant organs, the relationship between the cell cycle and developmental events is not well established in embryos. Zygotes of fucoid algae, including Fucus and Pelvetia are particularly well suited for studying the interaction(s) between cell cycle progression and the early morphogenetic events, as the establishment of polarity and its morphogenetic expression, i.e. germination, and the first cell cycle are concomitant. We have previously demonstrated that, in Fucus zygotes, various aspects of cell cycle progression are tightly controlled by cyclin-dependent kinase (CDK)-like proteins, including two PSTAIRE CDK-like proteins, p34 and p32, which are synthesised after fertilisation. We show that specific inhibition of CDK-like proteins, either with purine derivatives such as olomoucine and amino-purvalanol or by microinjection of the CDK inhibitor p21(cip1), prevents germination and cell division. Whereas direct inhibition of DNA replication by aphidicolin did not affect polarised development, olomoucine, which has previously been shown to prevent entry in S phase, and other purine derivatives also inhibited photopolarisation. Early microinjection of a monoclonal anti-PSTAIRE antibody also prevented germination and cell division. Only p34 had affinity for amino-purvalanol, suggesting that among PSTAIRE CDKs, this protein is the main target of purine derivatives. Models to account for the simultaneous control of early cell cycle progression and polarisation are proposed.

  5. Alteration of gene expression profiles in skeletal muscle of rats exposed to microgravity during a spaceflight

    NASA Technical Reports Server (NTRS)

    Taylor, Wayne E.; Bhasin, Shalender; Lalani, Rukhsana; Datta, Anuj; Gonzalez-Cadavid, Nestor F.

    2002-01-01

    To clarify the mechanism of skeletal muscle wasting during spaceflights, we investigated whether intramuscular gene expression profiles are affected, by using DNA microarray methods. Male rats sent on the 17-day NASA STS-90 Neurolab spaceflight were sacrificed 24 hours after return to earth (MG group). Ground control rats were maintained for 17 days in flight-simulated cages (CS group). Spaceflight induced a 19% and 23% loss of tibialis anterior and gastrocnemius muscle mass, respectively, as compared to ground controls. Muscle RNA was analyzed by the Clontech Atlas DNA expression array in four rats, with two MG/ CS pairs for the tibialis anterior, and one pair for the gastrocnemius. Alterations in gene expression were verified for selected genes by reverse-transcription PCR. In both muscles of MG rats, mRNAs for 12 genes were up-regulated by over 2-fold, and 38 were down-regulated compared to controls. There was inhibition of genes for cell proliferation and growth factor cascades, including cell cycle genes and signal transduction proteins, such as p21 Cip1, retinoblastoma (Rb), cyclins G1/S, -E and -D3, MAP kinase 3, MAD3, and ras related protein RAB2. These data indicate that following exposure to microgravity, there is downregulation of genes involved in regulation of muscle satellite cell replication.

  6. Breast Cancer Stem Cells

    PubMed Central

    Velasco-Velázquez, Marco A.; Homsi, Nora; De La Fuente, Marisol; Pestell, Richard G.

    2012-01-01

    Breast cancer stem cells (BCSCs) constitute a subpopulation of tumor cells that express stem cell-associated markers and have a high capacity for tumor generation in vivo. Identification of BCSCs from tumor samples or breast cancer cell lines has been based mainly on CD44+/CD24−/low or ALDH+ phenotypes. BCSCs isolation has allowed the analysis of the molecular mechanisms involved in their origin, self-renewal, differentiation into tumor cells, resistance to radiation therapy and chemotherapy, and invasiveness and metastatic ability. Molecular genetic analysis using knockout animals and inducible transgenics have identified NF-κB, c-Jun, p21CIP1, and Forkhead-like-protein Dach1 in BCSC expansion and fate. Clinical analyses of BCSCs in breast tumors have found a correlation between the proportion of BCSCs and poor prognosis. Therefore, new therapies that specifically target BCSCs are an urgent need. We summarize recent evidence that partially explain the biological characteristics of BCSCs. PMID:22249027

  7. The activity and stability of the intrinsically disordered Cip/Kip protein family are regulated by non-receptor tyrosine kinases

    PubMed Central

    Otieno, Steve; Lelli, Moreno; Kriwacki, Richard W.

    2014-01-01

    The Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors includes p21Cip1, p27Kip1 and p57Kip2. Their kinase inhibitory activities are mediated by a homologous N-terminal kinase-inhibitory domain (KID). The Cdk inhibitory activity and stability of p27 have been shown to be regulated by a two-step phosphorylation mechanism involving a tyrosine residue within the KID and a threonine residue within the flexible C-terminus. We show that these residues are conserved in p21 and p57, suggesting that a similar phosphorylation cascade regulates these Cdk inhibitors. However, the presence of a cyclin binding motif within its C-terminus alters the regulatory interplay between p21 and Cdk2/cyclin A, and its responses to tyrosine phosphorylation and altered p21:Cdk2/cyclin A stoichiometry. We also show that the Cip/Kip proteins can be phosphorylated in vitro by representatives of many non-receptor tyrosine kinase (NRTK) sub-families, suggesting that NRTKs may generally regulate the activity and stability of these Cdk inhibitors. Our results further suggest that the Cip/Kip proteins integrate signals from various NRTK pathways and cell cycle regulation. PMID:25463440

  8. RPLP1, a crucial ribosomal protein for embryonic development of the nervous system.

    PubMed

    Perucho, Laura; Artero-Castro, Ana; Guerrero, Sergi; Ramón y Cajal, Santiago; LLeonart, Matilde E; Wang, Zhao-Qi

    2014-01-01

    Ribosomal proteins are pivotal to development and tissue homeostasis. RP Large P1 (Rplp1) overexpression is associated with tumorigenesis. However, the physiological function of Rplp1 in mammalian development remains unknown. In this study, we disrupted Rplp1 in the mouse germline and central nervous system (Rplp1CNSΔ). Rplp1 heterozygosity caused body size reductions, male infertility, systemic abnormalities in various tissues and a high frequency of early postnatal death. Rplp1CNSΔ newborn mice exhibited perinatal lethality and brain atrophy with size reductions of the neocortex, midbrain and ganglionic eminence. The Rplp1 knockout neocortex exhibited progenitor cell proliferation arrest and apoptosis due to the dysregulation of key cell cycle and apoptosis regulators (cyclin A, cyclin E, p21CIP1, p27KIP1, p53). Similarly, Rplp1 deletion in pMEFs led to proliferation arrest and premature senescence. Importantly, Rplp1 deletion in primary mouse embryonic fibroblasts did not alter global protein synthesis, but did change the expression patterns of specific protein subsets involved in protein folding and the unfolded protein response, cell death, protein transport and signal transduction, among others. Altogether, we demonstrated that the translation "fine-tuning" exerted by Rplp1 is essential for embryonic and brain development and for proper cell proliferation.

  9. TFAP2C controls hormone response in breast cancer cells through multiple pathways of estrogen signaling.

    PubMed

    Woodfield, George W; Horan, Annamarie D; Chen, Yizhen; Weigel, Ronald J

    2007-09-15

    Breast cancers expressing estrogen receptor-alpha (ERalpha) are associated with a favorable biology and are more likely to respond to hormonal therapy. In addition to ERalpha, other pathways of estrogen response have been identified including ERbeta and GPR30, a membrane receptor for estrogen, and the key mechanisms regulating expression of ERs and hormone response remain controversial. Herein, we show that TFAP2C is the key regulator of hormone responsiveness in breast carcinoma cells through the control of multiple pathways of estrogen signaling. TFAP2C regulates the expression of ERalpha directly by binding to the ERalpha promoter and indirectly via regulation of FoxM1. In so doing, TFAP2C controls the expression of ERalpha target genes, including pS2, MYB, and RERG. Furthermore, TFAP2C controlled the expression of GPR30. In distinct contrast, TFAP2A, a related factor expressed in breast cancer, was not involved in estrogen-mediated pathways but regulated expression of genes controlling cell cycle arrest and apoptosis including p21(CIP1) and IGFBP-3. Knockdown of TFAP2C abrogated the mitogenic response to estrogen exposure and decreased hormone-responsive tumor growth of breast cancer xenografts. We conclude that TFAP2C is a central control gene of hormone response and is a novel therapeutic target in the design of new drug treatments for breast cancer.

  10. Pleurotus ostreatus inhibits proliferation of human breast and colon cancer cells through p53-dependent as well as p53-independent pathway

    PubMed Central

    JEDINAK, ANDREJ; SLIVA, DANIEL

    2009-01-01

    In spite of the global consumption of mushrooms, only two epidemiological studies demonstrated an inverse correlation between mushroom intake and the risk of cancer. Therefore, in the present study we evaluated whether extracts from edible mushrooms Agaricus bisporus (portabella), Flammulina velutipes (enoki), Lentinula edodes (shiitake) and Pleurotus ostreatus (oyster) affect the growth of breast and colon cancer cells. Here, we identified as the most potent, P. ostreatus (oyster mushroom) which suppressed proliferation of breast cancer (MCF-7, MDA-MB-231) and colon cancer (HT-29, HCT-116) cells, without affecting proliferation of epithelial mammary MCF-10A and normal colon FHC cells. Flow cytometry revealed that the inhibition of cell proliferation by P. ostreatus was associated with the cell cycle arrest at G0/G1 phase in MCF-7 and HT-29 cells. Moreover, P. ostreatus induced the expression of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21(CIP1/WAF1), whereas inhibited the phosphorylation of retinoblastoma Rb protein in MCF-7 cells. In addition, P. ostreatus also up-regulated expression of p21 and inhibited Rb phosphorylation in HT-29 cells, suggesting that that P. ostreatus suppresses the proliferation of breast and colon cancer cells via p53-dependent as well as p53-independent pathway. In conclusion, our results indicated that the edible oyster mushroom has potential therapeutic/preventive effects on breast and colon cancer. PMID:19020765

  11. The role of p21 in regulating mammalian regeneration.

    PubMed

    Arthur, Larry Matthew; Heber-Katz, Ellen

    2011-06-29

    The MRL (Murphy Roths Large) mouse has provided a unique model of adult mammalian regeneration as multiple tissues show this important phenotype. Furthermore, the healing employs a blastema-like structure similar to that seen in amphibian regenerating tissue. Cells from the MRL mouse display DNA damage, cell cycle G2/M arrest, and a reduced level of p21CIP1/WAF. A functional role for p21 was confirmed when tissue injury in an adult p21-/- mouse showed a healing phenotype that matched the MRL mouse, with the replacement of tissues, including cartilage, and with hair follicle formation and a lack of scarring. Since the major canonical function of p21 is part of the p53/p21 axis, we explored the consequences of p53 deletion. A regenerative response was not seen in a p53-/- mouse and the elimination of p53 from the MRL background had no negative effect on the regeneration of the MRL.p53-/- mouse. An exploration of other knockout mice to identify p21-dependent, p53-independent regulatory pathways involved in the regenerative response revealed another significant finding showing that elimination of transforming growth factor-β1 displayed a healing response as well. These results are discussed in terms of their effect on senescence and differentiation.

  12. The role of p21 in regulating mammalian regeneration

    PubMed Central

    2011-01-01

    The MRL (Murphy Roths Large) mouse has provided a unique model of adult mammalian regeneration as multiple tissues show this important phenotype. Furthermore, the healing employs a blastema-like structure similar to that seen in amphibian regenerating tissue. Cells from the MRL mouse display DNA damage, cell cycle G2/M arrest, and a reduced level of p21CIP1/WAF. A functional role for p21 was confirmed when tissue injury in an adult p21-/- mouse showed a healing phenotype that matched the MRL mouse, with the replacement of tissues, including cartilage, and with hair follicle formation and a lack of scarring. Since the major canonical function of p21 is part of the p53/p21 axis, we explored the consequences of p53 deletion. A regenerative response was not seen in a p53-/- mouse and the elimination of p53 from the MRL background had no negative effect on the regeneration of the MRL.p53-/- mouse. An exploration of other knockout mice to identify p21-dependent, p53-independent regulatory pathways involved in the regenerative response revealed another significant finding showing that elimination of transforming growth factor-β1 displayed a healing response as well. These results are discussed in terms of their effect on senescence and differentiation. PMID:21722344

  13. Apamin inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration through suppressions of activated Akt and Erk signaling pathway.

    PubMed

    Kim, Jung-Yeon; Kim, Kyung-Hyun; Lee, Woo-Ram; An, Hyun-Jin; Lee, Sun-Jae; Han, Sang-Mi; Lee, Kwang-Gill; Park, Yoon-Yub; Kim, Kee-Sik; Lee, Young-Soo; Park, Kwan-Kyu

    2015-07-01

    The increased proliferation and migration of vascular smooth muscle cells (VSMC) are key process in the development of atherosclerosis lesions. Platelet-derived growth factor (PDGF) initiates a multitude of biological effects that contribute to VSMC proliferation and migration. Apamin, a component of bee venom, has been known to block the Ca(2+)-activated K(+) channels. However, the effects of apamin in the regulation PDGF-BB-induced VSMC proliferation and migration has not been identified. In this study, we investigate the inhibitory effect of apamin on PDGF-BB-induced VSMC proliferation and migration. Apamin suppressed the PDGF-BB-induced VSMC proliferation and migration with no apparent cytotoxic effect. In accordance with these findings, apamin induced the arrest of cell cycle progression at G0/G1 phase. Apamin also decreased the expressions of G0/G1 specific regulatory proteins including proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinases (CDK) 4, cyclin E and CDK2, as well as increased the expression of p21(Cip1) in PDGF-BB-induced VSMC. Moreover, apamin inhibited PDGF-BB-induced phosphorylation of Akt and Erk1/2. These results suggest that apamin plays an important role in prevention of vascular proliferation and migration through the G0/G1 cell cycle arrest by PDGF signaling pathway. Thus, apamin may be a promising candidate for the therapy of atherosclerosis.

  14. Hepatocyte growth factor (HGF) inhibits skeletal muscle cell differentiation: a role for the bHLH protein twist and the cdk inhibitor p27.

    PubMed

    Leshem, Y; Spicer, D B; Gal-Levi, R; Halevy, O

    2000-07-01

    Hepatocyte growth factor (HGF) plays a crucial role in regulating the differentiation of both fetal and adult skeletal myoblasts. This study aimed at defining the intracellular factors that mediate the effect of HGF on adult myoblast differentiation. HGF increased Twist expression while decreasing p27(kip1) protein levels and not affecting the induction of p21(Cip1/Waf1) in satellite cells. Like HGF, overexpression of Twist did not affect p21 expression while inhibiting muscle-specific proteins. Both ectopic Twist-antisense (Twist-AS) and p27 partially rescued the effects of HGF on bromodeoxyuridine (BrdU) incorporation and myosin heavy chain (MHC) expression in muscle satellite cells; the two plasmids together effected full rescue, suggesting that HGF independently regulates these two factors to mediate its effects. Ectopic p27 promoted differentiation in the presence of HGF by blocking the induction of Twist. Using Twist-AS to lower Twist levels restored the HGF-dependent reduction of p27 and MHC. In the presence of ectopic HGF, satellite cells formed thin mononuclear myotubes. Neither ectopic p27, Twist-AS, or their combination reversed this change in cell morphology, suggesting that HGF acts through additional mediators to inhibit downstream events during myogenesis. Taken together, the results suggest that the effects of HGF on muscle cell proliferation and differentiation are mediated through changes in the expression levels of the myogenic-inhibitory basic helix-loop-helix (bHLH) protein Twist and the cell-cycle inhibitor p27.

  15. The p53 tumor suppressor protein protects against chemotherapeutic stress and apoptosis in human medulloblastoma cells

    PubMed Central

    Parasido, Erika; Tricoli, Lucas; Sivakumar, Angiela; Mikhaiel, John P.; Yenugonda, Venkata; Rodriguez, Olga C.; Karam, Sana D.; Rood, Brian R.; Avantaggiati, Maria Laura; Albanese, Chris

    2015-01-01

    Medulloblastoma (MB), a primitive neuroectodermal tumor, is the most common malignant childhood brain tumor and remains incurable in about a third of patients. Currently, survivors carry a significant burden of late treatment effects. The p53 tumor suppressor protein plays a crucial role in influencing cell survival in response to cellular stress and while the p53 pathway is considered a key determinant of anti-tumor responses in many tumors, its role in cell survival in MB is much less well defined. Herein, we report that the experimental drug VMY-1-103 acts through induction of a partial DNA damage-like response as well induction of non-survival autophagy. Surprisingly, the genetic or chemical silencing of p53 significantly enhanced the cytotoxic effects of both VMY and the DNA damaging drug, doxorubicin. The inhibition of p53 in the presence of VMY revealed increased late stage apoptosis, increased DNA fragmentation and increased expression of genes involved in apoptosis, including CAPN12 and TRPM8, p63, p73, BIK, EndoG, CIDEB, P27Kip1 and P21cip1. These data provide the groundwork for additional studies on VMY as a therapeutic drug and support further investigations into the intriguing possibility that targeting p53 function may be an effective means of enhancing clinical outcomes in MB. PMID:26540407

  16. Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma

    PubMed Central

    Chen, Yulong; Terajima, Masahiko; Yang, Yanan; Sun, Li; Ahn, Young-Ho; Pankova, Daniela; Puperi, Daniel S.; Watanabe, Takeshi; Kim, Min P.; Blackmon, Shanda H.; Rodriguez, Jaime; Liu, Hui; Behrens, Carmen; Wistuba, Ignacio I.; Minelli, Rosalba; Scott, Kenneth L.; Sanchez-Adams, Johannah; Guilak, Farshid; Pati, Debananda; Thilaganathan, Nishan; Burns, Alan R.; Creighton, Chad J.; Martinez, Elisabeth D.; Zal, Tomasz; Grande-Allen, K. Jane; Yamauchi, Mitsuo; Kurie, Jonathan M.

    2015-01-01

    Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde–derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde–derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma. PMID:25664850

  17. Phosphorylation of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) by Akt promotes stability and mitogenic function of S-phase kinase-associated protein-2 (Skp2).

    PubMed

    Song, Gyun Jee; Leslie, Kristen L; Barrick, Stacey; Mamonova, Tatyana; Fitzpatrick, Jeremy M; Drombosky, Kenneth W; Peyser, Noah; Wang, Bin; Pellegrini, Maria; Bauer, Philip M; Friedman, Peter A; Mierke, Dale F; Bisello, Alessandro

    2015-01-30

    The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.

  18. Regulation of miR106b cluster through the RB pathway: mechanism and functional targets.

    PubMed

    Thangavel, Chellappagounder; Boopathi, Ettickan; Ertel, Adam; Lim, Meng; Addya, Sankar; Fortina, Paolo; Witkiewicz, Agnieszka K; Knudsen, Erik S

    2013-01-01

    The RB pathway plays a critical role in proliferation control that is commonly subverted in tumor development. However, restoration of RB pathway function can be elicited in many tumor cells by the inhibition of CDK4/6 activity that leads to dephosphorylation of RB and subsequent repression of E2F-mediated transcription. In this context, active RB/E2F complexes inhibit the expression of a critical program of coding genes that promote cell cycle progression. However, the non-coding RNA target genes downstream from RB that could be relevant for tumor biology remain obscure. Here, miRNA gene expression profiling identified the miR106b cluster as being efficiently repressed with CDK4/6 inhibition in an E2F and RB-dependent manner. Importantly, the miR106B-cluster is intragenic of MCM7, and through a series of functional studies, the basis of MCM7 regulation and concordant expression of the miRNA species within the 106b cluster was determined. Importantly, RB-mediated repression of the 106b cluster enhances the transcript levels of p21Cip1 and PTEN. These data provide a mechanistic basis for cross-talk between the RB pathway and p21 and PTEN through the regulation of the MCM7/miR106b locus.

  19. Bortezomib induces apoptosis and growth suppression in human medulloblastoma cells, associated with inhibition of AKT and NF-ĸB signaling, and synergizes with an ERK inhibitor.

    PubMed

    Yang, Fan; Jove, Veronica; Chang, Shirley; Hedvat, Michael; Liu, Lucy; Buettner, Ralf; Tian, Yan; Scuto, Anna; Wen, Wei; Yip, M L Richard; Van Meter, Timothy; Yen, Yun; Jove, Richard

    2012-04-01

    Medulloblastoma is the most common brain tumor in children. Here, we report that bortezomib, a proteasome inhibitor, induced apoptosis and inhibited cell proliferation in two established cell lines and a primary culture of human medulloblastomas. Bortezomib increased the release of cytochrome c to cytosol and activated caspase-9 and caspase-3, resulting in cleavage of PARP. Caspase inhibitor (Z-VAD-FMK) could rescue medulloblastoma cells from the cytotoxicity of bortezomib. Phosphorylation of AKT and its upstream regulator mTOR were reduced by bortezomib treatment in medulloblastoma cells. Bortezomib increased the expression of Bad and Bak, pro-apoptotic proteins, and p21Cip1 and p27Kip1, negative regulators of cell cycle progression, which are associated with the growth suppression and induction of apoptosis in these tumor cells. Bortezomib also increased the accumulation of phosphorylated IĸBα, and decreased nuclear translocation of NF-ĸB. Thus, NF-ĸB signaling and activation of its downstream targets are suppressed. Moreover, ERK inhibitors or downregulating ERK with ERK siRNA synergized with bortezomib on anticancer effects in medulloblastoma cells. Bortezomib also inhibited the growth of human medulloblastoma cells in a mouse xenograft model. These findings suggest that proteasome inhibitors are potentially promising drugs for treatment of pediatric medulloblastomas.

  20. Induction of G2/M phase arrest and apoptosis by the flavonoid tamarixetin on human leukemia cells.

    PubMed

    Nicolini, Fabio; Burmistrova, Olga; Marrero, María Teresa; Torres, Fernando; Hernández, Cristina; Quintana, José; Estévez, Francisco

    2014-12-01

    Flavonoids are naturally occurring polyphenolic compounds which display a vast array of biological activities. In this study, we investigated the effects of tamarixetin on viability of human tumor cell lines and found that it was cytotoxic against leukemia cells and in particular P-glycoprotein-overexpressing K562/ADR cells. This compound inhibited proliferation in a concentration- and time-dependent manner, induced apoptosis and blocked cell cycle progression at G2 -M phase. This was associated with the accumulation of cyclin B1, Bub1 and p21(Cip1/Waf-1), changes in the phosphorylation status of cyclin B1, Cdk1, Cdc25C and MPM-2, and inhibition of tubulin polymerization. Moreover, cell death was found to be associated with cytochrome c release and cleavage of caspases and of poly(ADP-ribose) polymerase, and completely abrogated by the free-radical scavenger N-acetyl-L-cysteine. The sensitivity of leukemic cells to tamarixetin suggests that it should be considered for further preclinical and in vivo testing.

  1. Effects of digoxin on cell cycle, apoptosis and NF-κB pathway in Burkitt's lymphoma cells and animal model.

    PubMed

    Wang, Ting; Xu, Peipei; Wang, Fan; Zhou, Di; Wang, Ruju; Meng, Li; Wang, Xiaohui; Zhou, Min; Chen, Bing; Ouyang, Jian

    2017-07-01

    Digoxin has potential antitumor properties. This study investigated whether digoxin suppressed Burkitt's lymphoma (BL) cells. Raji and NAMALWA cells were exposed to digoxin, followed by assay of cell viability, apoptosis and cell cycle. Western blotting was used to analyze NF-κB activity. A xenograft model was established for therapeutic efficacy evaluation. Digoxin inhibited cell growth and resulted in apoptosis and cell cycle arrest (G0/G1 for Raji cells; G2/M for NAMALWA cells). Digoxin inhibited DNA synthesis and induced morphological apoptotic characteristics. Besides, digoxin inhibited NF-κB and TNF-α-stimulated NF-κB activity, and suppressed NF-κB initiating genes (Bcl-2, Bcl-xL, cyclin D1, and c-myc), however, increased p21(cip1). Digoxin activated caspase-9/3. Furthermore, digoxin inhibited xenograft tumors growth and reduced Ki-67 and c-myc. Digoxin exerted antitumor effects on BL cells in vitro and in vivo might through regulating NF-κB and caspase pathway. These outcomes highlight the potential of digoxin as a therapeutic agent for BL.

  2. In vivo antitumor effects of 4,7-dimethoxy-5-methyl-1,3-benzodioxole isolated from the fruiting body of Antrodia camphorata through activation of the p53-mediated p27/Kip1 signaling pathway.

    PubMed

    Tu, Shih-Hsin; Wu, Chih-Hsiung; Chen, Li-Ching; Huang, Ching-Shui; Chang, Hui-Wen; Chang, Chien-Hsi; Lien, Hsiu-Man; Ho, Yuan-Soon

    2012-04-11

    In this study, 4,7-dimethoxy-5-methyl-1,3-benzodioxole (SY-1) was isolated from three different sources of dried Antrodia camphorata (AC) fruiting bodies. AC is a medicinal mushroom that grows on the inner heartwood wall of Cinnamomum kanehirai Hay (Lauraceae), which is an endemic species that is used in Chinese medicine for its antitumor properties. We demonstrated that SY-1 [given as a 1-30 mg/kg body weight intraperitoneal (ip) injection three times per week] profoundly decreased the growth of COLO-205 human colon cancer cell tumor xenografts in an athymic nude mouse model. We further demonstrated that significant AC extract-mediated antitumor effects were observed at the highest concentration (5 g/kg body weight/day). No gross toxicity signs were observed (i.e., body weight changes, general appearance, or individual organ effects). Frozen COLO-205 xenograft tumors were pulverized in liquid N(2), and the expression of cell cycle regulatory proteins was detected by immunoblotting. We found that the p53-mediated p27/Kip1 protein was significantly induced in the low-dose (1 mg/kg body weight) SY-1-treated tumors, whereas the p21/Cip1 protein levels did not change. The G0/G1 phase cell cycle regulators induced by SY-1 were also associated with a significant decrease in cyclins D1, D3, and A. These results provide further evidence that SY-1 may have significance for cancer chemotherapy.

  3. Enhanced malignant tumorigenesis in Cdk4 transgenic mice.

    PubMed

    Miliani de Marval, Paula L; Macias, Everardo; Conti, Claudio J; Rodriguez-Puebla, Marcelo L

    2004-03-11

    In a previous study, we reported that overexpression of cyclin-dependent kinase-4 (CDK4) in mouse epidermis results in epidermal hyperplasia, hypertrophy and severe dermal fibrosis. In this study, we have investigated the susceptibility to skin tumor formation by forced expression of CDK4. Skin tumors from transgenic mice showed a dramatic increase in the rate of malignant progression to squamous cell carcinomas (SCC) in an initiation-promotion protocol. Histopathological analysis of papillomas from transgenic mice showed an elevated number of premalignant lesions characterized by dysplasia and marked atypia. Interestingly, transgenic mice also developed tumors in initiated but not promoted skin, demonstrating that CDK4 replaced the action of tumor promoters. These results suggest that expression of cyclin D1 upon ras activation synergizes with CDK4 overexpression. However, cyclin D1 transgenic mice and double transgenic mice for cyclin D1 and CDK4 did not show increased malignant progression in comparison to CDK4 transgenic mice. Biochemical analysis of tumors showed that CDK4 sequesters the CDK2 inhibitors p27Kip1 and p21Cip1, suggesting that indirect activation of CDK2 plays an important role in tumor development. These results indicate that, contrary to the general assumption, the catalytic subunit, CDK4, has higher oncogenic activity than cyclin D1, revealing a potential use of CDK4 as therapeutic target.

  4. Galectin-3 regulates p21 stability in human prostate cancer cells

    PubMed Central

    Wang, Yi; Balan, Vitaly; Kho, Dhonghyo; Hogan, Victor; Nangia-Makker, Pratima; Raz, Avraham

    2014-01-01

    Galectin-3 (Gal-3) is a multifunctional protein involved in cancer through regulation of cell adhesion, cell growth, apoptosis, and metastasis, while p21 (Cip1/WAF1) is a negative regulator of the cell cycle, involved in apoptosis, transcription, DNA repair and metastasis. The results presented here demonstrate for the first time that the level of Gal-3 protein is associated with the level of p21 protein expression in human prostate cancer cells and the effects of Gal-3 on cell growth and apoptosis were reversed by modulating p21 expression level. Furthermore, Gal-3 regulates p21 expression at the post-translational level by stabilizing p21 protein via the carbohydrate-recognition domain (CRD). This is the first report suggesting a molecular function not yet described for Gal-3 as the regulator of p21 protein stability. This study provides a unique insight into the relationship of these two molecules during prostate cancer progression, and may provide a novel therapeutic target. PMID:23160381

  5. Synthesis of N-methylarylnitrones derived from alkyloxybenzaldehydes and antineoplastic effect on human cancer cell lines.

    PubMed

    Costa, Débora S S; Martino, Thiago; Magalhães, Fernanda C; Justo, Graça; Coelho, Marsen G P; Barcellos, Julio C F; Moura, Victor B; Costa, Paulo R R; Sabino, Kátia C C; Dias, Ayres G

    2015-05-01

    New O-isoprenylated-N-methylarylnitrones derived from isomeric o, m and p-hydroxybenzaldehydes have been prepared and the antineoplastic effects on human cancer cell lines were evaluated. The O-geranylated nitrone LQB-278 (1b) and its isomers 2b and 3b inhibited the NO production, but the anti-leukemic activity was drastically dependent on nitrone isomer, with the 1b being the most effective one (IC₅₀ of 6.7 μM) on Jurkat leukemia cell, by MTT assay. In addition, 1b up-regulated p21CIP1/WAF1/Sdi1 protein expression (flow cytometry), a cell cycle inhibitor, reduced cell growth, and induced DNA fragmentation (increased sub-G1 phase cells) and phosphatidylserine externalization in plasmatic membrane (increased annexin V positive cells). Finally, the 1b up-regulation of p21 expression and apoptosis induction seem to be the mechanisms by which it promotes its anti-leukemic effects, making this new molecular architecture a promising prototype for leukemia intervention.

  6. Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

    PubMed Central

    Geng, Deyu; Zhang, Zhixia; Guo, Huarong

    2012-01-01

    p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl) phthalate (DEHP), a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner. PMID:25585933

  7. Role of PI3K/AKT/mTOR signaling in the cell cycle progression of human prostate cancer.

    PubMed

    Gao, Ning; Zhang, Zhuo; Jiang, Bing-Hua; Shi, Xianglin

    2003-10-31

    Prostate cancer is one of the most common cancers among men. Recent studies demonstrated that PI3K signaling is an important intracellular mediator which is involved in multiple cellular functions including proliferation, differentiation, anti-apoptosis, tumorigenesis, and angiogenesis. In the present study, we demonstrate that the inhibition of PI3K activity by LY294002, inhibited prostate cancer cell proliferation and induced the G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins including cyclin D1, CDK4, and Rb phosphorylation at Ser780, Ser795, and Ser807/811, whereas expression of CDK6 and beta-actin was not affected by LY294002. The expression of cyclin kinase inhibitor, p21(CIP1/WAF1), was induced by LY294002, while levels of p16(INK4) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation and p70(S6K), but not MAPK. PI3K regulates cell cycle through AKT, mTOR to p70(S6K). The mTOR inhibitor rapamycin has similar inhibitory effects on G(1) cell cycle progression and expression of cyclin D1, CDK4, and Rb phosphorylation. These results suggest that PI3K mediates G(1) cell cycle progression and cyclin expression through the activation of AKT/mTOR/p70(S6K) signaling pathway in the prostate cancer cells.

  8. PRMT7 Preserves Satellite Cell Regenerative Capacity.

    PubMed

    Blanc, Roméo Sébastien; Vogel, Gillian; Chen, Taiping; Crist, Colin; Richard, Stéphane

    2016-02-16

    Regeneration of skeletal muscle requires the continued presence of quiescent muscle stem cells (satellite cells), which become activated in response to injury. Here, we report that whole-body protein arginine methyltransferase PRMT7(-/-) adult mice and mice conditionally lacking PRMT7 in satellite cells using Pax7-CreERT2 both display a significant reduction in satellite cell function, leading to defects in regenerative capacity upon muscle injury. We show that PRMT7 is preferentially expressed in activated satellite cells and, interestingly, PRMT7-deficient satellite cells undergo cell-cycle arrest and premature cellular senescence. These defects underlie poor satellite cell stem cell capacity to regenerate muscle and self-renew after injury. PRMT7-deficient satellite cells express elevated levels of the CDK inhibitor p21CIP1 and low levels of its repressor, DNMT3b. Restoration of DNMT3b in PRMT7-deficient cells rescues PRMT7-mediated senescence. Our findings define PRMT7 as a regulator of the DNMT3b/p21 axis required to maintain muscle stem cell regenerative capacity.

  9. p18(Hamlet) mediates different p53-dependent responses to DNA-damage inducing agents.

    PubMed

    Lafarga, Vanesa; Cuadrado, Ana; Nebreda, Angel R

    2007-10-01

    Cells organize appropriate responses to environmental cues by activating specific signaling networks. Two proteins that play key roles in coordinating stress responses are the kinase p38alpha (MAPK14) and the transcription factor p53 (TP53). Depending on the nature and the extent of the stress-induced damage, cells may respond by arresting the cell cycle or by undergoing cell death, and these responses are usually associated with the phosphorylation of particular substrates by p38alpha as well as the activation of specific target genes by p53. We recently characterized a new p38alpha substrate, named p18(Hamlet) (ZNHIT1), which mediates p53-dependent responses to different genotoxic stresses. Thus, cisplatin or UV light induce stabilization of the p18(Hamlet) protein, which then enhances the ability of p53 to bind to and activate the promoters of pro-apoptotic genes such as NOXA and PUMA leading to apoptosis induction. In a similar way, we report here that p18(Hamlet) can also mediate the cell cycle arrest induced in response to gamma-irradiation, by participating in the p53-dependent upregulation of the cell cycle inhibitor p21(Cip1) (CDKN1A).

  10. Viscolin Inhibits In Vitro Smooth Muscle Cell Proliferation and Migration and Neointimal Hyperplasia In Vivo

    PubMed Central

    Chen, Chin-Chuan; Liang, Chan-Jung; Leu, Yann-Lii; Chen, Yuh-Lien; Wang, Shu-Huei

    2016-01-01

    Viscolin, an extract of Viscum coloratum, has anti-inflammatory and anti-proliferative properties against harmful stimuli. The aim of the study was to examine the anti-proliferative effects of viscolin on platelet derived growth factor-BB (PDGF)-treated human aortic smooth muscle cells (HASMCs) and identify the underlying mechanism responsible for these effects. Viscolin reduced the PDGF-BB-induced HASMC proliferation and migration in vitro; it also arrested HASMCs in the G0/G1 phase by decreasing the protein expression of Cyclin D1, CDK2, Cyclin E, CDK4, and p21Cip1 as detected by Western blot analysis. These effects may be mediated by reduced PDGF-induced phosphorylation of ERK1/2, JNK, and P38, but not AKT as well as inhibition of PDGF-mediated nuclear factor (NF)-κB p65 and activator protein 1 (AP-1)/c-fos activation. Furthermore, viscolin pre-treatment significantly reduced neointimal hyperplasia of an endothelial-denuded femoral artery in vivo. Taken together, viscolin attenuated PDGF–BB-induced HASMC proliferation in vitro and reduced neointimal hyperplasia in vivo. Thus, viscolin may represent a therapeutic candidate for the prevention and treatment of vascular proliferative diseases. PMID:27977759

  11. Molecular control of brain size: Regulators of neural stem cell life, death and beyond

    SciTech Connect

    Joseph, Bertrand; Hermanson, Ola

    2010-05-01

    The proper development of the brain and other organs depends on multiple parameters, including strictly controlled expansion of specific progenitor pools. The regulation of such expansion events includes enzymatic activities that govern the correct number of specific cells to be generated via an orchestrated control of cell proliferation, cell cycle exit, differentiation, cell death etc. Certain proteins in turn exert direct control of these enzymatic activities and thus progenitor pool expansion and organ size. The members of the Cip/Kip family (p21Cip1/p27Kip1/p57Kip2) are well-known regulators of cell cycle exit that interact with and inhibit the activity of cyclin-CDK complexes, whereas members of the p53/p63/p73 family are traditionally associated with regulation of cell death. It has however become clear that the roles for these proteins are not as clear-cut as initially thought. In this review, we discuss the roles for proteins of the Cip/Kip and p53/p63/p73 families in the regulation of cell cycle control, differentiation, and death of neural stem cells. We suggest that these proteins act as molecular interfaces, or 'pilots', to assure the correct assembly of protein complexes with enzymatic activities at the right place at the right time, thereby regulating essential decisions in multiple cellular events.

  12. Melanoma dormancy in a mouse model is linked to GILZ/FOXO3A-dependent quiescence of disseminated stem-like cells

    PubMed Central

    Touil, Yasmine; Segard, Pascaline; Ostyn, Pauline; Begard, Severine; Aspord, Caroline; El Machhour, Raja; Masselot, Bernadette; Vandomme, Jerome; Flamenco, Pilar; Idziorek, Thierry; Figeac, Martin; Formstecher, Pierre; Quesnel, Bruno; Polakowska, Renata

    2016-01-01

    Metastatic cancer relapses following the reactivation of dormant, disseminated tumour cells; however, the cells and factors involved in this reactivation are just beginning to be identified. Using an immunotherapy-based syngeneic model of melanoma dormancy and GFP-labelled dormant cell-derived cell lines, we determined that vaccination against melanoma prevented tumour growth but did not prevent tumour cell dissemination or eliminate all tumour cells. The persistent disseminated melanoma tumour cells were quiescent and asymptomatic for one year. The quiescence/activation of these cells in vitro and the dormancy of melanoma in vivo appeared to be regulated by glucocorticoid-induced leucine zipper (GILZ)-mediated immunosuppression. GILZ expression was low in dormant cell-derived cultures, and re-expression of GILZ inactivated FOXO3A and its downstream target, p21CIP1. The ability of dormancy-competent cells to re-enter the cell cycle increased after a second round of cellular dormancy in vivo in association with shortened tumour dormancy period and faster and more aggressive melanoma relapse. Our data indicate that future cancer treatments should be adjusted according to the stage of disease progression. PMID:27465291

  13. Rho-associated kinase (ROCK) inhibition reverses low cell activity on hydrophobic surfaces.

    PubMed

    Tian, Yu Shun; Kim, Hyun Jung; Kim, Hyun-Man

    2009-08-28

    Hydrophobic polymers do not offer an adequate scaffold surface for cells to attach, migrate, proliferate, and differentiate. Thus, hydrophobic scaffolds for tissue engineering have traditionally been physicochemically modified to enhance cellular activity. However, modifying the surface by chemical or physical treatment requires supplementary engineering procedures. In the present study, regulation of a cell signal transduction pathway reversed the low cellular activity on a hydrophobic surface without surface modification. Inhibition of Rho-associated kinase (ROCK) by Y-27632 markedly enhanced adhesion, migration, and proliferation of osteoblastic cells cultured on a hydrophobic polystyrene surface. ROCK inhibition regulated cell-cycle-related molecules on the hydrophobic surface. This inhibition also decreased expression of the inhibitors of cyclin-dependent kinases such as p21(cip1) and p27(kip1) and increased expression of cyclin A and D. These results indicate that defective cellular activity on the hydrophobic surface can be reversed by the control of a cell signal transduction pathway without physicochemical surface modification.

  14. The Rho GTPase effector ROCK regulates cyclin A, cyclin D1, and p27Kip1 levels by distinct mechanisms.

    PubMed

    Croft, Daniel R; Olson, Michael F

    2006-06-01

    The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by studies utilizing ROCK-selective inhibitors such as Y-27632. However, the mechanisms by which ROCK signaling promotes cell cycle progression have not been thoroughly characterized. Using a conditionally activated ROCK-estrogen receptor fusion protein, we found that ROCK activation is sufficient to stimulate G1/S cell cycle progression in NIH 3T3 mouse fibroblasts. Further analysis revealed that ROCK acts via independent pathways to alter the levels of cell cycle regulatory proteins: cyclin D1 and p21(Cip1) elevation via Ras and the mitogen-activated protein kinase pathway, increased cyclin A via LIM kinase 2, and reduction of p27(Kip1) protein levels. Therefore, the influence of ROCK on cell cycle regulatory proteins occurs by multiple independent mechanisms.

  15. Identifying candidate agents for lung adenocarcinoma by walking the human interactome

    PubMed Central

    Sun, Yajiao; Zhang, Ranran; Jiang, Zhe; Xia, Rongyao; Zhang, Jingwen; Liu, Jing; Chen, Fuhui

    2016-01-01

    Despite recent advances in therapeutic strategies for lung cancer, mortality is still increasing. Therefore, there is an urgent need to identify effective novel drugs. In the present study, we implement drug repositioning for lung adenocarcinoma (LUAD) by a bioinformatics method followed by experimental validation. We first identified differentially expressed genes between LUAD tissues and nontumor tissues from RNA sequencing data obtained from The Cancer Genome Atlas database. Then, candidate small molecular drugs were ranked according to the effect of their targets on differentially expressed genes of LUAD by a random walk with restart algorithm in protein–protein interaction networks. Our method identified some potentially novel agents for LUAD besides those that had been previously reported (eg, hesperidin). Finally, we experimentally verified that atracurium, one of the potential agents, could induce A549 cells death in non-small-cell lung cancer-derived A549 cells by an MTT assay, acridine orange and ethidium bromide staining, and electron microscopy. Furthermore, Western blot assays demonstrated that atracurium upregulated the proapoptotic Bad and Bax proteins, downregulated the antiapoptotic p-Bad and Bcl-2 proteins, and enhanced caspase-3 activity. It could also reduce the expression of p53 and p21Cip1/Waf1 in A549 cells. In brief, the candidate agents identified by our approach may provide greater insights into improving the therapeutic status of LUAD. PMID:27729798

  16. Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling

    PubMed Central

    Bivik, Caroline; MacDonald, Ryan B.; Gunnar, Erika; Mazouni, Khalil; Schweisguth, Francois; Thor, Stefan

    2016-01-01

    The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems. PMID:27070787

  17. CBP loss cooperates with PTEN haploinsufficiency to drive prostate cancer: implications for epigenetic therapy

    PubMed Central

    Ding, Liya; Chen, Shuai; Liu, Ping; Pan, Yunqian; Zhong, Jian; Regan, Kevin M.; Wang, Liguo; Yu, Chunrong; Rizzardi, Tony; Cheng, Liang; Zhang, Jun; Schmechel, Stephen C.; Cheville, John C.; van Deursen, Jan; Tindall, Donald J.; Huang, Haojie

    2014-01-01

    Despite the high incidence and mortality of prostate cancer, the etiology of this disease is not fully understood. In this study, we develop functional evidence for CBP and PTEN interaction in prostate cancer based on findings of their correlate expression in the human disease. Cbppc−/−;Ptenpc+/− mice exhibited higher cell proliferation in the prostate and an early onset of high-grade prostatic intraepithelial neoplasia. Levels of EZH2 methyltransferase were increased along with its Thr350 phosphorylation in both mouse Cbp−/−;Pten+/− and human prostate cancer cells. CBP loss and PTEN deficiency cooperated to trigger a switch from K27-acetylated histone H3 to K27-trimethylated bulk histones, in a manner associated with decreased expression of the growth inhibitory EZH2 target genes DAB2IP, p27KIP1 and p21CIP1. Conversely, treatment with the histone deacetylase inhibitor panobinostat reversed this switch, in a manner associated with tumor suppression in Cbppc−/−;Ptenpc+/− mice. Our findings show how CBP and PTEN interact to mediate tumor suppression in the prostate, establishing a central role for histone modification in the etiology of prostate cancer and providing a rationale for clinical evaluation of epigenetic targeted therapy in prostate cancer patients. PMID:24491799

  18. Stat3-dependent acute Rantes production in vascular smooth muscle cells modulates inflammation following arterial injury in mice

    PubMed Central

    Kovacic, Jason C.; Gupta, Rohit; Lee, Angela C.; Ma, Mingchao; Fang, Fang; Tolbert, Claire N.; Walts, Avram D.; Beltran, Leilani E.; San, Hong; Chen, Guibin; St. Hilaire, Cynthia; Boehm, Manfred

    2009-01-01

    Inflammation is a key component of arterial injury, with VSMC proliferation and neointimal formation serving as the final outcomes of this process. However, the acute events transpiring immediately after arterial injury that establish the blueprint for this inflammatory program are largely unknown. We therefore studied these events in mice and found that immediately following arterial injury, medial VSMCs upregulated Rantes in an acute manner dependent on Stat3 and NF-κB (p65 subunit). This led to early T cell and macrophage recruitment, processes also under the regulation of the cyclin-dependent kinase inhibitor p21Cip1. Unique to VSMCs, Rantes production was initiated by Tnf-α, but not by Il-6/gp130. This Rantes production was dependent on the binding of a p65/Stat3 complex to NF-κB–binding sites within the Rantes promoter, with shRNA knockdown of either Stat3 or p65 markedly attenuating Rantes production. In vivo, acute NF-κB and Stat3 activation in medial VSMCs was identified, with acute Rantes production after injury substantially reduced in Tnfa–/– mice compared with controls. Finally, we generated mice with SMC-specific conditional Stat3 deficiency and confirmed the Stat3 dependence of acute Rantes production by VSMCs. Together, these observations unify inflammatory events after vascular injury, demonstrating that VSMCs orchestrate the arterial inflammatory response program via acute Rantes production and subsequent inflammatory cell recruitment. PMID:20038813

  19. Cytotoxic Effect of Ethanol Extract of Microalga, Chaetoceros calcitrans, and Its Mechanisms in Inducing Apoptosis in Human Breast Cancer Cell Line

    PubMed Central

    Ebrahimi Nigjeh, Siyamak; Yusoff, Fatimah Md; Mohamed Alitheen, Noorjahan Banu; Rasoli, Mehdi; Keong, Yeap Swee; Omar, Abdul Rahman bin

    2013-01-01

    Marine microalgae have been prominently featured in cancer research. Here, we examined cytotoxic effect and apoptosis mechanism of crude ethanol extracts of an indigenous microalga, Chaetoceros calcitrans (UPMAAHU10) on human breast cell lines. MCF-7 was more sensitive than MCF-10A with IC50 value of 3.00 ± 0.65, whilst the IC50 value of Tamoxifen against MCF-7 was 12.00 ± 0.52 μg/mL after 24 hour incubation. Based on Annexin V/Propidium iodide and cell cycle flow cytometry analysis, it was found that inhibition of cell growth by EEC on MCF-7 cells was through the induction of apoptosis without cell cycle arrest. The apoptotic cells at subG0/G1 phase in treated MCF-7 cells at 48 and 72 hours showed 34 and 16 folds increased compared to extract treated MCF-10A cells which showed only 6 and 7 folds increased at the same time points, respectively. Based on GeXP study, EEC induced apoptosis on MCF-7 cells via modulation of CDK2, MDM2, p21Cip1, Cyclin A2, Bax and Bcl-2. The EEC treated MCF-7 cells also showed an increase in Bax/Bcl-2 ratio that in turn activated the caspase-dependent pathways by activating caspase 7. Thus, marine microalga, Chaetoceros calcitrans may be considered a good candidate to be developed as a new anti-breast cancer drug. PMID:23509778