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Sample records for p15ink4b p16ink4a p21cip1

  1. Gene Expression Status and Methylation Pattern in Promoter of P15INK4b and P16INK4a in Cord Blood CD34+ Stem Cells

    PubMed Central

    Azad, Mehdi; Kaviani, Saeid; Noruzinia, Mehrdad; Mortazavi, Yousef; Mobarra, Naser; Alizadeh, Shaban; Shahjahani, Mohammad; Skandari, Fatemeh; Ahmadi, Mohammad Hosein; Atashi, Amir; Abroun, Saeid; Zonoubi, Zahra

    2013-01-01

    Objective(s) : Stem cell differentiation into different cell lineages depends upon several factors, cell cycle control elements and intracellular signaling elements, including P15INK4b and P16INK4a genes. Epigenetics may be regarded as a control mechanism which is affected by these factors with respect to their promoter structure. Materials and Methods : The CD34 + cord blood stem cells were purified, isolated and then expanded. The undifferentiated day genome was isolated from part of the cultured cells, and the seventh day differentiated genome was isolated from the other part after differentiation to erythroid lineage. The procedure was followed by a separate Real-Time PCR for the two genes using the obtained cDNA. The processed DNA of the former stages was used for MSP (Methylation Specific PCR) reaction. Finally, pre- and post differentiation results were compared.  Results : After performing MSP for each gene, it became clear that P15INK4b gene has undergone methylation and expression in predifferentiation stage. In addition, its status has not been changed after differentiation. P15INK4b gene expression was reduced after the differentiation. The other gene, P16INK4a, showed no predifferentiation methylation. Itwas completely expressed methylated and underwent reduced expression after differentiation. Conclusion : Specific predifferentiation expression of P15INK4b and P16INK4a genes along with reduction in their expression after erythroid differentiation indicated animportant role for these two genes in biology of CD34+ cells in primary stages and before differentiation. In addition, both genes are capable of epigenetic modifications due to the structure of their promoters. PMID:23997911

  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. Mechanisms of MEOX1 and MEOX2 Regulation of the Cyclin Dependent Kinase Inhibitors p21CIP1/WAF1 and p16INK4a in Vascular Endothelial Cells

    PubMed Central

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

    2011-01-01

    Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21CIP1/WAF1 and p16INK4a govern the G1/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21CIP1/WAF1 and p16INK4a. MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21CIP1/WAF1 and p16INK4a expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21CIP1/WAF1 and p16INK4a. In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16INK4a in a DNA binding dependent manner, whereas they induce p21CIP1/WAF1 in a DNA binding independent manner. PMID:22206000

  4. Transforming growth factor beta stabilizes p15INK4B protein, increases p15INK4B-cdk4 complexes, and inhibits cyclin D1-cdk4 association in human mammary epithelial cells.

    PubMed Central

    Sandhu, C; Garbe, J; Bhattacharya, N; Daksis, J; Pan, C H; Yaswen, P; Koh, J; Slingerland, J M; Stampfer, M R

    1997-01-01

    The effects of transforming growth factor beta (TGF-beta) were studied in closely related human mammary epithelial cells (HMEC), both finite-life-span 184 cells and immortal derivatives, 184A1S, and 184A1L5R, which differ in their cell cycle responses to TGF-beta but express type I and type II TGF-beta receptors and retain TGF-beta induction of extracellular matrix. The arrest-resistant phenotype was not due to loss of cyclin-dependent kinase (cdk) inhibitors. TGF-beta was shown to regulate p15INK4B expression at at least two levels: mRNA accumulation and protein stability. In TGF-beta-arrested HMEC, there was not only an increase in p15 mRNA but also a major increase in p5INK4B protein stability. As cdk4- and cdk6-associated p15INK4B increased during TGF-beta arrest of sensitive cells, there was a loss of cyclin D1, p21Cip1, and p27Kip1 from these kinase complexes, and cyclin E-cdk2-associated p27Kip1 increased. In HMEC, p15INK4B complexes did not contain detectable cyclin. p15INK4B from both sensitive and resistant cells could displace in vitro cyclin D1, p21Cip1, and p27Kip1 from cdk4 isolated from sensitive cells. Cyclin D1 could not be displaced from cdk4 in the resistant 184A1L5R cell lysates. Thus, in TGF-beta arrest, p15INK4B may displace already associated cyclin D1 from cdks and prevent new cyclin D1-cdk complexes from forming. Furthermore, p27Kip1 binding shifts from cdk4 to cyclin E-cdk2 during TGF-beta-mediated arrest. The importance of posttranslational regulation of p15INK4B by TGF-beta is underlined by the observation that in TGF-beta-resistant 184A1L5R, although the p15 transcript increased, p15INK4B protein was not stabilized and did not accumulate, and cyclin D1-cdk association and kinase activation were not inhibited. PMID:9111314

  5. Overexpression of p16(INK4a) in Mastocytosis (Urticarial Pigmentosa).

    PubMed

    Tsujita, Jun; Doi, Kazuko; Nakahara, Makiko; Nakahara, Takeshi; Kaku, Yumiko; Nishio, Kiichiro; Kan, Nagisa; Sato, Yuki; Nagata, Shoko; Nakao, Asako; Yoshida, Maiko; Uchi, Hiroshi; Furue, Masutaka

    2016-01-01

    The expression of p16(INK4a) has been reported to induce cell-cycle arrest and cellular senescence. The p16(INK4a) expression has never been examined in human mast cells and mastocytosis. We immunohistologically examined the expression of p16(INK4a) and tryptase in 5 normal human skin and 4 mastocytosis. In normal mast cells, only 5.9 ± 3.4 (mean ± standard deviation) % of tryptase-positive mast cells coexpressed p16(INK4a). However, significantly higher percentage (86.0 ± 14.1%) of tryptase-positive tumor cells was immunoreactive to p16(INK4a) in all of 4 mastocytosis. The p16(INK4a) overexpression may induce the senescence of neoplastic mast cells to undergo spontaneous regression of mastocytosis. PMID:27333655

  6. The Molecular Balancing Act of p16INK4a in Cancer and Aging

    PubMed Central

    LaPak, Kyle M.; Burd, Christin E.

    2013-01-01

    Located on chromosome 9p21.3, p16INK4a seems lost amongst a cluster of neighboring tumor suppressor genes. While best known for inhibiting cyclin dependent kinase (CDK) activity, p16INK4a is not a one trick pony. Long term p16INK4a expression pushes cells to enter senescence, an irreversible cell cycle arrest that prevents the growth of would-be cancer cells, but also contributes to aging. Loss of p16INK4a is one of the most frequent events in human tumors and allows pre-cancerous lesions to bypass senescence. Therefore, precise regulation of p16INK4a is essential to tissue homeostasis, maintaining a tight balance between tumor suppression and aging. Here, we outline the pathways required for proper p16INK4a regulation and highlight the critical functions of p16INK4a in cancer, aging and human physiology that make this gene special. PMID:24136988

  7. Evaluation of cervical cone biopsies for coexpression of p16INK4a and Ki-67 in epithelial cells.

    PubMed

    Reuschenbach, Miriam; Seiz, Mirjam; von Knebel Doeberitz, Christina; Vinokurova, Svetlana; Duwe, Alexander; Ridder, Ruediger; Sartor, Heike; Kommoss, Friedrich; Schmidt, Dietmar; von Knebel Doeberitz, Magnus

    2012-01-15

    Diffuse overexpression of p16(INK4a) in basal and parabasal cells of cervical epithelium is a hallmark of human papillomavirus-mediated transformation. Focal p16(INK4a) expression is occasionally observed in nondysplastic epithelium. In normal cells, expression of p16(INK4a) triggers cell cycle arrest. However, cells undergoing transformation in intraepithelial lesions actively proliferate. To prove that the different expression patterns of p16(INK4a) , i.e., focal versus diffuse, reflect biologically different entities, we hypothesized that p16(INK4a) -positive cells in epithelia displaying focal p16(INK4a) expression pattern do not coexpress proliferation-associated Ki-67 protein, while p16(INK4a) -positive cells in lesions with diffuse p16(INK4a) expression may do. A total of 138 cervical cone biopsies were stained for the expression of p16(INK4a) and Ki-67 using a primary antibody cocktail. All metaplastic lesions (n = 21) displayed focal staining for p16(INK4a) , and in all of these lesions p16(INK4a) -positive cells were found to be negative for Ki-67 expression. Diffuse expression of p16(INK4a) was observed in 12/21 (57.1%) cervical intraepithelial neoplasia (CIN) 1 lesions, all of them simultaneously showed Ki-67 immunoreactivity in a large proportion of p16(INK4a) -positive cells. Seventeen of 23 (73.9%) CIN2 lesions and all 27 (100%) CIN3/carcinoma in situ (CIS) as well as all 46 (100%) carcinoma cases displayed diffuse and combined expression of p16(INK4a) and Ki-67. Coexpression of Ki-67 and p16(INK4a) in the same cell is entirely restricted to cervical lesions displaying diffuse p16(INK4a) expression, whereas in lesions with focal p16(INK4a) expression, p16(INK4a) -expressing cells are negative for Ki-67. Thus, diffuse expression of p16(INK4a) reflects lesions with proliferation-competent cells, while p16(INK4a) -expressing cells associated with focal expression patterns are cell cycle arrested.

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

    PubMed

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

    2015-04-01

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

  9. 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

  10. p16INK4a reporter mice reveal age-promoting effects of environmental toxicants

    PubMed Central

    Sorrentino, Jessica A.; Krishnamurthy, Janakiraman; Tilley, Stephen; Alb, James G.; Burd, Christin E.; Sharpless, Norman E.

    2013-01-01

    While murine-based systems to identify cancer-promoting agents (carcinogens) are established, models to identify compounds that promote aging (gerontogens) have not been described. For this purpose, we exploited the transcription of p16INK4a, which rises dynamically with aging and correlates with age-associated disease. Activation of p16INK4a was visualized in vivo using a murine strain that harbors a knockin of the luciferase gene into the Cdkn2a locus (p16LUC mice). We exposed p16LUC mice to candidate gerontogens, including arsenic, high-fat diet, UV light, and cigarette smoke and serially imaged animals to monitor senescence induction. We show that exposure to a high-fat diet did not accelerate p16INK4a expression, whereas arsenic modestly augmented, and cigarette smoke and UV light potently augmented, activation of p16INK4a-mediated senescence. This work provides a toxicological platform to study mammalian aging and suggests agents that directly damage DNA promote molecular aging. PMID:24334456

  11. 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

  12. Immunohistological Expression of p16INK4a is Commonly Present Both in Benign and Malignant Sweat Gland Neoplasias.

    PubMed

    Tsujita, Jun; Kaku, Yumiko; Ichiki, Toshio; Eto, Ayaka; Maemura, Hiromi; Otsuka, Akiko; Nakaie, Risa; Kitagawa, Noriko; Morioka, Yuka; Matsuda, Tomoyo; Yoshida, Maiko; Furue, Masutaka

    2015-12-01

    The expression of p16INK4a has been reported to be a significant marker for malignant transformation of epidermal tumors. However, little is known about sweat gland tumors. We examined the immunohistological expression of p16INK4a in benign and malignant sweat gland tumors. The ductal and acrosyringial portion of normal eccrine glands were positively stained with p16INK4a while it was negative in the normal epidermis. Moderate to strong expression of p16INK4a was found in 16 of 17 eccrine poromas, 4 of 5 hidradenomas, 3 of 3 syringocystadenoma papilliferums, 2 of 2 mixed tumors, and 3 of 3 syringomas. The p16INK4a expression was observed focally or diffusely in 4 of 4 porocarcinomas, 4 of 4 apocrine carcinomas and 12 of 17 extramammary Paget's diseases. We conclude that the p16INK4a expression is not a good marker for dictating malignant transformation of sweat gland tumors.

  13. Downregulation of HO-1 promoted apoptosis induced by decitabine via increasing p15INK4B promoter demethylation in myelodysplastic syndrome.

    PubMed

    Ma, D; Fang, Q; Wang, P; Gao, R; Sun, J; Li, Y; Hu, X Y; Wang, J S

    2015-04-01

    Decitabine, which reverses hypermethylation of the p15(INK4B) gene in vitro, has been used to relieve cytopenias and blast excess in over 50% of patients with high-risk myelodysplastic syndrome (MDS). In this study, heme oxygenase-1 (HO-1) was overexpressed in MDS cell line SKM-1, which was closely related to resistance to decitabine-induced apoptosis. We aimed to further investigate the role of HO-1 in apoptosis induced by low-dose decitabine in SKM-1 cells. Upregulation of HO-1 by transfecting it into SKM-1 cells with lentivirus vector promoted cell proliferation and protected them against apoptosis. In contrast, downregulation of HO-1 enhanced decitabine-induced apoptosis but reduced accumulation of the S phase in cell cycle. To explore the mechanism, the expressions of cell cycle-related proteins were detected after the cells were treated by decitabine in each group. p15(INK4B) and CDK4 were overexpressed in SKM-1 cells in which HO-1 was inhibited, and the expression-depending apoptosis was related to the caspase-3 pathway. Even though HO-1 was silenced, the apoptotic rate never increased as the caspase-3 pathway was blocked. It is well known that p15(INK4B) dominantly regulates the S phase of the cell cycle. p15(INK4B) was herein demethylated more evidently in the group of SKM-1 cells in which HO-1 was downregulated, as well as in the mononuclear cells of patients suffering from MDS. In the case of poor prognosis, the mRNA level of HO-1 was raised. In conclusion, overexpression of HO-1 indicated resistance to demethylation of p15(INK4B) induced by decitabine.

  14. Association between P16INK4a Promoter Methylation and Ovarian Cancer: A Meta-Analysis of 12 Published Studies

    PubMed Central

    Niu, Xun; Shi, Hao; Zhong, Yi

    2016-01-01

    Background Ovarian cancer is the primary cause of death in women diagnosed with gynecological malignancies worldwide. Absence of early symptoms prevents prompt diagnosis or successful therapeutic intervention. P16INK4a is a well-known tumor suppressor gene (TSG). Aberrant methylation of TSG promoter is an important epigenetic silencing mechanism leading to ovarian cancer progression. Studies have reported differences in methylation frequencies of the p16INK4a promoter between ovarian cancer and the corresponding control group. However, the association between p16INK4a promoter methylation and ovarian cancer remains unclear and controversial. Therefore, a meta-analysis was conducted to clarify the relationship between p16INK4a promoter methylation and ovarian cancer. Methods PubMed, Web of Science, EMBASE and CNKI were searched to identify eligible studies for the evaluation of the association between p16INK4a promoter methylation and ovarian cancer. Odds ratio (ORs) and 95% confidence intervals (95%CI) were calculated to determine the strength of association between p16INK4a promoter methylation and ovarian cancer. Results A total of 612 ovarian cancer patients and 289 controls from 12 eligible studies were included in the meta-analysis. Overall, a significant association was observed between p16INK4a methylation status and ovarian cancer risk using a fixed-effects model (OR = 2.02, 95% CI = 1.39–2.94). Conclusion The results of our meta-analysis show that aberrant methylation of p16INK4a promoter was significantly associated with ovarian cancer. It may represent a promising molecular marker to monitor the disease and provides new insights into the treatment of human ovarian cancer. PMID:27648827

  15. Immunohistological Expression of p16INK4a is Commonly Present Both in Benign and Malignant Sweat Gland Neoplasias.

    PubMed

    Tsujita, Jun; Kaku, Yumiko; Ichiki, Toshio; Eto, Ayaka; Maemura, Hiromi; Otsuka, Akiko; Nakaie, Risa; Kitagawa, Noriko; Morioka, Yuka; Matsuda, Tomoyo; Yoshida, Maiko; Furue, Masutaka

    2015-12-01

    The expression of p16INK4a has been reported to be a significant marker for malignant transformation of epidermal tumors. However, little is known about sweat gland tumors. We examined the immunohistological expression of p16INK4a in benign and malignant sweat gland tumors. The ductal and acrosyringial portion of normal eccrine glands were positively stained with p16INK4a while it was negative in the normal epidermis. Moderate to strong expression of p16INK4a was found in 16 of 17 eccrine poromas, 4 of 5 hidradenomas, 3 of 3 syringocystadenoma papilliferums, 2 of 2 mixed tumors, and 3 of 3 syringomas. The p16INK4a expression was observed focally or diffusely in 4 of 4 porocarcinomas, 4 of 4 apocrine carcinomas and 12 of 17 extramammary Paget's diseases. We conclude that the p16INK4a expression is not a good marker for dictating malignant transformation of sweat gland tumors. PMID:27159948

  16. Examining how p16(INK4a) expression levels are linked to handgrip strength in the elderly.

    PubMed

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

    2016-01-01

    Although many studies have shown that p16(INK4a) is more highly expressed in the human body during senescence, studies on its relevance to handgrip strength among old adults, are relatively sparse. We enrolled 205 community-dwelling old adults aged 65 years and older without specific medical conditions. Handgrip strength of the dominant hand was measured. Low handgrip strength was defined as the lowest quartile of handgrip strength among the participants. RNA was extracted from peripheral white blood cells. Use quantitative polymerase chain reaction to estimate the p16(INK4a) mRNA expression level. The average handgrip strength was 25.22 ± 8.98 kg, and gender difference was observed. In the linear regression model, the p16(INK4a) mRNA expression level was significantly negatively associated with handgrip strength in men but not in women. The β coefficient, representing the change of handgrip strength for each increment in the p16(INK4a) mRNA expression level, was -0.208 (p = 0.024) among old men. The negative association remained after additional covariates adjustment. In the multiple logistic regression model among old men, the odds ratio (OR) of low handgrip strength was 1.246 (p = 0.032). In this study, we observed the p16(INK4a) mRNA expression level was negative associated with handgrip strength among community-dwelling old men.

  17. Examining how p16(INK4a) expression levels are linked to handgrip strength in the elderly.

    PubMed

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

    2016-01-01

    Although many studies have shown that p16(INK4a) is more highly expressed in the human body during senescence, studies on its relevance to handgrip strength among old adults, are relatively sparse. We enrolled 205 community-dwelling old adults aged 65 years and older without specific medical conditions. Handgrip strength of the dominant hand was measured. Low handgrip strength was defined as the lowest quartile of handgrip strength among the participants. RNA was extracted from peripheral white blood cells. Use quantitative polymerase chain reaction to estimate the p16(INK4a) mRNA expression level. The average handgrip strength was 25.22 ± 8.98 kg, and gender difference was observed. In the linear regression model, the p16(INK4a) mRNA expression level was significantly negatively associated with handgrip strength in men but not in women. The β coefficient, representing the change of handgrip strength for each increment in the p16(INK4a) mRNA expression level, was -0.208 (p = 0.024) among old men. The negative association remained after additional covariates adjustment. In the multiple logistic regression model among old men, the odds ratio (OR) of low handgrip strength was 1.246 (p = 0.032). In this study, we observed the p16(INK4a) mRNA expression level was negative associated with handgrip strength among community-dwelling old men. PMID:27549351

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

    PubMed Central

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

    2016-01-01

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

  19. 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

  20. p16INK4A and p14ARF Tumor Suppressor Pathways Are Deregulated in Malignant Rhabdoid Tumors

    PubMed Central

    Venneti, Sriram; Le, Paul; Martinez, Daniel; Eaton, Katherine W.; Shyam, Nikhil; Jordan-Sciutto, Kelly L.; Pawel, Bruce; Biegel, Jaclyn A.; Judkins, Alexander R.

    2011-01-01

    Malignant rhabdoid tumors (MRT) are aggressive tumors associated with mutations in the SMARCB1 gene. In experimental systems, the loss of SMARCB1 is hypothesized to alter p16INK4A pathways resulting in repression of tumor suppressors. To determine whether these pathways are deregulated in human MRT, we used immunohistochemistry on tissue microarrays to evaluate p16INK4A/E2F1/RB and p14ARF/MDM2/p53 pathways in 25 atypical teratoid/ rhabdoid tumors (AT/RT) and 11 non-CNS MRT. p16INK4A was negative or showed focal weak expression. The p16INK4A downstream targets CDK4/cyclin D1/ppRB were variably expressed at moderate to low levels; E2F1 was negative. Unexpectedly, p14ARF expression was seen in many cases, which correlated positively with p53 and inversely with MDM2 immunostaining in AT/RT. TP53 mutational analysis in 19/25 AT/RT and 8/11 non-CNS MRT cases showed point mutations in only 3 AT/RT cases, suggesting that p53 expression was driven mainly by p14ARF. Finally, nucleophosmin, a protein that stabilizes p53, was positive in the majority of cases and colocalized with p53. Together, these data suggest that in MRT there is deregulation of not only p16INK4A, but also the p14ARF pathway. These results provide insights into cell cycle deregulation in the pathogenesis of human MRT and may aid in the design and evaluation of potential therapies for these tumors. PMID:21666498

  1. 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

  2. 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. PMID:24307508

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

    PubMed

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

    2012-04-01

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

  4. UVA-induced epigenetic regulation of P16(INK4a) in human epidermal keratinocytes and skin tumor derived cells.

    PubMed

    Chen, I-Peng; Henning, Stefan; Faust, Alexandra; Boukamp, Petra; Volkmer, Beate; Greinert, Rüdiger

    2012-01-01

    UVA-radiation (315-400 nm) has been demonstrated to be capable of inducing DNA damage and is regarded as a carcinogen. While chromosomal aberrations found in UVA-irradiated cells and skin tumors provided evidence of the genetic involvement in UVA-carcinogenesis, its epigenetic participation is still illusive. We thus analysed the epigenetic patterns of 5 specific genes that are involved in stem cell fate (KLF4, NANOG), telomere maintenance (hTERT) and tumor suppression in cell cycle control (P16(INK4a), P21(WAFI/CIPI)) in chronically UVA-irradiated HaCaT human keratinocytes. A striking reduction of the permissive histone mark H3K4me3 has been detected in the promoter of P16(INK4a) (4-fold and 9-fold reduction for 10 and 15 weeks UVA-irradiated cells, respectively), which has often been found deregulated in skin cancers. This alteration in histone modification together with a severe promoter hypermethylation strongly impaired the transcription of P16(INK4a) (20-fold and 40-fold for 10 weeks and 15 weeks UVA-irradiation, respectively). Analysis of the skin tumor-derived cells revealed the same severe impairment of the P16(INK4a) transcription attributed to promoter hypermethylation and enrichment of the heterochromatin histone mark H3K9me3 and the repressive mark H3K27me3. Less pronounced UVA-induced epigenetic alterations were also detected for the other genes, demonstrating for the first time that UVA is able to modify transcription of skin cancer associated genes by means of epigenetic DNA and histone alterations. PMID:21986889

  5. 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

  6. A comprehensive evaluation of human papillomavirus positive status and p16INK4a overexpression as a prognostic biomarker in head and neck squamous cell carcinoma.

    PubMed

    Deng, Zeyi; Hasegawa, Masahiro; Aoki, Kazuo; Matayoshi, Sen; Kiyuna, Asanori; Yamashita, Yukashi; Uehara, Takayuki; Agena, Shinya; Maeda, Hiroyuki; Xie, Minqiang; Suzuki, Mikio

    2014-07-01

    Head and neck squamous cell carcinoma (HNSCC) patients with human papillomavirus (HPV) infection have better prognosis than those without HPV infection. Although p16(INK4a) expression is used as a surrogate marker for HPV infection, there is controversy as to whether p16(INK4a) reliably indicates HPV infection. Here, to evaluate the accuracy of p16(INK4a) expression for determining HPV infection and the prognostic value of HPV infection and p16(INK4a) expression for HNSCC survival, especially oropharyngeal squamous cell carcinoma (OPSCC) survival, 150 fresh-frozen HNSCC samples were analyzed for HPV DNA, E6/E7 mRNA and p16(INK4a) expression by polymerase chain reaction and immunohistochemistry. p16(INK4a) expression was scored from 0 to 4 according to the percentage of p16(INK4a)-positive cells, with overexpression defined as >40% positive cells. Of the 150 tumor samples tested, 10 tumors were nasopharyngeal, 53 oropharyngeal, 39 hypopharyngeal, 24 laryngeal and 24 were located in the oral cavity. HPV DNA was detected in 47 (31.3%) samples, but only 21 also exhibited HPV mRNA expression. Inter-rater agreement was low between p16(INK4a) expression and HPV DNA presence and between p16(INK4a) expression and HPV mRNA expression, but was good between the combination of HPV DNA status and p16(INK4a) overexpression and HPV mRNA expression. Three-year recurrence-free survival was significantly higher for OPSCC patients who were HPV DNA-positive than for OPSCC patients who were HPV DNA-negative (P=0.008) and for OPSCC patients overexpressing p16(INK4a) than for without overexpressing p16(INK4a) (P=0.034). Multivariate analysis revealed that T1-3 stage and the combination of HPV DNA positivity and p16(INK4a) overexpression predicted significantly better recurrence-free survival. This combination is a more accurate marker for active HPV infection in HNSCC than HPV DNA status or general p16(INK4a)-positive status alone and offers a useful and reliable method for detecting and

  7. Inactivation of p16INK4a (inhibitor of cyclin-dependent kinase 4A) immortalizes primary human keratinocytes by maintaining cells in the stem cell compartment.

    PubMed

    Maurelli, Riccardo; Zambruno, Giovanna; Guerra, Liliana; Abbruzzese, Claudia; Dimri, Goberdhan; Gellini, Mara; Bondanza, Sergio; Dellambra, Elena

    2006-07-01

    Replicative senescence of human keratinocytes is determined by a progressive decline of clonogenic and dividing cells, and its timing is controlled by clonal evolution (i.e., the transition from stem cells to transient amplifying and postmitotic cells). Progressive increase of p16INK4a (inhibitor of cyclin-dependent kinase 4A) expression has been shown to correlate with keratinocyte clonal evolution. Thus, the aim of our study is to understand whether p16INK4a accumulation is a triggering mechanism of epidermal clonal evolution or a secondary event. We show that inactivation of p16INK4a, by an antisense strategy, allows primary human keratinocytes to escape replicative senescence. Specifically, p16INK4a inactivation alone blocks clonal evolution and maintains keratinocytes in the stem cell compartment. Antisense excision is followed by keratinocyte senescence, confirming that persistent p16INK4a inactivation is required for maintenance of clonal evolution block. Immortalization is accompanied by resumption of B-Cell Specific Moloney murine leukemia virus site 1 (Bmi-1) expression and telomerase activity, hallmarks of tissue regenerative capacity. In turn, Bmi-1 expression is necessary to maintain the impairment of clonal evolution induced by p16INK4a inactivation. Finally, p16INK4a down-regulation in transient amplifying keratinocytes does not affect clonal evolution, and cells undergo senescence. Thus, p16INK4a inactivation appears to selectively prevent clonal conversion in cells endowed with a high proliferative potential. These data indicate that p16INK4a regulates keratinocyte clonal evolution and that inactivation of p16INK4a in epidermal stem cells is necessary for maintaining stemness. PMID:16754749

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

    PubMed Central

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

    2011-01-01

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

  9. P16INK4a: a potential diagnostic adjunct for prediction of high-grade cervical lesions in liquid-based cytology: with HPV testing and histological correlation.

    PubMed

    Wong, Y P; Abdul Raub, S H; Mohd Dali, A Z H; Kassim, F; Visvalingam, V; Zakaria, Z; Kamaluddin, M A; Noor Akmal, S

    2016-08-01

    Human papillomavirus (HPV) is a necessary cause of cervical cancer and its precursors. Increased expression of high-risk hrHPV viral oncogenes in abnormal cells might increase the expression of p16INK4a. We aimed to determine the role of p16INK4a in detecting hrHPV-transformed epithelial cells in liquid-based cervical cytology, and compared the results with hrHPV DNA testing by realtime polymerase chain reaction (RT-PCR). Fifty-seven cytological samples were tested for p16INK4a immunomarker and hrHPV DNA. Test performance of both tests was determined by comparing sensitivity, specificity and predictive values using available histological follow-up data as gold standard. Of 57 samples, 36 (63.2%) showed immunoreactivity for p16INK4a and 43 (75.4%) were hrHPV-infected. A fairly low concordance rate (k = 0.504) between p16INK4a immunolabelling and hrHPV DNA status was noted. For prediction of cervical intraepithelial neoplasia (CIN) II and worse lesions, p16INK4a had a sensitivity and specificity of 93.5% and 60%; whereas hrHPV DNA testing had a sensitivity and specificity of 100% and 20%. Dual testing by combining p16INK4a and hrHPV showed sensitivity and specificity of 100% and 33.3%. In conclusion, p16INK4a is useful in predicting severity of the cytological abnormalities. Although p16INK4a is more specific but less sensitive than hrHPV in detecting high-grade cervical lesions, a combination of both tests failed to demonstrate significant improvement in diagnostic sensitivity, specificity and predictive value. Larger-scale prospective studies are required to assess further whether this biomarker should be routinely used as primary screening tool independently or in combination with hrHPV testing to improve diagnostic accuracy in cervical cytology. PMID:27568665

  10. P16INK4a: a potential diagnostic adjunct for prediction of high-grade cervical lesions in liquid-based cytology: with HPV testing and histological correlation.

    PubMed

    Wong, Y P; Abdul Raub, S H; Mohd Dali, A Z H; Kassim, F; Visvalingam, V; Zakaria, Z; Kamaluddin, M A; Noor Akmal, S

    2016-08-01

    Human papillomavirus (HPV) is a necessary cause of cervical cancer and its precursors. Increased expression of high-risk hrHPV viral oncogenes in abnormal cells might increase the expression of p16INK4a. We aimed to determine the role of p16INK4a in detecting hrHPV-transformed epithelial cells in liquid-based cervical cytology, and compared the results with hrHPV DNA testing by realtime polymerase chain reaction (RT-PCR). Fifty-seven cytological samples were tested for p16INK4a immunomarker and hrHPV DNA. Test performance of both tests was determined by comparing sensitivity, specificity and predictive values using available histological follow-up data as gold standard. Of 57 samples, 36 (63.2%) showed immunoreactivity for p16INK4a and 43 (75.4%) were hrHPV-infected. A fairly low concordance rate (k = 0.504) between p16INK4a immunolabelling and hrHPV DNA status was noted. For prediction of cervical intraepithelial neoplasia (CIN) II and worse lesions, p16INK4a had a sensitivity and specificity of 93.5% and 60%; whereas hrHPV DNA testing had a sensitivity and specificity of 100% and 20%. Dual testing by combining p16INK4a and hrHPV showed sensitivity and specificity of 100% and 33.3%. In conclusion, p16INK4a is useful in predicting severity of the cytological abnormalities. Although p16INK4a is more specific but less sensitive than hrHPV in detecting high-grade cervical lesions, a combination of both tests failed to demonstrate significant improvement in diagnostic sensitivity, specificity and predictive value. Larger-scale prospective studies are required to assess further whether this biomarker should be routinely used as primary screening tool independently or in combination with hrHPV testing to improve diagnostic accuracy in cervical cytology.

  11. 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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

    The INK4 family of cyclin-dependent kinase inhibitors (CKI) encode important cell cycle regulators that tightly control cell cycle during G1 to S phase. These related genes are considered tumor suppressors as loss of function contributes to the malignant phenotype. Expression of CKIs p16, p14ARF, or p15 were defective in six different canine mammary tumor (CMT) cell lines compared to normal thoracic canine fibroblasts. This suggests CKI defects are frequently responsible for neoplastic transformation in canine mammary carcinomas. p16 and p14ARF are two alternatively spliced products derived from the canine p16/INK4A/p14ARF gene locus. Despite omissions in the published p16 transcript and canine genome and the presence of GC-rich repeats, we determined the complete coding sequence of canine p16 revealing a deletion and frameshift mutation in p16 exon 1α in CMT28 cells. In addition, we determined canine p14ARF mRNA and protein sequences. Mapping of these mutations uncovered important aspects of p16 and p14ARF expression and defects in CMT28 cells shifting the p16 reading frame into p14ARF making a fusion protein that was predicted to be truncated, unstable and devoid of structural and functional integrity. This data describes an important neoplastic mechanism in the p16/INK4A/p14ARF locus in a spontaneous canine model of breast cancer. PMID:22833492

  14. Aurora B prevents delayed DNA replication and premature mitotic exit by repressing p21Cip1

    PubMed Central

    Trakala, Marianna; Fernández-Miranda, Gonzalo; Pérez de Castro, Ignacio; Heeschen, Christopher; Malumbres, Marcos

    2013-01-01

    Aurora kinase B is a critical component of the chromosomal passenger complex, which is involved in the regulation of microtubule-kinetochore attachments and cytokinesis. By using conditional knockout cells and chemical inhibition, we show here that inactivation of Aurora B results in delayed G1/S transition and premature mitotic exit. Aurora B deficiency results in delayed DNA replication in cultured fibroblasts as well as liver cells after hepatectomy. This is accompanied by increased transcription of the cell cycle inhibitor p21Cip1. Lack of Aurora B does not prevent mitotic entry but results in a premature exit from prometaphase in the presence of increased p21Cip1-Cdk1 inactive complexes. Aurora B-null cells display reduced degradation of cyclin B1, suggesting the presence of phenomenon known as adaptation to the mitotic checkpoint, previously described in yeast. Elimination of p21Cip1 rescues Cdk1 activity and prevents premature mitotic exit in Aurora B-deficient cells. These results suggest that Aurora B represses p21Cip1, preventing delayed DNA replication, Cdk inhibition and premature mitotic exit. The upregulation of p21Cip1 observed after inhibition of Aurora B may have important implications in cell cycle progression, tetraploidy, senescence or cancer therapy. PMID:23428904

  15. The lymphoma-associated NPM-ALK oncogene elicits a p16INK4a/pRb-dependent tumor-suppressive pathway.

    PubMed

    Martinelli, Paola; Bonetti, Paola; Sironi, Cristina; Pruneri, Giancarlo; Fumagalli, Caterina; Raviele, Paola Rafaniello; Volorio, Sara; Pileri, Stefano; Chiarle, Roberto; McDuff, Fiona Kate Elizabeth; Tusi, Betsabeh Khoramian; Turner, Suzanne D; Inghirami, Giorgio; Pelicci, Pier Giuseppe; Colombo, Emanuela

    2011-06-16

    Oncogene-induced senescence (OIS) is a barrier for tumor development. Oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and, accordingly, ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes, however, inhibit ARF/p53 and only infrequently select for ARF or p53 mutations, suggesting the involvement of other tumor-suppressive pathways. We report that NPM-ALK, the initiating oncogene of anaplastic large cell lymphomas (ALCLs), induces DNA damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is caused by activation of the p16INK4a/pRb tumor-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in premalignant lesions and decreased tumor latency in the absence of p16INK4a. Accordingly, human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a, which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence, and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumors.

  16. Nuclear co-expression of p14ARF and p16INK4A in uterine cervical cancer-derived cell lines containing HPV.

    PubMed

    Vázquez-Vega, Salvador; Sánchez-Suárez, Lilia Patricia; Contreras-Paredes, Adriana; Castellanos-Juárez, Emilio; Peñarroja-Flores, Rubicelia; Lizano-Soberón, Marcela; Andrade-Cruz, Rafael; García-Carrancá, Alejandro; Benítez-Bribiesca, Luis

    The Papanicolaou test (Pap) has been responsible for a significant reduction of cervical cancer-related morbimortality. In order to increase its sensitivity and specificity new markers have been studied and incorporated to cytological and histological methods for diagnosis for cervical cancer, such as p16INK4A that has been considered the immunocytochemical marker of choice for detection of HPV related cancers. We considered that p14ARF could be a complementary marker in order to improve the accuracy of cytological diagnosis because its genetic proximity to p16INK4A. We performed a systematic analysis of several putative cervical cancer markers in order to evaluate their performance in the detection of malignancy, in comparison with p16INK4A and p14ARF, using immunocytochemistry (ICC), immunofluorescence (IF) and Western blot analyses. Most markers were non-specific and could not discriminate HPV infected cancer cell lines from other non HPV malignant. In contrast, nuclear co-expression of p16INK4A and p14ARF was observed only in HPV-transformed cancer cell lines. Notably, in C-33A cervical cancer cells (HPV negative), p14ARF was present in the nucleoli, but p16INK4A was conspicuously absent from the nuclei of these cells. We conclude that both markers; p16INK4A and p14ARF are complementary and should be evaluated jointly in order to improve the accuracy of cytological diagnosis of cervical cancer.

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2015-06-15

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

  19. 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.

  20. Effect of Iodine Intake on p14ARF and p16INK4a Expression in Thyroid Papillary Carcinoma in Rats

    PubMed Central

    Sun, Ruimei; Wang, Jinde; Li, Xiaojiang; Li, Lei; Yang, Jie; Ren, Yanxin; Xi, Yan; Sun, Chuanzheng

    2015-01-01

    Background Iodine intake is related to thyroid disease. This study investigated the effect of the amount of iodine intake on p14ARF and p16INK4a expression of thyroid papillary carcinoma in rats. Material/Methods A cohort of 240 SD rats were randomly divided into control group, low iodine, normal iodine, and high iodine groups (n=60 per group). We inoculated 2×105 papillary thyroid carcinoma (PTC) cells on the left side of the thyroid gland. After 6 and 12 weeks, serum thyroid hormone level and urine iodine level were measured in addition to morphological observations of tumor tissues. Expression of p14ARF, p16INK4a was detected by immunohistochemical staining. Results The expression of p14ARF, p16INK4a, FT3, and FT4 levels in all iodine-treated animals were significantly lower than in the control group, while TSH level was significantly higher (P<0.05). Compared to the normal iodine group, the low and high groups had lower p14ARF and p16INK4a expression, lower FT3 and FT4 levels, higher TSH levels, and heavier tumors (P<0.05). In a further between-group comparison, p14ARF and p16INK4a expression and FT3 and FT4 levels at 12 weeks were lower than at 6 weeks. Expression of p14ARF and p16INK4a were positively correlated with FT3 and FT4, and negatively correlated with TSH and tumor weight. Conclusions Low and high iodine diet intake could reduce p14ARF and p16INK4a expressions and promote tumor development. PMID:26248224

  1. Elevated p16ink4a Expression in Human Labial Salivary Glands as a Potential Correlate of Cognitive Aging in Late Midlife

    PubMed Central

    Sørensen, Christiane Elisabeth; Tritsaris, Katerina; Reibel, Jesper; Lauritzen, Martin; Mortensen, Erik Lykke; Osler, Merete; Pedersen, Anne Marie Lynge

    2016-01-01

    Background The cell-cycle inhibitor and tumor suppressor cyclin dependent kinase inhibitor, p16ink4a, is one of the two gene products of the ink4a/ARF (cdkn2a) locus on chromosome 9q21. Up-regulation of p16ink4a has been linked to cellular senescence, and findings from studies on different mammalian tissues suggest that p16ink4a may be a biomarker of organismal versus chronological age. Objective The aim of this study was to examine the immunolocalization pattern of p16ink4a in human labial salivary gland (LSG) tissue, and to analyze whether its expression level in LSGs is a peripheral correlate of cognitive decline in late midlife. Methods The present study was a part of a study of causes and predictors of cognitive decline in middle-aged men in a Danish birth cohort. It is based on data from 181 male participants from the Danish Metropolit birth cohort, born in 1953, who were examined for age-associated alterations in cognition, dental health, and morphological and autonomic innervation characteristics of the LSGs. The participants were allocated to two groups based on the relative change in cognitive performance from young adulthood to late midlife. LSG biopsies were analyzed by qRT-PCR for the expression level of p16ink4a. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of LSGs. Results p16ink4a immunoreactivity was observed in LSG ductal, myoepithelial, and stromal cells, but not in acinar cells. The mean relative expression of p16ink4a in LSGs was higher in the group of participants with decline in cognitive performance. A logistic regression analysis revealed that the relative p16 expression was predictive of the participant’s group assignment. A negative correlation was found between relative p16ink4a expression and the participant’s standardized regression residuals from early adulthood to late midlife cognitive performance scores. Conclusions p16ink4a expression in human LSGs may constitute a potential peripheral

  2. CDK4/6 and IGF1 receptor inhibitors synergize to suppress the growth of p16INK4A-deficient pancreatic cancers

    PubMed Central

    Heilmann, Andreas M.; Perera, Rushika M.; Ecker, Veronika; Nicolay, Brandon N.; Bardeesy, Nabeel; Benes, Cyril H.; Dyson, Nicholas J.

    2014-01-01

    Loss-of-function mutations in p16INK4A (CDKN2A) occur in approximately 80% of sporadic pancreatic ductal adenocarcinoma (PDAC), contributing to its early progression. While this loss activates the cell cycle-dependent kinases CDK4/6, which have been considered as drug targets for many years, p16INK4A-deficient PDAC cells are inherently resistant to CDK4/6 inhibitors. This study searched for targeted therapies that might synergize with CDK4/6 inhibition in this setting. We report that the IGF1R/IR inhibitor BMS-754807 cooperated with the CDK4/6 inhibitor PD-0332991 to strongly block proliferation of p16INK4A-deficient PDAC cells in vitro and in vivo. Sensitivity to this drug combination correlated with reduced activity of the master cell growth regulator mTORC1. Accordingly, replacing the IGF1R/IR inhibitor with the rapalog inhibitor temsirolimus broadened the sensitivity of PDAC cells to CDK4/6 inhibition. Our results establish targeted therapy combinations with robust cytostatic activity in p16INK4A-deficient PDAC cells and possible implications for improving treatment of a broad spectrum of human cancers characterized by p16INK4A loss. PMID:24986516

  3. Contrasting behavior of the p18INK4c and p16INK4a tumor suppressors in both replicative and oncogene-induced senescence.

    PubMed

    Gagrica, Sladjana; Brookes, Sharon; Anderton, Emma; Rowe, Janice; Peters, Gordon

    2012-01-01

    The cyclin-dependent kinase (CDK) inhibitors, p18(INK4c) and p16(INK4a), both have the credentials of tumor suppressors in human cancers and mouse models. For p16(INK4a), the underlying rationale is its role in senescence, but the selective force for inactivation of p18(INK4c) in incipient cancer cells is less clear. Here, we show that in human fibroblasts undergoing replicative or oncogene-induced senescence, there is a marked decline in the levels of p18(INK4c) protein and RNA, which mirrors the accumulation of p16(INK4a). Downregulation of INK4c is not dependent on p16(INK4a), and RAS can promote the loss of INK4c without cell-cycle arrest. Downregulation of p18(INK4c) correlates with reduced expression of menin and E2F1 but is unaffected by acute cell-cycle arrest or inactivation of the retinoblastoma protein (pRb). Collectively, our data question the idea that p18(INK4c) acts as a backup for loss of p16(INK4a) and suggest that the apparent activation of p18(INK4c) in some settings represents delayed senescence rather than increased expression. We propose that the contrasting behavior of the two very similar INK4 proteins could reflect their respective roles in senescence versus differentiation.

  4. Human Papillomavirus Genotyping and p16INK4a Expression in Cervical Lesions: A Combined Test to Avoid Cervical Cancer Progression

    PubMed Central

    Zouheir, Yassine; Fechtali, Taoufiq; Elgnaoui, Nadia

    2016-01-01

    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 p16INK4a 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 p16INK4a 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 p16INK4a protein was observed in 72.9% (70/96) of the biopsies. In addition, p16INK4a protein detection was closely correlated with recovery of HR HPV. Our result showed that p16INK4a expression level is correlated with HR-HPV status. PMID:27390742

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2013-09-01

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

  7. 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

  8. T cell tolerance induced by histone deacetylase inhibitor is mediated by P21cip1.

    PubMed

    Gilbert, Kathleen M; Boger, Susan R; Price, Peter; Fifer, E Kim

    2005-01-01

    MEB [n-butyrate 2-(4-morpholinyl) ethyl butyrate hydrochloride], a histone deacetylase inhibitor and G1 blocker, has been shown to induce unresponsiveness in antigen-activated Th1 cells. MEB was tested for here for its ability to inactivate naive alloantigen-specific T cells from DBA/2 and C57BL/10 mice. Since T cells from these two strains of mice have been shown to differ in their cell cycle regulation, it we hoped that this comparison would provide information concerning the role of cycle regulatory proteins in mediating MEB-induced T cell unresponsiveness. MEB inhibited proliferation in a one-way mixed lymphocyte reaction (MLR) in which spleen cells from DBA/2 mice (H-2d) or C57BL/10 mice (H-2b) were stimulated with spleen cells from C57BL/10 or DBA/2 mice, respectively. C57BL/10 responder T cells isolated from the MEB-treated primary MLR remained unresponsive to alloantigen following restimulation in a secondary MLR that did not contain MEB. T cells from DBA/2 mice were less sensitive to MEB-induced unresponsiveness and required a longer exposure or pretreatment with IL-2 to become tolerant. In all cases responsiveness to MEB-induced tolerance in the alloantigen-stimulated T cells corresponded with the levels of the cyclin-dependent kinase inhibitor p21cip1. Additional experiments showed that T cells from p21cip1-deficient mice, unlike T cells from p21cip1 wild-type littermates, were resistant to MEB-induced tolerance. These results underscore the role of p21cip1 in mediating T cell tolerance induced by the histone deacetylase inhibitor MEB.

  9. 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

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

    PubMed

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

    2012-10-25

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

  11. p16INK4a immunocytochemistry versus HPV testing for triage of women with minor cytological abnormalities: A systematic review and meta-analysis

    PubMed Central

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

    2014-01-01

    Background The best method to identify women with minor cervical lesions that require diagnostic work-up remains unclear. We performed a meta-analysis to assess the accuracy of p16INK4a immunocytochemistry compared to hrHPV DNA testing with hybrid capture II (HC2) to detect cervical intraepithelial neoplasia (CIN2+ and CIN3+) in women with a cervical cytology showing atypical squamous cells of undetermined significance (ASC-US) or low-grade cervical lesions (LSIL). Methods A literature search was performed in three electronic databases to identify studies eligible for this meta-analysis. Results Seventeen studies were included in the meta-analysis. The pooled sensitivity of p16INK4a to detect CIN2+ was 83.2% (95%CI: 76.8–88.2%) and 83.8% (95%CI: 73.5–90.6%) in ASC-US and LSIL cervical cytology respectively; pooled specificities were 71.0% (95%CI: 65.0–76.4%) and 65.7% (95%CI: 54.2–75.6%). Eight studies provided both HC2 and p16INK4a triage data. p16INK4a and HC2 have a similar sensitivity and p16INK4a has significantly higher specificity in the triage of women with ASC-US (relative sensitivity: 0.95 (95%CI: 0.89–1.01); relative specificity: 1.82 (95%CI: 1.57–2.12)). In the triage of LSIL, p16INK4a has a significantly lower sensitivity but higher specificity compared to HC2 (relative sensitivity: 0.87 (95%CI: 0.81–0.94); relative specificity: 2.74 (1.99–3.76)). Conclusion The published literature indicates an improved accuracy of p16INK4a compared to HC2 testing in the triage of ASC-US. In LSIL triage p16INK4a is more specific but less sensitive. PMID:22700382

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

    PubMed

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

    2015-02-01

    The cyclin-dependent kinase (Cdk) inhibitor p16(Ink4a) (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

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

    PubMed Central

    Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adopted for a wide range of biological applications including genome editing. In some cases, dissection of genome functions requires allele-specific genome editing, but the use of CRISPR for this purpose has not been studied in detail. In this study, using the p16INK4a gene in HCT116 as a model locus, we investigated whether chromatin states, such as CpG methylation, or a single-nucleotide gap form in a target site can be exploited for allele-specific locus binding and genome editing by CRISPR in vivo. First, we showed that allele-specific locus binding and genome editing could be achieved by targeting allele-specific CpG-methylated regions, which was successful for one, but not all guide RNAs. In this regard, molecular basis underlying the success remains elusive at this stage. Next, we demonstrated that an allele-specific single-nucleotide gap form could be employed for allele-specific locus binding and genome editing by CRISPR, although it was important to avoid CRISPR tolerance of a single nucleotide mismatch brought about by mismatched base skipping. Our results provide information that might be useful for applications of CRISPR in studies of allele-specific functions in the genomes. PMID:27465215

  15. Extracellular Signal-Regulated Kinase 2-Dependent Phosphorylation Induces Cytoplasmic Localization and Degradation of p21Cip1▿ †

    PubMed Central

    Hwang, Chae Young; Lee, Cheolju; Kwon, Ki-Sun

    2009-01-01

    p21Cip1 is an inhibitor of cell cycle progression that promotes G1-phase arrest by direct binding to cyclin-dependent kinase and proliferating cell nuclear antigen. Here we demonstrate that mitogenic stimuli, such as epidermal growth factor treatment and oncogenic Ras transformation, induce p21Cip1 downregulation at the posttranslational level. This downregulation requires the sustained activation of extracellular signal-regulated kinase 2 (ERK2), which directly interacts with and phosphorylates p21Cip1, promoting p21Cip1 nucleocytoplasmic translocation and ubiquitin-dependent degradation, thereby resulting in cell cycle progression. ERK1 is not likely involved in this process. Phosphopeptide analysis of in vitro ERK2-phosphorylated p21Cip1 revealed two phosphorylation sites, Thr57 and Ser130. Double mutation of these sites abolished ERK2-mediated p21Cip1 translocation and degradation, thereby impairing ERK2-dependent cell cycle progression at the G1/S transition. These results indicate that ERK2 activation transduces mitogenic signals, at least in part, by downregulating the cell cycle inhibitory protein p21Cip1. PMID:19364816

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

    PubMed Central

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

    2008-01-01

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

  17. 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

  18. Ah Receptor–Mediated Suppression of Liver Regeneration through NC-XRE–Driven p21Cip1 Expression

    PubMed Central

    Jackson, Daniel P.; Li, Hui; Mitchell, Kristen A.; Joshi, Aditya D.

    2014-01-01

    Previous studies in hepatocyte-derived cell lines and the whole liver established that the aryl hydrocarbon receptor (AhR) can disrupt G1-phase cell cycle progression following exposure to persistent AhR agonists, such as TCDD (dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin). Growth arrest was attributed to inhibition of G1-phase cyclin-dependent kinase 2 (CDK2) activity. The present study examined the effect of TCDD exposure on liver regeneration following 70% partial hepatectomy in mice lacking the Cip/Kip inhibitors p21Cip1 or p27Kip1 responsible for regulating CDK2 activity. Assessment of the regenerative process in wild-type, p21Cip1 knockout, and p27Kip1 knockout mice confirmed that TCDD-induced inhibition of liver regeneration is entirely dependent on p21Cip1 expression. Compared with wild-type mice, the absence of p21Cip1 expression completely abrogated the TCDD inhibition, and accelerated hepatocyte progression through G1 phase during the regenerative process. Analysis of the transcriptional response determined that increased p21Cip1 expression during liver regeneration involved an AhR-dependent mechanism. Chromatin immunoprecipitation studies revealed that p21Cip1 induction required AhR binding to the newly characterized nonconsensus xenobiotic response element, in conjunction with the tumor suppressor protein Kruppel-like factor 6 functioning as an AhR binding partner. The evidence also suggests that AhR functionality following partial hepatectomy is dependent on a p21Cip1-regulated signaling process, intimately linking AhR biology to the G1-phase cell cycle program. PMID:24431146

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

    PubMed

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

    2015-04-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    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.

  2. Less understood issues: p21(Cip1) in mitosis and its therapeutic potential.

    PubMed

    Kreis, N-N; Louwen, F; Yuan, J

    2015-04-01

    p21(Cip1) is a multifunctional protein and a key player in regulating different cellular processes. The transcription of p21 is regulated by p53-dependent and -independent pathways. The expression of p21 is increased in response to various cellular stresses to arrest the cell cycle and ensure genomic stability. p21 has been shown to be a tumor suppressor and an oncogene as well. The function of p21 in mitosis has been proposed but not systematically studied. We have recently shown that p21 binds to and inhibits the activity of Cdk1/cyclin B1, and is important for a fine-tuned mitotic progression. Loss of p21 prolongs the duration of mitosis and results in severe mitotic defects like chromosome segregation and cytokinesis failures promoting consequently genomic instability. Moreover, p21 is dramatically stabilized in mitotic tumor cells upon treatment with mitotic agents like paclitaxel or mitotic kinase inhibitors. Increased p21 is mainly localized in the cytoplasm and associates with cell survival indicating a crucial role of p21 in susceptibility to mitotic agents in tumor cells. In this review we will briefly summarize the structure and general physiological functions as well as regulation of p21, discuss in detail its role in mitosis and its potential to serve as a therapeutic target.

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

    PubMed

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

    1999-10-01

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

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

    PubMed

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

    1999-10-01

    Primary mediastinal B-cell lymphoma (PMBL) shows chromosome 9p anomalies in 50% of cases. Based on reports that p16INK4A gene, located on this chromosomal arm, is frequently altered in aggressive lymphomas, we analysed for alterations of this gene in 27 cases of PMBL, which were part of a series of 32 PMBL cases that have been characterized for alterations in c-myc, p53, N-ras, bcl-1, bcl-2, bcl-6 and for Epstein-Barr virus (EBV) infection. Four cases showed p16INK4A gene anomalies, including three with promoter methylation and one homozygous deletion. Eight PMBLs showed c-myc rearrangements. Three additional cases showed sequence variations in the c-myc P2 promoter, two of which consisted of the same germline variation involving a novel polymorphic XhoI site. Four tumours contained p53 gene mutations and three had clonal EBV infection. One case had a bcl-6 rearrangement. In conclusion, our study shows that p16INK4, c-myc and p53 alterations occur in 15%, 25% and 13% of PMBLs, respectively. EBV monoclonality was found in 9% of cases, whereas no abnormality was detected in bcl-1, bcl-2 and N-ras. Thus, none of the common genetic aberrations seen in other types of non-Hodgkin's lymphomas appears to be stringently involved in the pathogenesis of this unique lymphoma type. PMID:10520030

  5. Centrosome aberrations in human mammary epithelial cells driven by cooperative interactions between p16INK4a deficiency and telomere-dependent genotoxic stress

    PubMed Central

    Domínguez, Daniel; Feijoo, Purificación; Bernal, Aina; Ercilla, Amaia; Agell, Neus; Genescà, Anna; Tusell, Laura

    2015-01-01

    Virtually all human cancers display chromosome instability (CIN), a condition in which chromosomes are gained or lost at a high rate. CIN occurs early in cancer development where it may undermine the advance of the neoplastic disease. With the aim of establishing the mechanisms underlying CIN in cancer, we investigated possible links between telomere-dysfunction and centrosome defects, which were seen to coincide in early in breast carcinogenesis using human mammary epithelial cells (HMECs). In this study, we show that TP53 proficient vHMECs cells develop centrosome aberrations when telomere-dysfunction genotoxic stress is produced in the presence of a defective p16INK4a setting and in parallel with an activation of the DNA damage checkpoint response. These aberrations consist of the accumulation of centrosomes in polyploid vHMECs, plus centriole overduplication in both diploid and polyploid cells, thus reflecting that distinct mechanisms underlie the generation of centrosome aberrations in vHMECs. Transduction of vHMEC with hTERT, which rescued the telomere dysfunction phenotype and consequently reduced DNA damage checkpoint activation, led to a progressive reduction of centrosome aberrations with cell culture, both in diploid and in polyploid vHMECs. Radiation-induced DNA damage also raised centrosome aberrations in vHMEC-hTERT. Collectively, our results, using vHMECs define a model where p16INK4a deficiency along with short dysfunctional telomeres cooperatively engenders centrosome abnormalities before p53 function is compromised. PMID:26318587

  6. Investigating Diagnostic Problems of CIN1 and CIN2 Associated With High-risk HPV by Combining the Novel Molecular Biomarker PanHPVE4 With P16INK4a.

    PubMed

    van Baars, Romy; Griffin, Heather; Wu, Zhonglin; Soneji, Yasmina J; van de Sandt, Miekel M; Arora, Rupali; van der Marel, Jacolien; ter Harmsel, Bram; Jach, Robert; Okon, Krzysztof; Huras, Hubert; Jenkins, David; Quint, Wim G V; Doorbar, John

    2015-11-01

    Grading cervical intraepithelial neoplasia (CIN) determines clinical management of women after abnormal cytology with potential for overdiagnosis and overtreatment. We studied a novel biomarker of human papillomavirus (HPV) life-cycle completion (panHPVE4), in combination with the minichromosome maintenance (MCM) protein cell-cycle marker and the p16INK4a transformation marker, to improve CIN diagnosis and categorization. Scoring these biomarkers alongside CIN grading by 3 pathologists was performed on 114 cervical specimens with high-risk (HR) HPV. Interobserver agreement for histopathology was moderate (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% confidence interval [CI]: 0.763-0.969; p16INK4a : κ=0.798; 95% CI: 0.712-0.884; MCM: κ=0.894; 95% CI: NC (this quantity cannot be calculated). Biomarker expression was studied by immunofluorescence and immunohistochemistry and was correlated with 104 final CIN diagnoses after histologic 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

  7. 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

  8. 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.

  9. Cables1 controls p21/Cip1 protein stability by antagonizing proteasome subunit alpha type 3.

    PubMed

    Shi, Z; Li, Z; Li, Z J; Cheng, K; Du, Y; Fu, H; Khuri, F R

    2015-05-01

    The cyclin-dependent kinase (CDK) inhibitor 1A, p21/Cip1, is a vital cell cycle regulator, dysregulation of which has been associated with a large number of human malignancies. One critical mechanism that controls p21 function is through its degradation, which allows the activation of its associated cell cycle-promoting kinases, CDK2 and CDK4. Thus delineating how p21 is stabilized and degraded will enhance our understanding of cell growth control and offer a basis for potential therapeutic interventions. Here we report a novel regulatory mechanism that controls the dynamic status of p21 through its interaction with Cdk5 and Abl enzyme substrate 1 (Cables1). Cables1 has a proposed role as a tumor suppressor. We found that upregulation of Cables1 protein was correlated with increased half-life of p21 protein, which was attributed to Cables1/p21 complex formation and supported by their co-localization in the nucleus. Mechanistically, Cables1 interferes with the proteasome (Prosome, Macropain) subunit alpha type 3 (PSMA3) binding to p21 and protects p21 from PSMA3-mediated proteasomal degradation. Moreover, silencing of p21 partially reverses the ability of Cables1 to induce cell death and inhibit cell proliferation. In further support of a potential pathophysiological role of Cables1, the expression level of Cables1 is tightly associated with p21 in both cancer cell lines and human lung cancer patient tumor samples. Together, these results suggest Cables1 as a novel p21 regulator through maintaining p21 stability and support the model that the tumor-suppressive function of Cables1 occurs at least in part through enhancing the tumor-suppressive activity of p21. PMID:24975575

  10. 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

  11. 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

  12. Concurrence of replicative senescence and elevated expression of p16(INK4A) with subculture-induced but not calcium-induced differentiation in normal human oral keratinocytes.

    PubMed

    Lee, G; Park, B S; Han, S E; Oh, J E; You, Y O; Baek, J H; Kim, G S; Min, B M

    2000-10-01

    Primary normal human oral keratinocytes (NHOKs) undergo differentiation in the presence of calcium concentrations higher than 0.15 mM in vitro, which is useful in investigating the mechanisms involved in the differentiation of epithelial cells. Serial subculture of NHOKs to the postmitotic stage also induces terminal differentiation. However, the detailed mechanisms of both differentiation processes remain substantially unknown. To investigate the molecular differences in these processes, NHOKs were induced to differentiate by exposure to 1.2 mM of calcium and by serial subculture to the postmitotic stage. To study whether the cells were induced to differentiate and to undergo replicative senescence, the amount of cellular involucrin and the expression of senescence-associated beta-galactosidase (SA-beta-gal) were measured respectively. The expression of replicative senescence-associated genes and the activity of telomerase from the differentiated cells were also determined. Both calcium treatment and serial subculture to the postmitotic stage notably elevated the cellular involucrin. The percentage of SA-beta-gal-positive cells was significantly elevated by the continued subculture, but such changes were not observed in keratinocytes exposed to calcium. The concentration of cellular p16(INK4A) protein was progressively increased by the continued subculture but was not changed by calcium treatment. On the other hand, the concentrations of cellular p53 were similar in both differentiation processes. However, telomerase activity was lost in NHOKs that had undergone differentiation by both calcium treatment and serial subculture. The results indicate that calcium-induced differentiation of NHOKs has similar characteristics to their serial subculture-induced differentiation, but that the differentiation processes are not identical, because calcium-induced differentiation does not concur with either replicative senescence or the gradually increased concentration of p16

  13. P16(INK4A) immunostaining is a strong indicator for high-risk-HPV-associated oropharyngeal carcinomas and dysplasias, but is unreliable to predict low-risk-HPV-infection in head and neck papillomas and laryngeal dysplasias.

    PubMed

    Mooren, Jeroen J; Gültekin, Sibel E; Straetmans, Jos M J A A; Haesevoets, Annick; Peutz-Kootstra, Carine J; Huebbers, Christian U; Dienes, Hans P; Wieland, Ulrike; Ramaekers, Frans C S; Kremer, Bernd; Speel, Ernst-Jan M; Klussmann, Jens P

    2014-05-01

    Human papillomavirus (HPV) is a risk factor for the development of benign and malignant mucosal head and neck lesions. P16(INK4A) is often used as a surrogate marker for HPV-infection, although there is still controversy with respect its reliability. Our aim was to determine if p16(INK4A) overexpression can accurately predict both high-risk and low-risk-HPV-presence in (pre)malignant and benign head and neck lesions. P16(INK4A) immunohistochemistry was performed on paraffin-embedded tissue sections of 162 oropharyngeal squamous cell carcinomas (OPSCC), 14 tonsillar and 23 laryngeal dysplasias, and 20 tonsillar and 27 laryngeal papillomas. PCR, enzyme-immunoassay and FISH analysis were used to assess HPV-presence and type. Of the 162 OPSCC and 14 tonsillar dysplasias, 51 (31%) and 10 (71%) were HPV16-positive, respectively. All tonsillar papillomas were HPV-negative and four laryngeal dysplasias and 26 laryngeal papillomas were positive for HPV6 or -11. P16(INK4A) immunohistochemistry revealed a strong nuclear and cytoplasmic staining in 50 out of 51 HPV16-positive and 5 out of 111 HPV-negative OPSCC (p < 0.0001) and in all HPV16-positive tonsillar dysplasias, whereas highly variable staining patterns were detected in the papillomas and laryngeal dysplasias, irrespective of the HPV-status. In addition, the latter lesions generally showed a higher nuclear than cytoplasmic p16(INK4A) immunostaining intensity. In conclusion, our data show that strong nuclear and cytoplasmic p16(INK4A) overexpression is a reliable surrogate indicator for HPV16 in OPSCC and (adjacent) dysplasias. For HPV6 or -11-positive and HPV-negative benign and premalignant lesions of the tonsil and larynx, however, p16(INK4A) immunostaining is highly variable and cannot be recommended to predict HPV-presence. PMID:24127203

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

    PubMed

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

    2014-11-01

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

  15. Diabetes-induced oxidative DNA damage alters p53-p21CIP1/Waf1 signaling in the rat testis.

    PubMed

    Kilarkaje, Narayana; 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 21(cyclin-dependent kinase interacting protein 1/wild-type p53-activated factor 1) (p21(CIP1/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 p21(CIP1/Waf1) was mainly cytoplasmic but also perinuclear in pachytene spermatocytes and round spermatids. The cytoplasmic localization of p21(CIP1/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 p21(CIP1/Waf1) signaling in testicular germ cells in association with alteration in p-p53ser315 expression, probably to counteract DNA damage-induced cell death.

  16. Cyclin D1, p16(INK) (4A) and p27(Kip1) in pancreatic adenocarcinoma: assessing prognostic implications through quantitative image analysis.

    PubMed

    Georgiadou, Despoina; Sergentanis, Theodoros N; Sakellariou, Stratigoula; Filippakis, George M; Zagouri, Flora; Vlachodimitropoulos, Dimitris; Psaltopoulou, Theodora; Lazaris, Andreas C; Patsouris, Efstratios; Zografos, George C

    2014-12-01

    The prognostic significance of cyclin D1, p16(INK) (4A) and p27(Kip1) expression has been documented in several human malignancies; however, their prognostic potential in pancreatic adenocarcinoma is still unclear. This study aimed to assess the correlation of the aforementioned molecules with clinicopathological parameters and prognosis. Sixty patients with pancreatic ductal adenocarcinoma underwent surgical resection at a single institution; immunohistochemical staining of the studied markers was quantified by Ιmage analysis system. Cyclin D1 overexpression was positively associated with grade, neural infiltration and vascular invasion, whereas p27 positively correlated with age. Higher cyclin D1 expression indicated poorer survival (adjusted HR = 9.75, 95%CI: 1.48-64.31, p = 0.018, increment: one unit in H-score), whereas a marginal trend toward an association between p16 positivity and improved survival was observed (adjusted HR = 0.58, 95%CI: 0.32-1.05, p = 0.072 regarding positive vs negative cases). No significant association with overall survival was noted regarding p27. In conclusion, cyclin D1 overexpression and possibly p16 loss of expression in pancreatic adenocarcinoma seem to be adverse prognostic factors, whereas p27 expression did not seem to possess such prognostic properties. Further validation of the present findings in studies encompassing larger samples seems to be needed.

  17. 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. PMID:27126662

  18. 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.

  19. 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.

  20. Frequency of genetic and epigenetic alterations of p14ARF and p16INK4A in head and neck cancer in a Hungarian population.

    PubMed

    Kis, Andrea; Tatár, Tímea Zsófia; Gáll, Tamás; Boda, Róbert; Tar, Ildikó; Major, Tamás; Redl, Pál; Gergely, Lajos; Szarka, Krisztina

    2014-10-01

    Occurrence of genetic and epigenetic alterations affecting p14ARF and p16INK4A were investigated in tumour samples of 37 oral (OSCC) and 28 laryngeal squamous cell cancer (LSCC) patients, and compared to exfoliated buccal epithelial cells of 68 healthy controls. Presence of deletions and mutations/polymorphisms affecting exons were examined using sequencing. Methylation status of promoters was assessed by methylation-specific PCR. Chi-square and Fisher's exact tests were used to compare frequency of events. Exon deletions were found in four controls, one OSCC and 22 LSCC patients; the latter significantly differed from controls (p < 0.001). Only two mutations (T24610A and C24702A) were in p16 exon 1 of two OSCC patients. Polymorphisms G28575A (Ala140Thr), G31292C (C540G) and G28608A were found in both patient groups. The p14 promoter was unmethylated in 86.7 % of OSCC and in 85.7 % of LSCC patients; for the p16 promoter these rates were 69.0 % and 76.2 % for OSCC and LSCC patients, respectively. Combining the two patient groups, unmethylated promoter was significantly less frequent in case of both p14 and p16 (p = 0.043 and p = 0.001, respectively) compared to the control group. In summary, exon deletion may be important in LSCC, while promoter methylation was relatively frequent in both patient groups. PMID:24710824

  1. Novel role of cortactin in G protein-coupled receptor agonist-induced nuclear export and degradation of p21Cip1

    PubMed Central

    Janjanam, Jagadeesh; Rao, Gadiparthi N.

    2016-01-01

    Monocyte chemotactic protein 1 (MCP1) stimulates phosphorylation of cortactin on Y421 and Y446 residues in a time-dependent manner and phosphorylation at Y446 but not Y421 residue is required for MCP1-induced CDK-interacting protein 1 (p21Cip1) nuclear export and degradation in facilitating human aortic smooth muscle cell (HASMC) proliferation. In addition, MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation are dependent on Fyn activation. Upstream to Fyn, MCP1 stimulated C-C chemokine receptor type 2 (CCR2) and Gi/o and inhibition of either one of these molecules using their specific antagonists or inhibitors attenuated MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation. Cortactin phosphorylation at Y446 residue is also required for another G protein-coupled receptor (GPCR) agonist, thrombin-induced p21Cip1 nuclear export and its degradation in promoting HASMC proliferation. Quite interestingly, the receptor tyrosine kinase (RTK) agonist, platelet-derived growth factor-BB (PDGF-BB)-induced p21Cip1 degradation and HASMC proliferation do not require cortactin tyrosine phosphorylation. Together, these findings demonstrate that tyrosine phosphorylation of cortactin at Y446 residue is selective for only GPCR but not RTK agonist-induced nuclear export and proteolytic degradation of p21Cip1 in HASMC proliferation. PMID:27363897

  2. p21(Cip1) up-regulated during histone deacetylase inhibitor-induced CD4(+) T-cell anergy selectively associates with mitogen-activated protein kinases.

    PubMed

    Selma Dagtas, Ayse; Gilbert, Kathleen M

    2010-04-01

    Histone deacetylase inhibitor n-butyrate induced proliferative unresponsiveness in antigen-stimulated murine CD4(+) T cells. T cells anergized by n-butyrate demonstrated reduced interleukin-2 (IL-2) secretion and decreased activating protein 1 (AP-1) activity upon restimulation. Mechanistic studies determined that the cyclin-dependent kinase (cdk) inhibitor p21(Cip1) was up-regulated in the anergic CD4(+) T cells. p21(Cip1) is known to inhibit the cell cycle through its interaction with cdk, proliferating cell nuclear antigen (PCNA) or c-Jun N-terminal kinase (JNK). p21(Cip1) did not preferentially associate with PCNA or cdk in anergic T helper type 1 (Th1) cells. Instead, among the three interaction partners, p21(Cip1) was found to interact with phospho-JNK and phospho-c-jun selectively in the anergic CD4(+) T cells. The activity of c-jun and downstream transcription factor AP-1 were suppressed in the anergic Th1 cells. In contrast, p21(Cip1) and the two phospho-proteins were never detected concurrently in the control CD4(+) T cells. The n-butyrate-induced p21(Cip1)-mediated inhibition of JNK and c-jun represents a novel potential mechanism by which proliferative unresponsiveness was maintained in CD4(+) T cells.

  3. The effect of acute dichlorodiphenyltrichloroethane exposure on hypermethylation status and down-regulation of p53 and p16INK4a genes in rat liver.

    PubMed

    Kostka, Grażyna; Urbanek-Olejnik, Katarzyna; Liszewska, Monika; Winczura, Alicja

    2016-05-01

    The aim of the study was to investigate the early effect of acute dichlorodiphenyltrichloroethane (DDT) exposure on the methylation status of the promoter region of two tumor suppressor genes: p53 and p16(INK4a) (p16) in rat liver. We analyzed their transcript and protein expression profiles concurrently with the examination of transcriptional and protein expression levels of DNA (cytosine-5)-methyltransferase 1 (Dnmt1). Male Wistar rats were treated with a single dose of DDT (57 mg kg(-1) of body weight) and the methylation status of p53 and p16 genes was examined after 24 h using methylation-sensitive restriction analysis-MSRA. The obtained results indicate that DDT induced alternations in methylation of the promoter region in both p53 and p16 genes. In all the tested samples, the promoter CpG islands of p53 (-261, -179, and -450) were methylated within 100% as compared to control samples (0%). The methylation status of the p16 promoter (-11 and +77) was also altered due to exposure to DDT. Methylated cytosines were detectable in 75% of the tested DNA samples. The Real-time PCR and western blot analyses showed a decrease in mRNA and protein levels of p53, respectively, which was related to the increase in DNA synthesis. These relationships were also observed for mRNA and protein expressions of p16, although to a slighter extent. We also showed that hypermethylation in the promoter region of both tumor suppressor genes was consistent with an increased Dnmt1 mRNA level, and this relationship was further confirmed at the protein level of DNMT1. Concluding, our data suggests that epigenetically mediated changes in gene expression may play an important role in the mechanism of DDT toxicity, including carcinogenic action.

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

    PubMed

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

    2015-12-15

    The p16(INK4a) (p16) is a cyclin-dependent kinase inhibitor, which has been evaluated in several studies as a diagnostic marker of cervical cancer. Immunostaining using p16 specific antibody has confirmed an over-expression of p16 protein in cervical cancer cells and its association with disease progression. This article reports an ultrasensitive electrochemical immunosensor for specific detection of p16 and demonstrates its performance for detection of solubilized p16 protein in cell lysates obtained from patients. Sandwich-based immunoreaction couple with double signal amplification strategy based on catalytic enlargement of particle tag was used for high sensitivity and specificity. The conditions were optimized to create an immunoassay protocol. Disposable screen-printed electrode modified with capture antibodies (Ab1) was selected for further implementation towards point-of-care diagnostics. Small gold nanoparticles (15 nm diameter) conjugated with detection antibodies (Ab2) were found to better serve as a detection label due to limited interference with antigen-antibody interaction. Double signal enhancement was performed by sequential depositions of gold and silver layers. This gave the sensitivity of 1.78 μA mL(ng GST-p16)(-1) cm(-2) and detection limit of 1.3 ng mL(-1) for GST-p16 protein which is equivalent to 0.49 ng mL(-1) for p16 protein and 28 cells for HeLa cervical cancer cells. In addition to purified protein, the proposed immunosensor effectively detected elevated p16 level in cervical swab samples obtained from 10 patients with positive result from standard Pap smear test, indicating that an electrochemical immunosensors hold an excellent promise for detection of cervical cancer in clinical setting. PMID:26201985

  5. Methylation of Adjacent CpG Sites Affects Sp1/Sp3 Binding and Activity in the p21Cip1 Promoter

    PubMed Central

    Zhu, Wei-Guo; Srinivasan, Kanur; Dai, Zunyan; Duan, Wenrui; Druhan, Lawrence J.; Ding, Haiming; Yee, Lisa; Villalona-Calero, Miguel A.; Plass, Christoph; Otterson, Gregory A.

    2003-01-01

    DNA methylation in the promoter of certain genes is associated with transcriptional silencing. Methylation affects gene expression directly by interfering with transcription factor binding and/or indirectly by recruiting histone deacetylases through methyl-DNA-binding proteins. In this study, we demonstrate that the human lung cancer cell line H719 lacks p53-dependent and -independent p21Cip1 expression. p53 response to treatment with gamma irradiation or etoposide is lost due to a mutation at codon 242 of p53 (C→W). Treatment with depsipeptide, an inhibitor of histone deacetylase, was unable to induce p53-independent p21Cip1 expression because the promoter of p21Cip1 in these cells is hypermethylated. By analyzing luciferase activity of transfected p21Cip1 promoter vectors, we demonstrate that depsipeptide functions on Sp1-binding sites to induce p21Cip1 expression. We hypothesize that hypermethylation may interfere with Sp1/Sp3 binding. By using an electrophoretic mobility shift assay, we show that, although methylation within the consensus Sp1-binding site did not reduce Sp1/Sp3 binding, methylation outside of the consensus Sp1 element induced a significant decrease in Sp1/Sp3 binding. Depsipeptide induced p21Cip1 expression was reconstituted when cells were pretreated with 5-aza-2′-deoxycytidine. Our data suggest, for the first time, that hypermethylation around the consensus Sp1-binding sites may directly reduce Sp1/Sp3 binding, therefore leading to a reduced p21Cip1 expression in response to depsipeptide treatment. PMID:12773551

  6. miR-299-5p promotes cell growth and regulates G1/S transition by targeting p21Cip1/Waf1 in acute promyelocytic leukemia

    PubMed Central

    WU, SHUN-QUAN; ZHANG, LANG-HUI; HUANG, HAO-BO; LI, YA-PING; NIU, WEN-YAN; ZHAN, RONG

    2016-01-01

    MicroRNAs (miRs) are often located in genomic breakpoint regions and are hypothesized to be important regulators involved in the regulation of critical cell processes, including cell apoptosis, proliferation and differentiation. miR-299 has been reported to be upregulated in acute promyelocytic leukemia (APL); however, the function and mechanistic role of miR-299 in APL remains unknown. The present study demonstrated mir-299 significantly induced cell growth and cell cycle progression at the G1/S transition in APL cells. Notably, the present study revealed that miR-299-5p induces these effects, whereas miR-299-3p does not. Additional studies demonstrated that in APL cells the tumor suppressor p21Cip1/Waf1 is a downstream target of miR-299; miR-299 binds directly to the 3′ untranslated region of p21Cip1/Waf1, and reduces protein, but not mRNA, levels of p21Cip1/Waf1. The present findings demonstrate that miR-299 exerts growth-promoting effects in APL cells through the suppression of p21Cip1/Waf1. Overall, the present study demonstrates that p21Cip1/Waf1 is a direct functional target of miR-299 in APL. PMID:27347210

  7. [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

  8. 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.

  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. The CRL4Cdt2 ubiquitin ligase targets the degradation of p21Cip1 to control replication licensing.

    PubMed

    Kim, Youngjo; Starostina, Natalia G; Kipreos, Edward T

    2008-09-15

    The faithful replication of genomic DNA is crucial for maintaining genome stability. In eukaryotes, DNA rereplication is prevented by the temporal regulation of replication licensing. Replication-licensing factors are required to form prereplicative complexes during G1 phase, but are inactivated in S phase to prevent rereplication. A vertebrate CUL4 CRL ubiquitin ligase (CRL4) complex containing Cdt2 as the substrate recognition subunit promotes proper DNA replication, in part, by degrading the replication-licensing factor Cdt1 during S phase. We show that the Caenorhabditis elegans CRL4(Cdt2) complex has a conserved role in degrading Cdt1. Furthermore, we show that CRL4(Cdt2) restrains replication licensing in both C. elegans and humans by targeting the degradation of the cyclin-dependent kinase (CDK) inhibitors CKI-1 and p21(Cip1), respectively. Human CRL4(Cdt2) targets the degradation of p21 in S phase, with the in vivo ubiquitylation of p21 by CRL4(Cdt2) dependent on p21 binding to PCNA. Inactivation of Cdt2 induces rereplication, which requires the presence of the CDK inhibitor p21. Strikingly, coinactivation of CRL4(Cdt2) and SCF(Skp2) (which redundantly targets p21 degradation) prevents the nuclear export of the replication-licensing factor Cdc6 during S phase, and the block on nuclear export is dependent on p21. Our work defines the degradation of p21 as a critical aspect of replication licensing in human cells. PMID:18794348

  11. Antibiotic drug tigecycline inhibits melanoma progression and metastasis in a p21CIP1/Waf1-dependent manner

    PubMed Central

    Tan, Peng; Zhang, Yanli; Liu, Lichao; Yang, Liqun; Liu, Yaling; Cui, Hongjuan

    2016-01-01

    Antibiotics are common durgs with low toxicity but high effectiveness. They have been suggested to be drug candidates for cancer therapy in recent years. Here, we tried to investigate the antitumour effect of tigecycline on malignant melanoma. We showed that tigecycline dramatically inhibited cell proliferation and induced cell cycle arrest at G0/G1 phase. At the same time, tigecycline suppressed cell invasion and migration through preventing epithelial-mesenchymal transition (EMT) process. In addition, tigecycline also significantly blocked tumor growth in vivo. Expression of cell cycle-related proteins were investigated and resulted in downregulation of G1/S checkpoint proteins, such as CDK2 and Cyclin E. However, cyclin-dependent kinase inhibitor 1 (CDKN1A, p21CIP1/Waf1) was downregulated after tigecycline treatment, which was not conformed to its conventional function. To explain this, we overexpressed p21 in melanoma cells. We found that p21 overexpression significantly rescued tigecycline-induced cell proliferation inhibition as well as migration and invasion suppression. Taken together, our results revealed that the essential role of p21 in the inhibitory effect of tigecycline on proliferation, migration and invasion of melanoma. Tigecycline might act as a candidate therapeutic drug for treatment of patients suffering from malignant melanoma. PMID:26621850

  12. Ex vivo targeting of p21Cip1/Waf1 permits relative expansion of human hematopoietic stem cells.

    PubMed

    Stier, Sebastian; Cheng, Tao; Forkert, Randolf; Lutz, Christoph; Dombkowski, David M; Zhang, Jie Lin; Scadden, David T

    2003-08-15

    Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling, we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor, p21Cip1/Waf1 (p21), have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling, we tested whether reduction of p21 alone in human adult-derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control, manipulated cells. Further, we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore, lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion. PMID:12702511

  13. Protein Kinase C Delta (PKCδ) Affects Proliferation of Insulin-Secreting Cells by Promoting Nuclear Extrusion of the Cell Cycle Inhibitor p21Cip1/WAF1

    PubMed Central

    Ranta, Felicia; Leveringhaus, Johannes; Theilig, Dorothea; Schulz-Raffelt, Gabriele; Hennige, Anita M.; Hildebrand, Dominic G.; Handrick, René; Jendrossek, Verena; Bosch, Fatima; Schulze-Osthoff, Klaus; Häring, Hans-Ulrich; Ullrich, Susanne

    2011-01-01

    Background High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ) in β-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions. Methodology and Principal Findings Using genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT) significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21Cip1/WAF1. This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21Cip1/WAF1 at Ser146. In kinase dead PKCδ (PKCδKN) overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21Cip1/WAF1 was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21Cip1/WAF1 with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic. Conclusions and Significance These observations disclose PKCδ as negative regulator of p21Cip1/WAF1, which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role. PMID:22216119

  14. p21Cip1 Protection against Hyperoxia Requires Bcl-XL and Is Uncoupled from Its Ability to Suppress Growth

    PubMed Central

    Vitiello, Peter F.; Staversky, Rhonda J.; Gehen, Sean C.; Johnston, Carl J.; Finkelstein, Jacob N.; Wright, Terry W.; O’Reilly, Michael A.

    2006-01-01

    The cyclin-dependent kinase inhibitor p21Cip1/Waf1/Sdi1 protects the lung against hyperoxia, but the mechanism of protection remains unclear because loss of p21 does not lead to aberrant cell proliferation. Because some members of the Bcl-2 gene family have been implicated in hyperoxia-induced cell death, the current study investigated their expression as well as p21-dependent growth suppression and cytoprotection. Conditional overexpression of full-length p21, its amino-terminal cyclin-binding (p211–82NLS) domain or its carboxy-terminal PCNA-binding (p2176–164) domain inhibited growth of human lung adenocarcinoma H1299 cells, but only the full-length protein was cytoprotective. Low levels of p21 inhibited cell proliferation, whereas higher levels were required for protection. Expression of the anti-apoptotic protein Bcl-XL declined during hyperoxia but was maintained in cells expressing p21. RNA interference (RNAi) knockdown of Bcl-XL enhanced hyperoxic death of cells expressing p21, whereas overexpression of Bcl-XL increased cell survival. Consistent with growth suppression and cytoprotection requiring different levels of p21, hyperoxia inhibited PCNA expression in p21+/+ and p21+/− mice but not in p21−/− mice. In contrast, p21 was haplo-insufficient for maintaining expression of Bcl-XL and protection against hyperoxia. Taken together, these data show that p21-mediated cytoprotection against hyperoxia involves regulation of Bcl-XL and is uncoupled from its ability to inhibit proliferation. PMID:16723699

  15. The effects of two polymorphisms on p21cip1 function and their association with Alzheimer's disease in a population of European descent.

    PubMed

    Yates, Sharon C; Zafar, Amen; Rabai, Erzsebet M; Foxall, James B; Nagy, Sheila; Morrison, Karen E; Clarke, Carl; Esiri, Margaret M; Christie, Sharon; Smith, A David; Nagy, Zsuzsanna

    2015-01-01

    With the exception of ApoE4, genome-wide association studies have failed to identify strong genetic risk factors for late-onset Alzheimer's disease, despite strong evidence of heritability, suggesting that many low penetrance genes may be involved. Additionally, the nature of the identified genetic risk factors and their relation to disease pathology is also largely obscure. Previous studies have found that a cancer-associated variant of the cell cycle inhibitor gene p21cip1 is associated with increased risk of Alzheimer's disease. The aim of this study was to confirm this association and to elucidate the effects of the variant on protein function and Alzheimer-type pathology. We examined the association of the p21cip1 variant with Alzheimer's disease and Parkinson's disease with dementia. The genotyping studies were performed on 719 participants of the Oxford Project to Investigate Memory and Ageing, 225 participants of a Parkinson's disease DNA bank, and 477 participants of the Human Random Control collection available from the European Collection of Cell Cultures. The post mortem studies were carried out on 190 participants. In the in-vitro study, human embryonic kidney cells were transfected with either the common or rare p21cip1 variant; and cytometry was used to assess cell cycle kinetics, p21cip1 protein expression and sub-cellular localisation. The variant was associated with an increased risk of Alzheimer's disease, and Parkinson's disease with dementia, relative to age matched controls. Furthermore, the variant was associated with an earlier age of onset of Alzheimer's disease, and a more severe phenotype, with a primary influence on the accumulation of tangle pathology. In the in-vitro study, we found that the SNPs reduced the cell cycle inhibitory and anti-apoptotic activity of p21cip1. The results suggest that the cancer-associated variant of p21cip1 may contribute to the loss of cell cycle control in neurons that may lead to Alzheimer

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

    PubMed

    Suzuki, Takehiro; Nohara, Keiko

    2013-09-01

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

  17. Cholesterol Retards Senescence in Bone Marrow Mesenchymal Stem Cells by Modulating Autophagy and ROS/p53/p21Cip1/Waf1 Pathway

    PubMed Central

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

    2016-01-01

    In the present study, we demonstrated that bone marrow mesenchymal stem cells (BMSCs) of the 3rd passage displayed the senescence-associated phenotypes characterized with increased activity of SA-β-gal, altered autophagy, and increased G1 cell cycle arrest, ROS production, and expression of p53 and p21Cip1/Waf1 compared with BMSCs of the 1st passage. Cholesterol (CH) reduced the number of SA-β-gal positive cells in a dose-dependent manner in aging BMSCs induced by H2O2 and the 3rd passage BMSCs. Moreover, CH inhibited the production of ROS and expression of p53 and p21Cip1/Waf1 in both cellular senescence models and decreased the percentage of BMSCs in G1 cell cycle in the 3rd passage BMSCs. CH prevented the increase in SA-β-gal positive cells induced by RITA (reactivation of p53 and induction of tumor cell apoptosis, a p53 activator) or 3-MA (3-methyladenine, an autophagy inhibitor). Our results indicate that CH not only is a structural component of cell membrane but also functionally contributes to regulating cellular senescence by modulating cell cycle, autophagy, and the ROS/p53/p21Cip1/Waf1 signaling pathway. PMID:27703600

  18. Ciz1, a p21 cip1/Waf1-interacting zinc finger protein and DNA replication factor, is a novel molecular partner for human enhancer of rudimentary homolog.

    PubMed

    Lukasik, Anna; Uniewicz, Katarzyna A; Kulis, Marta; Kozlowski, Piotr

    2008-01-01

    Enhancer of rudimentary homolog (Drosophila) (ERH) is a small, highly conserved, nuclear protein with a unique three-dimensional structure, whose gene has been identified in animals, plants and protists, but not in fungi. Involvement of ERH in fundamental processes such as regulation of pyrimidine metabolism, cell cycle progression, transcription and cell growth control has been suggested. Here, employing a yeast two-hybrid system, a glutathione S-transferase pull-down assay and tandem MS, we demonstrate that Ciz1 is a bona fide interactor of human ERH. Ciz1 is a nuclear zinc finger protein interacting with p21(Cip1/Waf1), a universal inhibitor of cyclin-dependent kinases, and is a DNA replication factor. The region of Ciz1 necessary for the interaction with ERH spans residues 531-644, encompassing its first zinc finger motif. This region overlaps the p21(Cip1/Waf1)-binding site, suggesting that the interaction with ERH could block the binding of p21(Cip1/Waf1) by Ciz1 in the cell. When ERH and Ciz1 are coexpressed in HeLa cells, Ciz1 recruits ERH to DNA replication foci.

  19. Gene expression of the p16(INK4a)-Rb and p19(Arf)-p53-p21(Cip/Waf1) signaling pathways in the regulation of hematopoietic stem cell aging by ginsenoside Rg1.

    PubMed

    Yue, Z; Rong, J; Ping, W; Bing, Y; Xin, Y; Feng, L D; Yaping, W

    2014-12-04

    The elucidation of the molecular mechanisms underlying the effects of traditional Chinese medicines in clinical practice is a key step toward their worldwide application, and this topic is currently a subject of intense research interest. Rg1, a component of ginsenoside, has recently been shown to perform several pharmacological functions; however, the underlying mechanisms of these effects remain unclear. In the present study, we investigated whether Rg1 has an anti-senescence effect on hematopoietic stem cells (HSCs) and the possible molecular mechanisms driving any effects. The results showed that Rg1 could effectively delay tert-butyl hydroperoxide (t-BHP)-induced senescence and inhibit gene expression in the p16(INK4a)-Rb and p19(Arf)-p53-p21(Cip/Waf1) signaling pathways in HSCs. Our study suggested that these two signaling pathways might be potential targets for elucidating the molecular mechanisms of the Rg1 anti-senescence effect.

  20. Glucose-Induced Oxidative Stress Reduces Proliferation in Embryonic Stem Cells via FOXO3A/β-Catenin-Dependent Transcription of p21(cip1).

    PubMed

    McClelland Descalzo, Darcie L; Satoorian, Tiffany S; Walker, Lauren M; Sparks, Nicole R L; Pulyanina, Polina Y; Zur Nieden, Nicole I

    2016-07-12

    Embryonic stem cells (ESCs), which are derived from a peri-implantation embryo, are routinely cultured in medium containing diabetic glucose (Glc) concentrations. While pregnancy in women with pre-existing diabetes may result in small embryos, whether such high Glc levels affect ESC growth remains uncovered. We show here that long-term exposure of ESCs to diabetic Glc inhibits their proliferation, thereby mimicking in vivo findings. Molecularly, Glc exposure increased oxidative stress and activated Forkhead box O3a (FOXO3a), promoting increased expression and activity of the ROS-removal enzymes superoxide dismutase and catalase and the cell-cycle inhibitors p21(cip1) and p27(kip1). Diabetic Glc also promoted β-catenin nuclear localization and the formation of a complex with FOXO3a that localized to the promoters of Sod2, p21(cip1), and potentially p27(kip1). Our results demonstrate an adaptive response to increases in oxidative stress induced by diabetic Glc conditions that promote ROS removal, but also result in a decrease in proliferation.

  1. Glucose-Induced Oxidative Stress Reduces Proliferation in Embryonic Stem Cells via FOXO3A/β-Catenin-Dependent Transcription of p21(cip1).

    PubMed

    McClelland Descalzo, Darcie L; Satoorian, Tiffany S; Walker, Lauren M; Sparks, Nicole R L; Pulyanina, Polina Y; Zur Nieden, Nicole I

    2016-07-12

    Embryonic stem cells (ESCs), which are derived from a peri-implantation embryo, are routinely cultured in medium containing diabetic glucose (Glc) concentrations. While pregnancy in women with pre-existing diabetes may result in small embryos, whether such high Glc levels affect ESC growth remains uncovered. We show here that long-term exposure of ESCs to diabetic Glc inhibits their proliferation, thereby mimicking in vivo findings. Molecularly, Glc exposure increased oxidative stress and activated Forkhead box O3a (FOXO3a), promoting increased expression and activity of the ROS-removal enzymes superoxide dismutase and catalase and the cell-cycle inhibitors p21(cip1) and p27(kip1). Diabetic Glc also promoted β-catenin nuclear localization and the formation of a complex with FOXO3a that localized to the promoters of Sod2, p21(cip1), and potentially p27(kip1). Our results demonstrate an adaptive response to increases in oxidative stress induced by diabetic Glc conditions that promote ROS removal, but also result in a decrease in proliferation. PMID:27411103

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

    PubMed Central

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

    2015-01-01

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

  3. The human protein p19ARF is not detected in hemopoietic human cell lines that abundantly express the alternative beta transcript of the p16INK4a/MTS1 gene.

    PubMed

    Della Valle, V; Duro, D; Bernard, O; Larsen, C J

    1997-11-13

    The p16/MTS1/CDKN2 gene on human chromosome band 9p21 encodes two unrelated proteins: p16INK4a, a specific inhibitor of the cyclin D-dependent kinases CKD4 and CDK6, and the structurally unrelated p19ARF protein that arrests cell growth in G1/S and also in G2/M. By use of polyclonal antibodies, the human p19ARF (hp19ARF) protein has been identified in the nucleus of various cells including normal cultured fibroblasts. The level of this protein did not fluctuate throughout the cell cycle and was more elevated in fibroblasts with limited or arrested growth, suggesting that p19ARF accumulated in presenescent or senescent cells. Interestingly, hp19ARF was not detected in several hemopoietic tumor cell lines (mainly of B-type lymphoid origin) that expressed abundant amounts of the p16beta transcript. This finding indicates that in certain tumors, the expression of hp19ARF RNA and protein may be uncoupled. Furthermore, it suggests that disruption of a translational mechanism may be involved in the inactivation of hp19ARF. PMID:9395243

  4. The cyclin-dependent kinase inhibitor p21(cip1/waf1) enhances the cytotoxicity of ganciclovir in HSV-tk transfected ovarian carcinoma cells.

    PubMed

    Ziller, Christelle; Lincet, Hubert; Muller, Christian D; Staedel, Cathy; Behr, Jean-Paul; Poulain, Laurent

    2004-08-20

    Suicide gene therapy could be an attractive addition to the treatment of ovarian carcinomas, for which acquired chemoresistance frequently results in treatment failure. Here we show that transfection of the HSV-tk gene, followed by incubation with up to 1 mM ganciclovir fails to induce cell death in SKOV3 chemoresistant human ovarian carcinoma cells. However, co-transfection of HSV-tk with Cip1/Waf1 encoding the p21(cip1/waf1) inhibitor of cdks, allows 100 microM ganciclovir to eradicate the population of tumor cells. Potentiation of a drug by co-transfer of HSV-tk with Cip1/Waf1could thus represent another therapeutic approach for tumours that are resistant to conventional therapy. PMID:15246560

  5. Human Cytomegalovirus pUL29/28 and pUL38 Repression of p53-Regulated p21CIP1 and Caspase 1 Promoters during Infection

    PubMed Central

    Savaryn, John P.; Reitsma, Justin M.; Bigley, Tarin M.; Halligan, Brian D.; Qian, Zhikang; Yu, Dong

    2013-01-01

    During infection by human cytomegalovirus (HCMV), the tumor suppressor protein p53, which promotes efficient viral gene expression, is stabilized. However, the expression of numerous p53-responsive cellular genes is not upregulated. The molecular mechanism used to manipulate the transcriptional activity of p53 during infection remains unclear. The HCMV proteins IE1, IE2, pUL44, and pUL84 likely contribute to the regulation of p53. In this study, we used a discovery-based approach to identify the protein targets of the HCMV protein pUL29/28 during infection. Previous studies have demonstrated that pUL29/28 regulates viral gene expression by interacting with the chromatin remodeling complex NuRD. Here, we observed that pUL29/28 also associates with p53, an additional deacetylase complex, and several HCMV proteins, including pUL38. We confirmed the interaction between p53 and pUL29/28 in both the presence and absence of infection. HCMV pUL29/28 with pUL38 altered the activity of the 53-regulatable p21CIP1 promoter. During infection, pUL29/28 and pUL38 contributed to the inhibition of p21CIP1 as well as caspase 1 expression. The expression of several other p53-regulating genes was not altered. Infection using a UL29-deficient virus resulted in increased p53 binding and histone H3 acetylation at the responsive promoters. Furthermore, expression of pUL29/28 and its interacting partner pUL38 contributed to an increase in the steady-state protein levels of p53. This study identified two additional HCMV proteins, pUL29/28 and pUL38, which participate in the complex regulation of p53 transcriptional activity during infection. PMID:23236067

  6. Cytosolic retention of phosphorylated extracellular signal-regulated kinase and a Rho-associated kinase-mediated signal impair expression of p21(Cip1/Waf1) in phorbol 12-myristate-13- acetate-induced apoptotic cells.

    PubMed

    Lai, Jin-Mei; Wu, Sulin; Huang, Duen-Yi; Chang, Zee-Fen

    2002-11-01

    In response to treatment with phorbol-12-myristate-13-acetate (PMA), the half-population of erythromyeloblast D2 cells, a cytokine-independent variant of TF-1 cells, displayed adhesion and differentiated into a monocyte/macrophage-like morphology, while the other half-population remained in suspension and underwent apoptosis. Expression of the cell cycle inhibitor p21(Cip1/Waf1) was induced after PMA treatment in the adherent cells but not in the proapoptotic cells. We investigated the mechanism responsible for the impairment of p21(Cip1/Waf1) induction in PMA-induced proapoptotic cells. We demonstrated that in PMA-induced adherent cells, upregulation of p21(Cip1/Waf1) requires the activation and nuclear translocation of phosphorylated extracellular signal-regulated kinase (phospho-ERK). Although ERK was phosphorylated to comparable levels in PMA-induced proapoptotic and adherent cells, nuclear distribution of phospho-ERK was seen only in the adherent, not in the proapoptotic cells. We also found that only PMA-induced proapoptotic cells contained the phosphorylated form of myosin light chain, which is dependent on Rho-associated kinase (ROCK) activation, and that expression of a dominant-active form of ROCK suppressed activation of the p21(Cip1/Waf1) promoter during PMA induction. Finally, we demonstrated that inhibition of ROCK restores nuclear distribution of phospho-ERK and activation of p21(Cip1/Waf1) expression. Based on these findings, we propose that a ROCK-mediated signal is involved in interfering with the process of ERK-mediated p21(Cip1/Waf1) induction in PMA-induced proapoptotic TF-1 and D2 cells.

  7. Conserved region 2 of adenovirus E1A has a function distinct from pRb binding required to prevent cell cycle arrest by p16INK4a or p27Kip1.

    PubMed

    Alevizopoulos, K; Sanchez, B; Amati, B

    2000-04-13

    Ectopic expression of the CDK inhibitors (CKIs) p16INK4a and p27Kip1 in Rat1 fibroblasts induces dephosphorylation and activation of Retinoblastoma-family proteins (pRb, p107 and p130), their association with E2F proteins, and cell cycle arrest in G1. The growth-inhibitory action of p16, in particular, is believed to be mediated essentially via pRb activation. The 12S E1A protein of human Adenovirus 5 associates with pRb-family proteins via residues in its Conserved Regions (CR) 1 and 2, in particular through the motif LXCXE in CR2. These interactions are required for E1A to prevent G1 arrest upon co-expression of CKIs. We show here that mutating either of two conserved motifs adjacent to LXCXE in CR2, GFP and SDDEDEE, also impairs the ability of E1A to overcome G1 arrest by p16 or p27. Strikingly, however, these mutations affect neither the association of E1A with pRb, p07 and p130, nor its ability to derepress E2F-1 transcriptional activity in transient transfection assays. One of the EIA mutants, however, is defective in derepressing several endogenous E2F target genes in the presence of p16 or p27. Thus, CR2 possesses an essential function besides pRb-binding. We speculate that this function might be required for the full derepression of E2F-regulated genes in their natural chromatin context. PMID:10803468

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

    PubMed

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

    2015-03-01

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

  9. Up-regulation of p27Kip1, p21WAF1/Cip1 and p16Ink4a is associated with, but not sufficient for, induction of squamous differentiation.

    PubMed

    Harvat, B L; Wang, A; Seth, P; Jetten, A M

    1998-05-01

    Irreversible growth arrest is an early and integral part of squamous cell differentiation in normal human epidermal keratinocytes (NHEKs) and is assumed to be linked to the control of expression of differentiation-specific genes. In this study, we examine the link between the molecular events associated with growth arrest and the expression of differentiation genes. NHEKs that have been induced to undergo growth arrest and differentiation by suspension culture contain populations in both G1 and G2/M of the cell cycle. The irreversible growth arrest state in NHEKs is characterized by an accumulation of the hypophosphorylated forms of Rb and p130, with subsequent down-regulation of levels of Rb, up-regulation of p130 and associated down-regulation of E2F-regulated genes such as cyclin A. These events correlate with an inhibition of G1 cdk activity, mediated in part by an increase in the cdk inhibitors p21(WAF1/Cip1), p27(Kip1) and p16(Ink4a). Flow cytometric and immunoblot analysis demonstrated that the timing of the up-regulation of p27, p16 and p130 corresponds closely with the induction of the squamous-specific genes cornifin alpha (SPRR-1) and transglutaminase type I, suggesting a close link between control of growth arrest and differentiation. However, growth arrest induced by over-expression of p27, p21 or p16 by recombinant adenovirus is not sufficient to induce expression of the differentiation genes, or to invoke the pattern of cell cycle regulatory protein expression characteristic of the differentiation-specific irreversible growth arrest. We conclude that growth arrest mediated by activation of the Rb pathway is not sufficient to trigger terminal squamous differentiation and additional signals which can be generated during suspension culture are required to promote the complete differentiation program. PMID:9547295

  10. Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells

    PubMed Central

    Wang, Hui-Chen; Hsu, Sung-Po; Lee, Wen-Sen

    2015-01-01

    Previously, we demonstrated that progesterone (P4) at physiologic levels (5-500 nM) inhibited proliferation in cultured rat aortic smooth muscle cells (RASMCs) through a P4 receptor (PR)-dependent pathway. We also showed that P4-induced cell cycle arrest in RASMCs occurs when the cyclin-CDK2 system is inhibited just as p21cip1 and p27kip1 protein levels are augmented. In the present study, we further investigated the molecular mechanism underlying P4-induced up-regulations of p21cip1 and p27kip1 in RASMCs. We used pharmacological inhibitors as well as dominant negative constructs and conducted Western blot analyses to delineate the signaling pathway involved. Our data suggest that P4 up-regulated the expression of p21cip1 and p27kip1 in RASMCs through increasing the level of p53 protein mediated by activating the cSrc/Kras/Raf-1/AKT/ERK/p38/IκBα/NFκB pathway. The findings of the present study highlight the molecular mechanism underlying P4-induced up-regulations in p21cip1 and p27kip1 in RASMCs. PMID:25932965

  11. 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.

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

    PubMed

    Omran, O M; AlSheeha, M

    2015-01-01

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

  13. Tumor suppressor gene p16/INK4A/CDKN2A-dependent regulation into and out of the cell cycle in a spontaneous canine model of breast cancer.

    PubMed

    Agarwal, Payal; Sandey, Maninder; DeInnocentes, Patricia; Bird, R Curtis

    2013-06-01

    p16/INK4A/CDKN2A is an important tumor suppressor gene that arrests cell cycle in G1 phase inhibiting binding of CDK4/6 with cyclin D1, leaving the Rb tumor suppressor protein unphosphorylated and E2F bound and inactive. We hypothesized that p16 has a role in exit from cell cycle that becomes defective in cancer cells. Well characterized p16-defective canine mammary cancer cell lines (CMT28, CMT27, and CMT12), derived stably p16-transfected CMT cell clones (CMT27A, CMT27H, CMT28A, and CMT28F), and normal canine fibroblasts (NCF), were used to investigate expression of p16 after serum starvation into quiescence followed by re-feeding to induce cell cycle re-entry. The parental CMT cell lines used lack p16 expression either at the mRNA or protein expression levels, while p27 and other p16-associated proteins, including CDK4, CDK6, cyclin D1, and Rb, were expressed. We have successfully demonstrated cell cycle arrest and relatively synchronous cell cycle re-entry in parental CMT12, CMT28 and NCF cells as well as p16 transfected CMT27A, CMT27H, CMT28A, and CMT28F cells and confirmed this by (3)H-thymidine incorporation and flow cytometric analysis of cell cycle phase distribution. p16-transfected CMT27A and CMT27H cells exited cell cycle post-serum-starvation in contrast to parental CMT27 cells. NCF, CMT27A, and CMT28F cells expressed upregulated levels of p27 and p16 mRNA, post-serum starvation, as cells exited cell cycle and entered quiescence. Because quiescence and differentiation are associated with increased levels of p27, our data demonstrating that p16 was upregulated along with p27 during quiescence, suggests a potential role for p16 in maintaining these non-proliferative states. PMID:23238983

  14. 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

    2009-05-01

    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. PMID:19244234

  15. p21(CIP1/WAF1)-dependent inhibition of cardiac hypertrophy in response to Angiotensin II involves Akt/Myc and pRb signaling.

    PubMed

    Hauck, Ludger; Grothe, Daniela; Billia, Filio

    2016-09-01

    The cyclin-dependent kinase inhibitor p21(CIP1/WAF1) (p21) is highly expressed in the adult heart. However, in response to stress, its expression is downregulated. Therefore, we investigated the role of p21 in the regulation of cardiac hypertrophic growth. At 2 months of age, p21 knockout mice (p21KO) lack an overt cardiac phenotype. In contrast, by 10 months of age, p21KO developed age-dependent cardiac hypertrophy and heart failure. After 3 weeks of trans-aortic banding (TAB), the heart/body weight ratio in 11 week old p21KO mice increased by 57%, as compared to 42% in wild type mice indicating that p21KO have a higher susceptibility to pressure overload-induced cardiac hypertrophy. We then chronically infused 8 week old wild type mice with Angiotensin II (2.0mg/kg/min) or saline subcutaneously by osmotic pumps for 14 days. Recombinant TAT conjugated p21 protein variants (10mg/kg body weight) or saline were intraperitoneally injected once daily for 14 days into Angiotensin II and saline-infused animals. Angiotensin II treated mice developed pathological cardiac hypertrophy with an average increase of 38% in heart/body weight ratios, as compared to saline-treated controls. Reconstitution of p21 function by TAT.p21 protein transduction prevented Angiotensin II-dependent development of cardiac hypertrophy and failure. Taken together, our genetic and biochemical data show an important function of p21 in the regulation of growth-related processes in the heart. PMID:27486069

  16. 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

    2009-05-01

    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.

  17. Proto-oncogene FBI-1 Represses Transcription of p21CIP1 by Inhibition of Transcription Activation by p53 and Sp1*S⃞

    PubMed Central

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

    2009-01-01

    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. PMID:19244234

  18. p21(CIP1/WAF1)-dependent inhibition of cardiac hypertrophy in response to Angiotensin II involves Akt/Myc and pRb signaling.

    PubMed

    Hauck, Ludger; Grothe, Daniela; Billia, Filio

    2016-09-01

    The cyclin-dependent kinase inhibitor p21(CIP1/WAF1) (p21) is highly expressed in the adult heart. However, in response to stress, its expression is downregulated. Therefore, we investigated the role of p21 in the regulation of cardiac hypertrophic growth. At 2 months of age, p21 knockout mice (p21KO) lack an overt cardiac phenotype. In contrast, by 10 months of age, p21KO developed age-dependent cardiac hypertrophy and heart failure. After 3 weeks of trans-aortic banding (TAB), the heart/body weight ratio in 11 week old p21KO mice increased by 57%, as compared to 42% in wild type mice indicating that p21KO have a higher susceptibility to pressure overload-induced cardiac hypertrophy. We then chronically infused 8 week old wild type mice with Angiotensin II (2.0mg/kg/min) or saline subcutaneously by osmotic pumps for 14 days. Recombinant TAT conjugated p21 protein variants (10mg/kg body weight) or saline were intraperitoneally injected once daily for 14 days into Angiotensin II and saline-infused animals. Angiotensin II treated mice developed pathological cardiac hypertrophy with an average increase of 38% in heart/body weight ratios, as compared to saline-treated controls. Reconstitution of p21 function by TAT.p21 protein transduction prevented Angiotensin II-dependent development of cardiac hypertrophy and failure. Taken together, our genetic and biochemical data show an important function of p21 in the regulation of growth-related processes in the heart.

  19. Down-regulation of both p21/Cip1 and p27/Kip1 produces a more aggressive prostate cancer phenotype

    PubMed Central

    Roy, Srirupa; Singh, Rana P.; Agarwal, Chapla; Siriwardana, Sunitha; Sclafani, Robert; Agarwal, Rajesh

    2009-01-01

    Roles of cyclin dependent kinase inhibitors, p21/Cip1 (p21) and p27/Kip1 (p27) in prostate cancer (PCa) progression is still not clear. Lower p27 protein expression in PCa tissues is often associated with poor prognosis, but prognostic significance of p21 is still controversial. Herein, we investigated the role of these molecules in determining PCa growth characteristics. We generated human PCa DU145 cell variants with knocked down levels of p21 (DU-p21) or p27 (DU-p27), or both (DU-p21+p27) via retroviral transduction of respective shRNAs and compared their various characteristics with empty vector-transduced DU145 (DU-EV) cells in vitro as well as in vivo. Knocking down either p21 or p27 did not show any significant change in doubling time, clonogenicity and cell cycle progression in DU145 cells, but simultaneous knock-down of both p21 and p27 significantly enhanced these parameters. In athymic mice, DU-p21+p27 tumors showed higher growth rate than the comparable growth of DU-EV, DU-p21 and DU-p27 tumors. Concurrently, DU-p21+p27 tumors had significantly higher proliferation rate, showing 54% and 48% increase in proliferating cell nuclear antigen (PCNA) and Ki-67-positive cells, respectively, compared to DU-EV tumors. DU-p21+p27 tumors also showed higher microvessel density and increased expression of vascular endothelial growth factor (VEGF). Proliferation and angiogenic status of DU-p21 and DU-p27 tumors was comparable to DU-EV tumors. Both in vitro and in vivo results implicate that p21 and p27 have compensatory roles in advanced prostate cancer cells, and ablation or down-modulation of both these molecules essentially enhances the aggressive prostate carcinoma phenotype. PMID:18583941

  20. 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.

  1. A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

    PubMed Central

    2012-01-01

    Introduction Tumor cell migration and invasion are critical initiation steps in the process of breast cancer metastasis, the primary cause of breast cancer morbidity and death. Here we investigated the role of p21Cip1 (p21), a member of the core cell cycle machinery, in transforming growth factor-beta (TGFβ)-mediated breast cancer cell migration and invasion. Methods A mammary fat pad xenograft mouse model was used to assess the mammary tumor growth and local invasion. The triple negative human breast cancer cell lines MDA-MB231 and its sub-progenies SCP2 and SCP25, SUM159PT, SUM149PT, SUM229PE and SUM1315MO2 were treated with 5 ng/ml TGFβ and the protein expression levels were measured by Western blot. Cell migration and invasion were examined using the scratch/wound healing and Transwell assay. TGFβ transcriptional activity was measured by a TGFβ/Smad reporter construct (CAGA12-luc) using luciferase assay. q-PCR was used for assessing TGFβ downstream target genes. The interactions among p21, p/CAF and Smad3 were performed by co-immunoprecipitation. In addition, Smad3 on DNA binding ability was measured by DNA immunoprecipitation using biotinylated Smad binding element DNA probes. Finally, the association among active TGFβ/Smad signaling, p21 and p/CAF with lymph node metastasis was examined by immunohistochemistry in tissue microarray containing 50 invasive ductal breast tumors, 25 of which are lymph node positive. Results We found p21 expression to correlate with poor overall and distant metastasis free survival in breast cancer patients. Furthermore, using xenograft animal models and in vitro studies, we found p21 to be essential for tumor cell invasion. The invasive effects of p21 were found to correlate with Smad3, and p/CAF interaction downstream of TGFβ. p21 and p/CAF regulates TGFβ-mediated transcription of pro-metastatic genes by controlling Smad3 acetylation, DNA binding and transcriptional activity. In addition, we found that active TGF

  2. 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 Central

    Su, Liang-Cheng; Jiang, Shih Sheng; Chan, Tzu-Min; Chang, Chung-Ho; Chen, Li-Tzong; Kung, Hsing-Jien; Wang, Horng-Dar; Chuu, Chih-Pin

    2015-01-01

    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. PMID:25788262

  3. 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.

  4. 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. PMID:24937448

  5. 8-60hIPP5(m)-induced G2/M cell cycle arrest involves activation of ATM/p53/p21(cip1/waf1) pathways and delayed cyclin B1 nuclear translocation.

    PubMed

    Zeng, Qi-Yan; Zeng, Lin-Jie; Huang, Yu; Huang, Yong-Qi; Zhu, Qi-Fang; Liao, Zhi-Hong

    2014-01-01

    Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. The active mutant IPP5 (8-60hIPP5(m)), the latest member of the inhibitory molecules for PP1, has been shown to inhibit the growth of human cervix carcinoma cells (HeLa). In order to elucidate the underlying mechanisms, the present study assessed overexpression of 8-60hIPP5(m) in HeLa cells. Flow cytometric and biochemical analyses showed that overexpression of 8-60hIPP5(m) induced G2/M-phase arrest, which was accompanied by the upregulation of cyclin B1 and phosphorylation of G2/M-phase proteins ATM, p53, p21(cip1/waf1) and Cdc2, suggesting that 8-60hIPP5(m) induces G2/M arrest through activation of the ATM/p53/p21(cip1/waf1)/Cdc2/ cyclin B1 pathways. We further showed that overexpression of 8-60hIPP5(m) led to delayed nuclear translocation of cyclin B1. 8-60hIPP5(m) also could translocate to the nucleus in G2/M phase and interact with pp1α and Cdc2 as demonstrated by co-precipitation assay. Taken together, our data demonstrate a novel role for 8-60hIPP5(m) in regulation of cell cycle in HeLa cells, possibly contributing to the development of new therapeutic strategies for cervix carcinoma.

  6. 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. PMID:19471103

  7. 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.

  8. Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21Cip1 expression and hampers tumour cell growth in vitro and in vivo

    PubMed Central

    Raimondi, L; Ciarapica, R; De Salvo, M; Verginelli, F; Gueguen, M; Martini, C; De Sio, L; Cortese, G; Locatelli, M; Dang, T P; Carlesso, N; Miele, L; Stifani, S; Limon, I; Locatelli, F; Rota, R

    2012-01-01

    Rhabdomyosarcoma (RMS) is a paediatric soft-tissue sarcoma arising from skeletal muscle precursors coexpressing markers of proliferation and differentiation. Inducers of myogenic differentiation suppress RMS tumourigenic phenotype. The Notch target gene HES1 is upregulated in RMS and prevents tumour cell differentiation in a Notch-dependent manner. However, Notch receptors regulating this phenomenon are unknown. In agreement with data in RMS primary tumours, we show here that the Notch3 receptor is overexpressed in RMS cell lines versus normal myoblasts. Notch3-targeted downregulation in RMS cells induces hyper-phosphorylation of p38 and Akt essential for myogenesis, resulting in the differentiation of tumour cells into multinucleated myotubes expressing Myosin Heavy Chain. These phenomena are associated to a marked decrease in HES1 expression, an increase in p21Cip1 level and the accumulation of RMS cells in the G1 phase. HES1-forced overexpression in RMS cells reverses, at least in part, the pro-differentiative effects of Notch3 downregulation. Notch3 depletion also reduces the tumourigenic potential of RMS cells both in vitro and in vivo. These results indicate that downregulation of Notch3 is sufficient to force RMS cells into completing a correct full myogenic program providing evidence that it contributes, partially through HES1 sustained expression, to their malignant phenotype. Moreover, they suggest Notch3 as a novel potential target in human RMS. PMID:22117196

  9. Epigenetic reactivation of p21CIP1/WAF1 and KLOTHO by a combination of bioactive dietary supplements is partially ERα-dependent in ERα-negative human breast cancer cells.

    PubMed

    Sinha, Sonam; Shukla, Samriddhi; Khan, Sajid; Tollefsbol, Trygve O; Meeran, Syed M

    2015-05-01

    Available treatment strategies against estrogen receptor (ER)-negative breast cancer patients are limited due to their poor response to hormonal therapy. We have shown previously that the combinations of green tea polyphenols (GTPs), a dietary DNA methyltransferase inhibitor, and sulforaphane (SFN), a dietary histone deacetylase inhibitor, reactivate ERα expression in ERα-negative MDA-MB-231 cells. Here, we investigated the functional significance of ERα reactivation in the reactivation of silenced tumor suppressor genes (TSGs) in ERα-negative human breast cancer cells. We found that the treatment of MDA-MB-231 cells with the combinations of GTPs and SFN leads to the reactivation of silenced TSGs such as p21(CIP1/WAF1) and KLOTHO through active chromatin modifications. Further, GTPs- and SFN-mediated reactivation of TSGs was, at least in part, dependent on ERα reactivation in ERα-negative MDA-MB-231 cells. Collectively, our findings suggest that a novel combination of bioactive dietary supplements could further be explored as an effective therapeutic option against hormonal refractory breast cancer.

  10. Essential interaction between the fission yeast DNA polymerase δ subunit Cdc27 and Pcn1 (PCNA) mediated through a C-terminal p21Cip1-like PCNA binding motif

    PubMed Central

    Reynolds, Nicola; Warbrick, Emma; Fantes, Peter A.; MacNeill, Stuart A.

    2000-01-01

    Direct interaction between DNA polymerase δ and its processivity factor proliferating cell nuclear antigen (PCNA) is essential for effective replication of the eukaryotic genome, yet the precise manner by which this occurs is unclear. We show that the 54 kDa subunit of DNA polymerase δ from Schizosaccharomyces pombe interacts directly with Pcn1 (PCNA) both in vivo and in vitro. Binding is effected via a short sequence at the C–terminus of Cdc27 with significant similarity to the canonical PCNA binding motif first identified in the mammalian p21Cip1 protein. This motif is both necessary and sufficient for binding of Pcn1 by Cdc27 in vitro and is essential for Cdc27 function in vivo. We also show that the Pcn1 binding motif in Cdc27 is distinct from its binding site for Cdc1, the 55 kDa B-subunit of polymerase δ, and present evidence that Cdc27 can bind to Pcn1 and Cdc1 simultaneously. Finally, we show that Cdc27 performs at least two distinct essential functions, one of which is independent of Pcn1 binding. PMID:10698951

  11. CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis

    PubMed Central

    Mao, Weiqun; Ahmed, Ahmed A.; Yang, Hailing; Zhou, Jinhua; Jennings, Nicholas; Rodriguez-Aguayo, Cristian; Lopez-Berestein, Gabriel; Miranda, Roberto; Qiao, Wei; Baladandayuthapani, Veera; Li, Zongfang; Sood, Anil K.; Liu, Jinsong; Le, Xiao-Feng; Bast, Robert C.

    2015-01-01

    Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27Kip1 as well as TP53-dependent transcriptional induction of p21Cip1 in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells. PMID:26146988

  12. CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis.

    PubMed

    Zhang, Shu; Lu, Zhen; Mao, Weiqun; Ahmed, Ahmed A; Yang, Hailing; Zhou, Jinhua; Jennings, Nicholas; Rodriguez-Aguayo, Cristian; Lopez-Berestein, Gabriel; Miranda, Roberto; Qiao, Wei; Baladandayuthapani, Veera; Li, Zongfang; Sood, Anil K; Liu, Jinsong; Le, Xiao-Feng; Bast, Robert C

    2015-01-01

    Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1) as well as TP53-dependent transcriptional induction of p21(Cip1) in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells. PMID:26146988

  13. MycN Is Critical for the Maintenance of Human Embryonic Stem Cell-Derived Neural Crest Stem Cells.

    PubMed

    Zhang, Jie Ting; Weng, Zhi Hui; Tsang, Kam Sze; Tsang, Lai Ling; Chan, Hsiao Chang; Jiang, Xiao Hua

    2016-01-01

    The biologic studies of human neural crest stem cells (hNCSCs) are extremely challenging due to the limited source of hNCSCs as well as ethical and technical issues surrounding isolation of early human embryonic tissues. On the other hand, vast majority of studies on MycN have been conducted in human tumor cells, thus, the role of MycN in normal human neural crest development is completely unknown. In the present study, we determined the role of MycN in hNCSCs isolated from in vitro-differentiating human embryonic stem cells (hESCs). For the first time, we show that suppression of MycN in hNCSCs inhibits cell growth and cell cycle progression. Knockdown of MycN in hNCSCs increases the expression of Cdkn1a, Cdkn2a and Cdkn2b, which encodes the cyclin-dependent kinases p21CIP1, p16 INK4a and p15INK4b. In addition, MycN is involved in the regulation of human sympathetic neurogenesis, as knockdown of MycN enhances the expression of key transcription factors involved in sympathetic neuron differentiation, including Phox2a, Phox2b, Mash1, Hand2 and Gata3. We propose that unlimited source of hNCSCs provides an invaluable platform for the studies of human neural crest development and diseases.

  14. 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...

  15. 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.

  16. Uropathogenic E. coli infection provokes epigenetic downregulation of CDKN2A (p16INK4A) in uroepithelial cells.

    PubMed

    Tolg, Cornelia; Sabha, Nesrin; Cortese, Rene; Panchal, Trupti; Ahsan, Alya; Soliman, Ashraf; Aitken, Karen J; Petronis, Arturas; Bägli, Darius J

    2011-06-01

    Host cell and bacterial factors determine severity and duration of infections. To allow for bacteria pathogenicity and persistence, bacteria have developed mechanisms that modify expression of host genes involved in cell cycle progression, apoptosis, differentiation and the immune response. Recently, Helicobacter pylori infection of the stomach has been correlated with epigenetic changes in the host genome. To identify epigenetic changes during Escherichia coli induced urinary tract infection (UTI), we developed an in vitro model of persistent infection of human uroepithelial cells with uropathogenic E. coli (UPEC), resulting in intracellular bacteria colonies. Cells inoculated with FimH-negative E. coli (N-UPEC) that are not internalized and non-inoculated cells were used as controls. UPEC infection significantly induced de novo methyltransferase (DNMT) activity (12.5-fold P=0.002 UPEC vs non-inoculated and 250-fold P=0.001 UPEC vs N-UPEC inoculated cells) and Dnmt1 RNA expression (6-fold P=0.04 UPEC vs non-inoculated cells) compared with controls. DNMT1 protein levels were significantly increased in three uroepithelial cell lines (5637, J82, HT-1197) in response to UPEC infection as demonstrated by confocal analysis. Real-time PCR analysis of candidate genes previously associated with bacteria infection and/or innate immunity, revealed UPEC-induced downregulation of the tumor suppressor gene CDKN2A (3.3-fold P=0.007 UPEC vs non-inoculated and 3.3-fold P=0.001 UPEC vs N-UPEC) and the DNA repair gene MGMT (9-fold P=0.03 UPEC vs non-inoculated). Expression of CDH1, MLH1, DAPK1 and TLR4 was not affected. Pyrosequencing of CDKN2A and MGMT CpG islands revealed increased methylation in CDKN2A exon 1 (3.8-fold P=0.04 UPEC vs N-UPEC and UPEC vs non-inoculated). Methylation of MGMT was not affected. UPEC-induced methylation of CDKN2A exon 1 may increase bladder cancer and presage UTI risk, and be useful as a biological marker for UTI susceptibility or recurrence.

  17. Focal adhesion kinase antagonizes doxorubicin cardiotoxicity via p21(Cip1.).

    PubMed

    Cheng, Zhaokang; DiMichele, Laura A; Rojas, Mauricio; Vaziri, Cyrus; Mack, Christopher P; Taylor, Joan M

    2014-02-01

    Clinical application of potent anthracycline anticancer drugs, especially doxorubicin (DOX), is limited by a toxic cardiac side effect that is not fully understood and preventive strategies are yet to be established. Studies in genetically modified mice have demonstrated that focal adhesion kinase (FAK) plays a key role in regulating adaptive responses of the adult myocardium to pathological stimuli through activation of intracellular signaling cascades that facilitate cardiomyocyte growth and survival. The objective of this study was to determine if targeted myocardial FAK activation could protect the heart from DOX-induced de-compensation and to characterize the underlying mechanisms. To this end, mice with myocyte-restricted FAK knock-out (MFKO) or myocyte-specific expression of an active FAK variant (termed SuperFAK) were subjected to DOX treatment. FAK depletion enhanced susceptibility to DOX-induced myocyte apoptosis and cardiac dysfunction, while elevated FAK activity provided remarkable cardioprotection. Our mec6hanistic studies reveal a heretofore unappreciated role for the protective cyclin-dependent kinase inhibitor p21 in the repression of the pro-apoptotic BH3-only protein Bim and the maintenance of mitochondrial integrity and myocyte survival. DOX treatment induced proteasomal degradation of p21, which exacerbated mitochondrial dysfunction and cardiomyocyte apoptosis. FAK was both necessary and sufficient for maintaining p21 levels following DOX treatment and depletion of p21 compromised FAK-dependent protection from DOX. These findings identify p21 as a key determinant of DOX resistance downstream of FAK in cardiomyocytes and indicate that cardiac-restricted enhancement of the FAK/p21 signaling axis might be an effective strategy to preserve myocardial function in patients receiving anthracycline chemotherapy. PMID:24342076

  18. 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.

  19. 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.

  20. p16INK4A immunohistochemistry is superior to HPV in situ hybridization for the detection of high-risk HPV in atypical squamous metaplasia.

    PubMed

    Kong, Christina S; Balzer, Bonnie L; Troxell, Megan L; Patterson, Bruce K; Longacre, Teri A

    2007-01-01

    In situ hybridization (ISH) assays for high-risk human papillomavirus (HR-HPV) and immunohistochemical (IHC) assays for surrogate markers such as p16 can be useful in detecting HR-HPV in cervical dysplasia, but the use of these markers in problematic cervical biopsies has not been well-established. We evaluated 3 chromogenic ISH assays (Ventana INFORM HPVII and HPVIII and DakoCytomation GenPoint) in conjunction with p16 IHC and HPV polymerase chain reaction in a study set consisting of 12 low-grade squamous intraepithelial lesions, 16 high-grade squamous intraepithelial lesions, and 30 benign cervix samples. A test set of 28 cases of atypical squamous metaplasia were also evaluated withVentana HPVIII ISH and p16 IHC. In the study set, the sensitivity of the DakoCytomation ISH assay (which detects HPV subtypes 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, and 68) was similar to the Ventana HPVII assay but less than that of the Ventana HPVIII ISH assay (both of which detect HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) and less than p16 IHC (55.6% vs. 53.6 vs. 69.2% vs. 82.1%). All HPV ISH assays exhibited 100% specificity. p16 reactivity consisted of 2 patterns: focal strong and diffuse strong. Because focal strong p16 reactivity was identified in benign squamous epithelium (6.7% cases) and dysplastic epithelium, it was considered an equivocal result and only diffuse strong reactivity was considered to be specific for the presence of HR-HPV. In the squamous intraepithelial lesions study set, the difference in sensitivity between Ventana HPVIII ISH and p16 was not statistically significant. However, in the atypical squamous metaplasia test set cases, p16 reactivity (focal strong and diffuse strong) was significantly more sensitive than Ventana HPVIII ISH in correlating with the presence of human papillomavirus as detected by polymerase chain reaction (83.3% vs. 33.3% P=0.004). Because focal strong p16 reactivity is less specific, cases with this staining pattern are considered atypical and require further evaluation by other means. Overall, p16 IHC is considered the best candidate for the initial assessment of cervical biopsies that are histologically indeterminate for dysplasia given its wide availability, comparative ease of interpretation, and high sensitivity and specificity.

  1. INK4 locus of the tumor-resistant rodent, the naked mole rat, expresses a functional p15/p16 hybrid isoform.

    PubMed

    Tian, Xiao; Azpurua, Jorge; Ke, Zhonghe; Augereau, Adeline; Zhang, Zhengdong D; Vijg, Jan; Gladyshev, Vadim N; Gorbunova, Vera; Seluanov, Andrei

    2015-01-27

    The naked mole rat (Heterocephalus glaber) is a long-lived and tumor-resistant rodent. Tumor resistance in the naked mole rat is mediated by the extracellular matrix component hyaluronan of very high molecular weight (HMW-HA). HMW-HA triggers hypersensitivity of naked mole rat cells to contact inhibition, which is associated with induction of the INK4 (inhibitors of cyclin dependent kinase 4) locus leading to cell-cycle arrest. The INK4a/b locus is among the most frequently mutated in human cancer. This locus encodes three distinct tumor suppressors: p15(INK4b), p16(INK4a), and ARF (alternate reading frame). Although p15(INK4b) has its own ORF, p16(INK4a) and ARF share common second and third exons with alternative reading frames. Here, we show that, in the naked mole rat, the INK4a/b locus encodes an additional product that consists of p15(INK4b) exon 1 joined to p16(INK4a) exons 2 and 3. We have named this isoform pALT(INK4a/b) (for alternative splicing). We show that pALT(INK4a/b) is present in both cultured cells and naked mole rat tissues but is absent in human and mouse cells. Additionally, we demonstrate that pALT(INK4a/b) expression is induced during early contact inhibition and upon a variety of stresses such as UV, gamma irradiation-induced senescence, loss of substrate attachment, and expression of oncogenes. When overexpressed in naked mole rat or human cells, pALT(INK4a/b) has stronger ability to induce cell-cycle arrest than either p15(INK4b) or p16(INK4a). We hypothesize that the presence of the fourth product, pALT(INK4a/b) of the INK4a/b locus in the naked mole rat, contributes to the increased resistance to tumorigenesis of this species.

  2. 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

  3. P21Cip1 Protects against Oxidative Stress by Suppressing ER Dependent Activation of Mitochondrial Death Pathways

    PubMed Central

    Vitiello, Peter F.; Wu, Yu-Chieh M.; Staversky, Rhonda J.; O’Reilly, Michael A.

    2009-01-01

    Although it is well established that the cell cycle inhibitor p21 protects against genotoxic stress by preventing the replication of damaged DNA, recent studies have shown cytoplasmic forms can also protect. It protects by delaying the loss of the anti-apoptotic proteins, Mcl-1 and Bcl-XL; however, the mechanism of regulation is unknown. Utilizing hyperoxia as a model of chronic oxidative stress and DNA damage, p21 was detected in the nucleus and cytoplasm and cytoplasmic expression of p21 was sufficient for cytoprotection. P21 was enriched in a subcellular fraction containing mitochondria and endoplasmic reticulum (ER), suggesting that it may be coordinating ER and mitochondrial stress pathways. Consistent with this, p21 suppressed hyperoxic downregulation of BiP and subsequent activation ER stress signaling which effected Mcl-1, but not Bcl-XL; though both inhibited hyperoxic cell death. Taken together, these data show that p21 integrates the DNA damage response with ER stress signaling which then regulates mitochondrial death pathways during chronic genotoxic stress. PMID:18948188

  4. Increased levels of p21((CIP1/WAF1)) correlate with decreased chondrogenic differentiation potential in synovial membrane progenitor cells.

    PubMed

    Masson, Anand Oliveira; Hess, Ricarda; O'Brien, Kate; Bertram, Karri L; Tailor, Pankaj; Irvine, Edward; Ren, Guomin; Krawetz, Roman J

    2015-07-01

    Cartilage injuries are a major concern in the field of orthopedics. They occur following trauma, as well as from a variety of pathological conditions including Osteoarthritis (OA). Although cartilage does not exhibit robust endogenous repair, it has been demonstrated that modulating the activity of p21 can increase the regenerative abilities of cartilage in vitro and in vivo. Since the synovial membrane is abundant with mesenchymal progenitor cells (MPCs) capable of differentiating into cartilage both in vitro and in vivo, we examined if p21 expression levels varied between MPCs derived from normal vs. OA knee joints. Analysis of p21 at the mRNA and protein levels within normal and OA MPCs demonstrated differential levels of expression between these two groups, with OA MPCs having higher p21 expression levels. The higher levels of p21 in OA MPCs are also correlated with a decreased chondrogenic differentiation capacity and synovial inflammation, however, there was no evidence of senescence in the OA cells. The results of this study suggest that cell cycle regulation in MPCs may be altered in OA and that modulation of this pathway may have therapeutic potential once the mechanism by which this regulates stem/progenitor cells is better understood.

  5. 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

  6. Role of stress-activated OCT4A in the cell fate decisions of embryonal carcinoma cells treated with etoposide.

    PubMed

    Huna, Anda; Salmina, Kristine; Erenpreisa, Jekaterina; Vazquez-Martin, Alejandro; Krigerts, Jekabs; Inashkina, Inna; Gerashchenko, Bogdan I; Townsend, Paul A; Cragg, Mark S; Jackson, Thomas R

    2015-01-01

    Tumor cellular senescence induced by genotoxic treatments has recently been found to be paradoxically linked to the induction of "stemness." This observation is critical as it directly impinges upon the response of tumors to current chemo-radio-therapy treatment regimens. Previously, we showed that following etoposide (ETO) treatment embryonal carcinoma PA-1 cells undergo a p53-dependent upregulation of OCT4A and p21Cip1 (governing self-renewal and regulating cell cycle inhibition and senescence, respectively). Here we report further detail on the relationship between these and other critical cell-fate regulators. PA-1 cells treated with ETO display highly heterogeneous increases in OCT4A and p21Cip1 indicative of dis-adaptation catastrophe. Silencing OCT4A suppresses p21Cip1, changes cell cycle regulation and subsequently suppresses terminal senescence; p21Cip1-silencing did not affect OCT4A expression or cellular phenotype. SOX2 and NANOG expression did not change following ETO treatment suggesting a dissociation of OCT4A from its pluripotency function. Instead, ETO-induced OCT4A was concomitant with activation of AMPK, a key component of metabolic stress and autophagy regulation. p16ink4a, the inducer of terminal senescence, underwent autophagic sequestration in the cytoplasm of ETO-treated cells, allowing alternative cell fates. Accordingly, failure of autophagy was accompanied by an accumulation of p16ink4a, nuclear disintegration, and loss of cell recovery. Together, these findings imply that OCT4A induction following DNA damage in PA-1 cells, performs a cell stress, rather than self-renewal, function by moderating the expression of p21Cip1, which alongside AMPK helps to then regulate autophagy. Moreover, this data indicates that exhaustion of autophagy, through persistent DNA damage, is the cause of terminal cellular senescence. PMID:26102294

  7. Total body irradiation selectively induces murine hematopoietic stem cell senescence.

    PubMed

    Wang, Yong; Schulte, Bradley A; LaRue, Amanda C; Ogawa, Makio; Zhou, Daohong

    2006-01-01

    Exposure to ionizing radiation (IR) and certain chemotherapeutic agents not only causes acute bone marrow (BM) suppression but also leads to long-term residual hematopoietic injury. This latter effect has been attributed to damage to hematopoietic stem cell (HSC) self-renewal. Using a mouse model, we investigated whether IR induces senescence in HSCs, as induction of HSC senescence can lead to the defect in HSC self-renewal. It was found that exposure of C57BL/6 mice to a sublethal dose (6.5 Gy) of total body irradiation (TBI) resulted in a sustained quantitative and qualitative reduction of LKS+ HSCs. In addition, LKS+ HSCs from irradiated mice exhibited an increased expression of the 2 commonly used biomarkers of cellular senescence, p16(Ink4a) and SA-beta-gal. In contrast, no such changes were observed in irradiated LKS- hematopoietic progenitor cells. These results provide the first direct evidence demonstrating that IR exposure can selectively induce HSC senescence. Of interest, the induction of HSC senescence was associated with a prolonged elevation of p21(Cip1/Waf1), p19(Arf), and p16(Ink4a) mRNA expression, while the expression of p27(Kip1) and p18(Ink4c) mRNA was not increased following TBI. This suggests that p21(Cip1/Waf1), p19(Arf), and p16(Ink4a) may play an important role in IR-induced senescence in HSCs.

  8. Evidence for a CDK4-dependent checkpoint in a conditional model of cellular senescence

    PubMed Central

    Brookes, Sharon; Gagrica, Sladjana; Sanij, Elaine; Rowe, Janice; Gregory, Fiona J; Hara, Eiji; Peters, Gordon

    2015-01-01

    Cellular senescence, the stable cell cycle arrest elicited by various forms of stress, is an important facet of tumor suppression. Although much is known about the key players in the implementation of senescence, including the pRb and p53 axes and the cyclin dependent kinase inhibitors p16INK4a and p21CIP1, many details remain unresolved. In studying conditional senescence in human fibroblasts that express a temperature sensitive SV40 large T-antigen (T-Ag), we uncovered an unexpected role for CDK4. At the permissive temperature, where pRb and p53 are functionally compromised by T-Ag, cyclin D-CDK4 complexes are disrupted by the high p16INK4a levels and reduced expression of p21CIP1. In cells arrested at the non-permissive temperature, p21CIP1 promotes reassembly of cyclin D-CDK4 yet pRb is in a hypo-phosphorylated state, consistent with cell cycle arrest. In exploring whether the reassembled cyclin D-CDK4-p21 complexes are functional, we found that shRNA-mediated knockdown or chemical inhibition of CDK4 prevented the increase in cell size associated with the senescent phenotype by allowing the cells to arrest in G1 rather than G2/M. The data point to a role for CDK4 kinase activity in a G2 checkpoint that contributes to senescence. PMID:25695870

  9. Melanoma candidate genes CDKN2A/p16/INK4A, p14ARF, and CDK4 sequencing in patients with uveal melanoma with relative high-risk for hereditary cancer predisposition.

    PubMed

    Abdel-Rahman, Mohamed H; Pilarski, Robert; Massengill, James B; Christopher, Benjamin N; Noss, Ryan; Davidorf, Frederick H

    2011-06-01

    The reported frequencies of germline mutations in the melanoma candidate genes are low in patients with uveal melanoma (UM). However, the number of families studied is limited and the majority of the published reports used low-sensitivity techniques for mutational screening. Identifying the frequency of alterations in any of the melanoma genes in patients with UM with increased hereditary cancer risk is important for proper counseling of these patients. We studied a total of 47 patients with UM including three with a family history of UM, 18 with a family and/or personal history of cutaneous melanoma (CM), three with early age at diagnosis (<30), 11 with increased risk for a known familial cancer syndrome, and 12 with a second primary tumor. Germline screening for mutations in CDKN2A, p14ARF, and exon 2 of CDK4 was carried out by direct sequencing. We identified a variant (IVS1-69 C>T) of uncertain significance in exon 1b of p14ARF in one of the patients with UM and his mother who also had UM. The variant was neither detected in any of the other patients with UM nor in 146 controls. We did not identify pathogenic mutations in CDKN2A nor exon 2 of CDK4 gene. Our study supports the low frequency of germline mutation of the CM candidate genes in patients with UM with family histories suggestive of a high risk for hereditary cancer. Germline testing for CDKN2A might be reserved for patients with UM with a family history of two or more CM.

  10. EGFR and mutant p53 expand esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors

    PubMed Central

    Ohashi, Shinya; Natsuizaka, Mitsuteru; Wong, Gabrielle S.; Michaylira, Carmen Z.; Grugan, Katharine D.; Stairs, Douglas B.; Kalabis, Jiri; Vega, Maria E.; Kalman, Ross A.; Nakagawa, Momo; Klein-Szanto, Andres J; Herlyn, Meenhard; Diehl, J. Alan; Rustgi, Anil K.; Nakagawa, Hiroshi

    2010-01-01

    Transforming growth factor (TGF)-β is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive as to which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-β. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to enrichment of an EMT-competent cellular subpopulation amongst telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by induction of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT upon TGF-β stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in induction of p15INK4B and p16INK4A, reactivating the EGFR-dependent senescence program. Importantly, TGF-β-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis. PMID:20424117

  11. Karyopherin α2 induces apoptosis in tongue squamous cell carcinoma CAL-27 cells through the p53 pathway.

    PubMed

    Gao, Li; Yu, Lei; Li, Chun-Ming; Li, Ying; Jia, Bao-Lin; Zhang, Bin

    2016-06-01

    Tumor onset and progression are associated with dysfunction of the nuclear transport machinery at the level of import and export receptors. However, the role of Karyopherin α2 (KPNA2) in human tongue squamous cell carcinoma (TSCC) remains unknown. We assessed the proliferation, apoptosis and migration of TSCC CAL-27 cells using wound healing, Transwell and MTT assays, western blotting, electron microscopy and acridine orange/ethidium bromide staining following knockdown of KPNA2. The results revealed the antiproliferative, proapoptotic and anti-migratory effects of KPNA2 silencing on the TSCC CAL-27 cells. Moreover, the knockdown of KPNA2 proved to be accompanied by the upregulation of active caspase-3, cytochrome c, Bax, Bad and decreased expression of Bcl-2, p-Bad and XIAP. KPNA2 activated the caspase-dependent pathway in the CAL-27 cells with upregulation of p53, p21Cip1/Waf1 and p16INK4a. Thus, the present study demonstrated that p53/p21Cip1/Waf1/p16INK4a may be an important pathway involved in the function of KPNA2 in TSCC CAL-27 cells. PMID:27109484

  12. 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.

  13. Induction of p21CIP1 protein and cell cycle arrest after inhibition of Aurora B kinase is attributed to aneuploidy and reactive oxygen species.

    PubMed

    Kumari, Geeta; Ulrich, Tanja; Krause, Michael; Finkernagel, Florian; Gaubatz, Stefan

    2014-06-01

    Cell cycle progression requires a series of highly coordinated events that ultimately lead to faithful segregation of chromosomes. Aurora B is an essential mitotic kinase, which is involved in regulation of microtubule-kinetochore attachments and cytokinesis. Inhibition of Aurora B results in stabilization of p53 and induction of p53-target genes such as p21 to inhibit proliferation. We have previously demonstrated that induction of p21 by p53 after inhibition of Aurora B is dependent on the p38 MAPK, which promotes transcriptional elongation of p21 by RNA Pol II. In this study, we show that a subset of p53-target genes are induced in a p38-dependent manner upon inhibition of Aurora B. We also demonstrate that inhibition of Aurora B results in down-regulation of E2F-mediated transcription and that the cell cycle arrest after Aurora B inhibition depends on p53 and pRB tumor suppressor pathways. In addition, we report that activation of p21 after inhibition of Aurora B is correlated with increased chromosome missegregation and aneuploidy but not with binucleation or tetraploidy. We provide evidence that p21 is activated in aneuploid cells by reactive oxygen species (ROS) and p38 MAPK. Finally, we demonstrate that certain drugs that act on aneuploid cells synergize with inhibitors of Aurora B to inhibit colony formation and oncogenic transformation. These findings provide an important link between aneuploidy and the stress pathways activated by Aurora B inhibition and also support the use of Aurora B inhibitors in combination therapy for treatment of cancer.

  14. Immunohistochemical examination of the INK4 and Cip inhibitors in the rat neonatal cerebellum: cellular localization and the impact of protein calorie malnutrition.

    PubMed

    Shambaugh, G E; Haines, G K; Koch, A; Lee, E J; Zhou, J n; Pestell, R

    2000-02-01

    Expression of the cyclin-dependent kinase inhibitors (CKIs) has been linked to the inhibition of cellular proliferation and the induction of differentiation. Based on structure function analysis, two distinct families of CDKIs, the INK4 and the Cip/Kip family have been identified. The INK4 family member p16(Ink4), and the Cip/Kip protein p27(Kip1) have been implicated in normal development of the CNS and cerebellum. Recent studies have suggested a functional inter-dependence between the CKI and the abundance of cyclin D1 in orchestrating growth factor-induced cellular proliferation. The neonatal rat cerebellum undergoes proliferative growth and differentiation, localized to distinct topographical regions and cell types. The cell type and the temporal profile of CKI expression during postnatal cerebellar development had not been described. The current studies determined the specific cerebellar cell types in which the CKIs were expressed during post natal development by co-staining for cell-type specific markers. p16(Ink4a) and p27(Kip1) immunostaining was identified in both neurons and glial cells, increasing progressively between postnatal days 6 to 13 into adulthood. By contrast, neuronal and glial cell p21(Cip1) staining was prominent at days 6-11 and decreased thereafter. Cyclin D1 was expressed in the proliferating external granular cells, with occassional staining in the molecular, and internal granular layers. Dual immunostaining demonstrated cyclin D1 within cells expressing CKI (p16(Ink4a), p21(Cip1),p27(Kip1)). Cerebellar cellular growth arrest, induced by protein-calorie malnutrition, inhibited cyclin D1 protein levels without affecting CKI immunostaining suggesting CKI do not mediate the developmental arrest. These results demonstrate that the CKIs are induced by differentiation cues in specific cell types with distinct kinetics in the developing cerebellum in vivo.

  15. Protective Effect of Ginsenoside Rg1 on Hematopoietic Stem/Progenitor Cells through Attenuating Oxidative Stress and the Wnt/β-Catenin Signaling Pathway in a Mouse Model of d-Galactose-induced Aging

    PubMed Central

    Li, Jing; Cai, Dachuan; Yao, Xin; Zhang, Yanyan; Chen, Linbo; Jing, Pengwei; Wang, Lu; Wang, Yaping

    2016-01-01

    Stem cell senescence is an important and current hypothesis accounting for organismal aging, especially the hematopoietic stem cell (HSC). Ginsenoside Rg1 is the main active pharmaceutical ingredient of ginseng, which is a traditional Chinese medicine. This study explored the protective effect of ginsenoside Rg1 on Sca-1+ hematopoietic stem/progenitor cells (HSC/HPCs) in a mouse model of d-galactose-induced aging. The mimetic aging mouse model was induced by continuous injection of d-gal for 42 days, and the C57BL/6 mice were respectively treated with ginsenoside Rg1, Vitamin E or normal saline after 7 days of d-gal injection. Compared with those in the d-gal administration alone group, ginsenoside Rg1 protected Sca-1+ HSC/HPCs by decreasing SA-β-Gal and enhancing the colony forming unit-mixture (CFU-Mix), and adjusting oxidative stress indices like reactive oxygen species (ROS), total anti-oxidant (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and malondialdehyde (MDA). In addition, ginsenoside Rg1 decreased β-catenin and c-Myc mRNA expression and enhanced the phosphorylation of GSK-3β. Moreover, ginsenoside Rg1 down-regulated advanced glycation end products (AGEs), 4-hydroxynonenal (4-HNE), phospho-histone H2A.X (r-H2A.X), 8-OHdG, p16Ink4a, Rb, p21Cip1/Waf1 and p53 in senescent Sca-1+ HSC/HPCs. Our findings indicated that ginsenoside Rg1 can improve the resistance of Sca-1+ HSC/HPCs in a mouse model of d-galactose-induced aging through the suppression of oxidative stress and excessive activation of the Wnt/β-catenin signaling pathway, and reduction of DNA damage response, p16Ink4a-Rb and p53-p21Cip1/Waf1 signaling. PMID:27294914

  16. SWI/SNF mediates polycomb eviction and epigenetic reprogramming of the INK4b-ARF-INK4a locus.

    PubMed

    Kia, Sima Kheradmand; Gorski, Marcin M; Giannakopoulos, Stavros; Verrijzer, C Peter

    2008-05-01

    Stable silencing of the INK4b-ARF-INK4a tumor suppressor locus occurs in a variety of human cancers, including malignant rhabdoid tumors (MRTs). MRTs are extremely aggressive cancers caused by the loss of the hSNF5 subunit of the SWI/SNF chromatin-remodeling complex. We found previously that, in MRT cells, hSNF5 is required for p16(INK4a) induction, mitotic checkpoint activation, and cellular senescence. Here, we investigated how the balance between Polycomb group (PcG) silencing and SWI/SNF activation affects epigenetic control of the INK4b-ARF-INK4a locus in MRT cells. hSNF5 reexpression in MRT cells caused SWI/SNF recruitment and activation of p15(INK4b) and p16(INK4a), but not of p14(ARF). Gene activation by hSNF5 is strictly dependent on the SWI/SNF motor subunit BRG1. SWI/SNF mediates eviction of the PRC1 and PRC2 PcG silencers and extensive chromatin reprogramming. Concomitant with PcG complex removal, the mixed lineage leukemia 1 (MLL1) protein is recruited and active histone marks supplant repressive ones. Strikingly, loss of PcG complexes is accompanied by DNA methyltransferase DNMT3B dissociation and reduced DNA methylation. Thus, various chromatin states can be modulated by SWI/SNF action. Collectively, these findings emphasize the close interconnectivity and dynamics of diverse chromatin modifications in cancer and gene control. PMID:18332116

  17. Islet biology, the CDKN2A/B locus and type 2 diabetes risk.

    PubMed

    Kong, Yahui; Sharma, Rohit B; Nwosu, Benjamin U; Alonso, Laura C

    2016-08-01

    Type 2 diabetes, fuelled by the obesity epidemic, is an escalating worldwide cause of personal hardship and public cost. Diabetes incidence increases with age, and many studies link the classic senescence and ageing protein p16(INK4A) to diabetes pathophysiology via pancreatic islet biology. Genome-wide association studies (GWASs) have unequivocally linked the CDKN2A/B locus, which encodes p16 inhibitor of cyclin-dependent kinase (p16(INK4A)) and three other gene products, p14 alternate reading frame (p14(ARF)), p15(INK4B) and antisense non-coding RNA in the INK4 locus (ANRIL), with human diabetes risk. However, the mechanism by which the CDKN2A/B locus influences diabetes risk remains uncertain. Here, we weigh the evidence that CDKN2A/B polymorphisms impact metabolic health via islet biology vs effects in other tissues. Structured in a bedside-to-bench-to-bedside approach, we begin with a summary of the evidence that the CDKN2A/B locus impacts diabetes risk and a brief review of the basic biology of CDKN2A/B gene products. The main emphasis of this work is an in-depth look at the nuanced roles that CDKN2A/B gene products and related proteins play in the regulation of beta cell mass, proliferation and insulin secretory function, as well as roles in other metabolic tissues. We finish with a synthesis of basic biology and clinical observations, incorporating human physiology data. We conclude that it is likely that the CDKN2A/B locus influences diabetes risk through both islet and non-islet mechanisms. PMID:27155872

  18. The Role of Hypoxia Inducible Factor-1 Alpha in Bypassing Oncogene-Induced Senescence

    PubMed Central

    Kilic Eren, Mehtap; Tabor, Vedrana

    2014-01-01

    Oncogene induced senescence (OIS) is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR), senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on RasV12-induced senescence in human diploid fibroblasts (HDFs). We showed here that hypoxia prevents execution of oncogene induced senescence (OIS), through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21CIP1 and p16INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α). In addition, hypoxia also decreased marks of H-RasV12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21CIP1 but not p16INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways. PMID:24984035

  19. Differential targeting of the cyclin-dependent kinase inhibitor, p21CIP1/WAF1, by chelators with anti-proliferative activity in a range of tumor cell-types

    PubMed Central

    Moussa, Rayan S.; Kovacevic, Zaklina; Richardson, Des R.

    2015-01-01

    Chelators such as 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) target tumor cell iron pools and inhibit proliferation. These agents also modulate multiple targets, one of which is the cyclin-dependent kinase inhibitor, p21. Hence, this investigation examined the mechanism of action of these compounds in targeting p21. All the chelators up-regulated p21 mRNA in the five tumor cell-types assessed. In contrast, examining their effect on total p21 protein levels, these agents induced either: (1) down-regulation in MCF-7 cells; (2) up-regulation in SK-MEL-28 and CFPAC-1 cells; or (3) had no effect in LNCaP and SK-N-MC cells. The nuclear localization of p21 was also differentially affected by the ligands depending upon the cell-type, with it being decreased in MCF-7 cells, but increased in SK-MEL-28 and CFPAC-1 cells. Further studies assessing the mechanisms responsible for these effects demonstrated that p21 expression was not correlated with p53 status, suggesting a p53-independent mechanism. Considering this, we examined proteins that modulate p21 independently of p53, namely NDRG1, MDM2 and ΔNp63. These studies demonstrated that a dominant negative MDM2 isoform (p75MDM2) closely resembled p21 expression in response to chelation in three cell lines. These data suggest MDM2 may be involved in the regulation of p21 by chelators. PMID:26335183

  20. Differential targeting of the cyclin-dependent kinase inhibitor, p21CIP1/WAF1, by chelators with anti-proliferative activity in a range of tumor cell-types.

    PubMed

    Moussa, Rayan S; Kovacevic, Zaklina; Richardson, Des R

    2015-10-01

    Chelators such as 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) target tumor cell iron pools and inhibit proliferation. These agents also modulate multiple targets, one of which is the cyclin-dependent kinase inhibitor, p21. Hence, this investigation examined the mechanism of action of these compounds in targeting p21. All the chelators up-regulated p21 mRNA in the five tumor cell-types assessed. In contrast, examining their effect on total p21 protein levels, these agents induced either: (1) down-regulation in MCF-7 cells; (2) up-regulation in SK-MEL-28 and CFPAC-1 cells; or (3) had no effect in LNCaP and SK-N-MC cells. The nuclear localization of p21 was also differentially affected by the ligands depending upon the cell-type, with it being decreased in MCF-7 cells, but increased in SK-MEL-28 and CFPAC-1 cells. Further studies assessing the mechanisms responsible for these effects demonstrated that p21 expression was not correlated with p53 status, suggesting a p53-independent mechanism. Considering this, we examined proteins that modulate p21 independently of p53, namely NDRG1, MDM2 and ΔNp63. These studies demonstrated that a dominant negative MDM2 isoform (p75(MDM2)) closely resembled p21 expression in response to chelation in three cell lines. These data suggest MDM2 may be involved in the regulation of p21 by chelators. PMID:26335183

  1. PDTC, metal chelating compound, induces G1 phase cell cycle arrest in vascular smooth muscle cells through inducing p21Cip1 expression: involvement of p38 mitogen activated protein kinase.

    PubMed

    Moon, Sung-Kwon; Jung, Sun-Young; Choi, Yung-Hyun; Lee, Young-Choon; Patterson, Cam; Kim, Cheorl-Ho

    2004-02-01

    Pyrrolidine dithiocarbamate (PDTC), a metal chelating compound, is known to induce cell death in vascular smooth muscle cells (VSMC). However, the molecular mechanism for PDTC-induced VSMC death is not well understood. Addition of PDTC reduced cell growth and DNA synthesis on VSMC in low density conditions. However, in serum depleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. Several metal chelators prevented the cell death induced by PDTC. In a serum-deprived condition, addition of exogenous metals, copper, iron, and zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as copper, iron, and zinc in serum may mediate the cytotoxic effect of PDTC. At low VSMC density in 10% FBS, treatment of PDTC, which induced a cell-cycle block in G1-phase, induced down-regulation of cyclins and CDKs and up-regulation of the CDK inhibitor p21 expression, whereas up-regulation of p27 or p53 by PDTC was not observed. Finally, we determined PDTC-mediated signaling pathway involved in VSMC death. Among relevant pathways, PDTC induced marked activation of p38MAPK and JNK. Expression of dominant negative p38MAPK and SB203580, a p38MAPK specific inhibitor, blocked PDTC-dependent p38MAPK, growth inhibition, and p21 expression. These data demonstrate that the p38MAPK pathway participates in p21 induction, which consequently leads to decrease of cyclin D1/cdk4 and cyclin E/cdk2 complexes and PDTC-dependent VSMC growth inhibition. In conclusion, an understanding of the molecular mechanisms of PDTC in VSMC provides a theoretical basis for clinical approaches using antioxidant therapies in atherosclerosis. PMID:14603533

  2. Solitary fibrous tumor of the orbit: morphological, cytogenetic and molecular features.

    PubMed

    Cerdá-Nicolás, Miguel; Löpez-Gines, Concha; Gil-Benso, Rosario; Benito, Rafael; Pellin, Antonio; Ruiz-Saurí, Amparo; Sanchos-Garcia, Juan; Roldan, Pedro; Talamantes, Fernando; Barberá, José

    2006-12-01

    Solitary fibrous tumor (SFT), a benign neoplasm arising in mesenchymal structures, was initially described in the pleura but subsequently has also been documented in other locations. It is uncommon in the orbit, where it closely resembles other benign spindle-shaped mesenchymal tumors of this area such as schwannoma, meningioma or hemangiopericytoma. We present a case of orbital SFT in a 34-year-old woman. The radiological study showed the presence of an enhanced uptake lesion measuring 2 cm in major diameter. The histopathological evaluation revealed alternating cellular and hypocellular areas with spindle-shaped cells. The cellular organization displayed a broad variety of irregular morphological patterns. The neoplastic cells were intensely positive for CD34 and vimentin, while S100, epithelial membrane antigen (EMA), Caldesmon, Calretinin and WT-1 proved negative. The pericellular matrix exhibited strong positivity for CD44 and collagen IV. Scarce mitotic figures, a Ki-67 nuclear labeling index of <5%, and focal expression of p53 were also observed. Measurement of DNA content revealed a DNA index of 1, indicating a diploid peak in 95% of the tumor cells. A normal 46,XX karyotype was present. No TP53 (exons 5-8) mutations or MDM2 and CDK4 amplifications were observed. No p14(ARF), p15(INK4B) and p16(INK4A) deletions or hypermethylation were observed in this benign tumor. Following surgical resection and radiotherapy, the patient showed no tumor relapse after one year of follow-up.

  3. Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation

    PubMed Central

    Kilic Eren, Mehtap; Kilincli, Ayten; Eren, Özkan

    2015-01-01

    The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol’s anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21CIP1 and p16INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21CIP1 and p16INK4A levels. Interestingly DNA damaging agent

  4. Protective Effect of Ginsenoside Rg1 on Hematopoietic Stem/Progenitor Cells through Attenuating Oxidative Stress and the Wnt/β-Catenin Signaling Pathway in a Mouse Model of d-Galactose-induced Aging.

    PubMed

    Li, Jing; Cai, Dachuan; Yao, Xin; Zhang, Yanyan; Chen, Linbo; Jing, Pengwei; Wang, Lu; Wang, Yaping

    2016-01-01

    Stem cell senescence is an important and current hypothesis accounting for organismal aging, especially the hematopoietic stem cell (HSC). Ginsenoside Rg1 is the main active pharmaceutical ingredient of ginseng, which is a traditional Chinese medicine. This study explored the protective effect of ginsenoside Rg1 on Sca-1⁺ hematopoietic stem/progenitor cells (HSC/HPCs) in a mouse model of d-galactose-induced aging. The mimetic aging mouse model was induced by continuous injection of d-gal for 42 days, and the C57BL/6 mice were respectively treated with ginsenoside Rg1, Vitamin E or normal saline after 7 days of d-gal injection. Compared with those in the d-gal administration alone group, ginsenoside Rg1 protected Sca-1⁺ HSC/HPCs by decreasing SA-β-Gal and enhancing the colony forming unit-mixture (CFU-Mix), and adjusting oxidative stress indices like reactive oxygen species (ROS), total anti-oxidant (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and malondialdehyde (MDA). In addition, ginsenoside Rg1 decreased β-catenin and c-Myc mRNA expression and enhanced the phosphorylation of GSK-3β. Moreover, ginsenoside Rg1 down-regulated advanced glycation end products (AGEs), 4-hydroxynonenal (4-HNE), phospho-histone H2A.X (r-H2A.X), 8-OHdG, p16(Ink4a), Rb, p21(Cip1/Waf1) and p53 in senescent Sca-1⁺ HSC/HPCs. Our findings indicated that ginsenoside Rg1 can improve the resistance of Sca-1⁺ HSC/HPCs in a mouse model of d-galactose-induced aging through the suppression of oxidative stress and excessive activation of the Wnt/β-catenin signaling pathway, and reduction of DNA damage response, p16(Ink4a)-Rb and p53-p21(Cip1/Waf1) signaling. PMID:27294914

  5. DNA damage causes TP53-dependent coupling of self-renewal and senescence pathways in embryonal carcinoma cells.

    PubMed

    Jackson, Thomas R; Salmina, Kristine; Huna, Anda; Inashkina, Inna; Jankevics, Eriks; Riekstina, Una; Kalnina, Zane; Ivanov, Andrey; Townsend, Paul A; Cragg, Mark S; Erenpreisa, Jekaterina

    2013-02-01

    Recent studies have highlighted an apparently paradoxical link between self-renewal and senescence triggered by DNA damage in certain cell types. In addition, the finding that TP53 can suppress senescence has caused a re-evaluation of its functional role in regulating these outcomes. To investigate these phenomena and their relationship to pluripotency and senescence, we examined the response of the TP53-competent embryonal carcinoma (EC) cell line PA-1 to etoposide-induced DNA damage. Nuclear POU5F1/OCT4A and P21CIP1 were upregulated in the same cells following etoposide-induced G 2M arrest. However, while accumulating in the karyosol, the amount of OCT4A was reduced in the chromatin fraction. Phosphorylated CHK2 and RAD51/γH2AX-positive nuclear foci, overexpression of AURORA B kinase and moderate macroautophagy were evident. Upon release from G 2M arrest, cells with repaired DNA entered mitoses, while the cells with persisting DNA damage remained at this checkpoint or underwent mitotic slippage and gradually senesced. Reduction of TP53 using sh- or si-RNA prevented the upregulation of OCT4A and P21CIP1 and increased DNA damage. Subsequently, mitoses, micronucleation and senescence were all enhanced after TP53 reduction with senescence confirmed by upregulation of CDKN2A/P16INK4A and increased sa-β-galactosidase positivity. Those mitoses enhanced by TP53 silencing were shown to be multicentrosomal and multi-polar, containing fragmented and highly deranged chromosomes, indicating a loss of genome integrity. Together, these data suggest that TP53-dependent coupling of self-renewal and senescence pathways through the DNA damage checkpoint provides a mechanism for how embryonal stem cell-like EC cells safeguard DNA integrity, genome stability and ultimately the fidelity of self-renewal. PMID:23287532

  6. Alterations of p16-pRb pathway and chromosome locus 9p21-22 in sporadic invasive breast carcinomas.

    PubMed Central

    Gorgoulis, V. G.; Koutroumbi, E. N.; Kotsinas, A.; Zacharatos, P.; Markopoulos, C.; Giannikos, L.; Kyriakou, V.; Voulgaris, Z.; Gogas, I.; Kittas, C.

    1998-01-01

    The p16-pRb pathway represents a vital cell-cycle checkpoint. In the present study we investigated the alterations of this G1-phase protein pathway using immunohistochemical and molecular methods in a series of 55 breast carcinomas and correlated the findings with clinicopathological features of the patients. Furthermore, we examined its relationship with the status of the chromosomal region 9p21-22 performing a deletion map analysis because there are indications that, in addition to CDKN2 and MTS2/p15(INK4B) tumor suppressor genes (TSGs), this area harbors other TSG(s). Aberrant expression (Ab) of p16 and pRb was observed in 26 (47%) and 16 (29%) of the carcinomas, respectively. A statistical trend pointing out an inverse relationship between p16 and pRb expression was found (p = 0.079). Analysis of the region that encodes for p16 by deletion mapping, a PCR-based methylation assay and PCR-SSCP, revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16(INK4A) inactivation in breast carcinomas. The results of deletion mapping also suggest that another TSG(s) may reside at the 9p21-22 area particularly at the D9S162 loci and that co-deletion of this putative gene with CDKN2/p16(INK4A) may play a role in breast carcinogenesis. In addition, microsatellite instability (MI), a marker of replication error phenotype (RER+), was observed with a frequency of 16% in the area examined and was inversely related with loss of heterozygosity (LOH). Interestingly, most cases with MI at the region encoding for p16 were aggregated in a subgroup of breast carcinomas with no other obvious genetic and/or epigenetic CDKN2/p16(INK4A) alterations. We speculate that there is an additional mechanism of CDKN2/p16(INK4A) inactivation. The relationship of p16 protein level pRb, status, the p16-pRb combined immunoprofiles, and the microsatellite alterations detected at the 9p21-22 locus with the patients' clinicopathological parameters

  7. Comparison of the cytotoxicity of cladribine and clofarabine when combined with fludarabine and busulfan in AML cells: Enhancement of cytotoxicity with epigenetic modulators.

    PubMed

    Valdez, Benigno C; Li, Yang; Murray, David; Ji, Jie; Liu, Yan; Popat, Uday; Champlin, Richard E; Andersson, Borje S

    2015-06-01

    Clofarabine (Clo), fludarabine (Flu), and busulfan (Bu) combinations are efficacious in hematopoietic stem cell transplantation for myeloid leukemia. We sought to determine whether the more affordable drug cladribine (Clad) can provide a viable alternative to Clo, with or without panobinostat (Pano) and 5-aza-2'-deoxycytidine (DAC). Both Clad+Flu+Bu and Clo+Flu+Bu combinations showed synergistic cytotoxicity in KBM3/Bu250(6), HL60, and OCI-AML3 cell lines. Cell exposure to these drug combinations resulted in 60%-80% inhibition of proliferation; activation of the ATM pathway; increase in histone modifications; decrease in HDAC3, HDAC4, HDAC5 and SirT7 proteins; decrease in mitochondrial membrane potential; activation of apoptosis and stress signaling pathways; and downregulation of the AKT pathway. These drug combinations activated DNA-damage response and apoptosis in primary cell samples from AML patients. At lower concentrations of Clad/Clo, Flu, and Bu, inclusion of Pano and DAC enhanced cell killing, increased histone modifications and DNA demethylation, and increased the levels of P16/INK4a, P15/INK4b and P21/Waf1/Cip1 proteins. The observed DNA demethylating activity of Clad and Clo may complement DAC activity; increase demethylation of the gene promoters for SFRP1, DKK3, and WIF1; and cause degradation of β-catenin in cells exposed to Clad/Clo+Flu+Bu+DAC+Pano. The overlapping activities of Clad/Clo+Flu+Bu, Pano, and DAC in DNA-damage formation and repair, histone modifications, DNA demethylation, and apoptosis may underlie their synergism. Our results provide a basis for supplanting Clo with Clad and for including epigenetic modifiers in the pre-hematopoietic stem cell transplantation conditioning regimen for myeloid leukemia patients. PMID:25704054

  8. Polycomb Mediated Epigenetic Silencing and Replication Timing at the INK4a/ARF Locus during Senescence

    PubMed Central

    Verthuy, Christophe; Chasson, Lionel; Serrano, Manuel; Djabali, Malek

    2009-01-01

    Background The INK4/ARF locus encodes three tumor suppressor genes (p15Ink4b, Arf and p16Ink4a) and is frequently inactivated in a large number of human cancers. Mechanisms regulating INK4/ARF expression are not fully characterized. Principal Findings Here we show that in young proliferating embryonic fibroblasts (MEFs) the Polycomb Repressive Complex 2 (PRC2) member EZH2 together with PRC1 members BMI1 and M33 are strongly expressed and localized at the INK4/ARF regulatory domain (RD) identified as a DNA replication origin. When cells enter senescence the binding to RD of both PRC1 and PRC2 complexes is lost leading to a decreased level of histone H3K27 trimethylation (H3K27me3). This loss is accompanied with an increased expression of the histone demethylase Jmjd3 and with the recruitment of the MLL1 protein, and correlates with the expression of the Ink4a/Arf genes. Moreover, we show that the Polycomb protein BMI1 interacts with CDC6, an essential regulator of DNA replication in eukaryotic cells. Finally, we demonstrate that Polycomb proteins and associated epigenetic marks are crucial for the control of the replication timing of the INK4a/ARF locus during senescence. Conclusions We identified the replication licencing factor CDC6 as a new partner of the Polycomb group member BMI1. Our results suggest that in young cells Polycomb proteins are recruited to the INK4/ARF locus through CDC6 and the resulting silent locus is replicated during late S-phase. Upon senescence, Jmjd3 is overexpressed and the MLL1 protein is recruited to the locus provoking the dissociation of Polycomb from the INK4/ARF locus, its transcriptional activation and its replication during early S-phase. Together, these results provide a unified model that integrates replication, transcription and epigenetics at the INK4/ARF locus. PMID:19462008

  9. Long noncoding RNA, polycomb, and the ghosts haunting INK4b-ARF-INK4a expression.

    PubMed

    Aguilo, Francesca; Zhou, Ming-Ming; Walsh, Martin J

    2011-08-15

    Polycomb group proteins (PcG) function as transcriptional repressors of gene expression. The important role of PcG in mediating repression of the INK4b-ARF-INK4a locus, by directly binding to the long noncoding RNA (lncRNA) transcript antisense noncoding RNA in the INK4 locus (ANRIL), was recently shown. INK4b-ARF-INK4a encodes 3 tumor-suppressor proteins, p15(INK4b), p14(ARF), and p16(INK4a), and its transcription is a key requirement for replicative or oncogene-induced senescence and constitutes an important barrier for tumor growth. ANRIL gene is transcribed in the antisense orientation of the INK4b-ARF-INK4a gene cluster, and different single-nucleotide polymorphisms are associated with increased susceptibility to several diseases. Although lncRNA-mediated regulation of INK4b-ARF-INK4a gene is not restricted to ANRIL, both polycomb repressive complex-1 (PRC1) and -2 (PRC2) interact with ANRIL to form heterochromatin surrounding the INK4b-ARF-INK4a locus, leading to its repression. This mechanism would provide an increased advantage for bypassing senescence, sustaining the requirements for the proliferation of stem and/or progenitor cell populations or inappropriately leading to oncogenesis through the aberrant saturation of the INK4b-ARF-INK4a locus by PcG complexes. In this review, we summarize recent findings on the underlying epigenetic mechanisms that link PcG function with ANRIL, which impose gene silencing to control cellular homeostasis as well as cancer development. PMID:21828241

  10. Long Noncoding RNA, Polycomb, and the Ghosts Haunting INK4b-ARF-INK4a Expression

    PubMed Central

    Aguilo, Francesca; Zhou, Ming-Ming; Walsh, Martin J.

    2012-01-01

    Polycomb group proteins (PcG) function as transcriptional repressors of gene expression. The important role of PcG in mediating repression of the INK4b-ARF-INK4a locus, by directly binding to the long noncoding RNA (lncRNA) transcript antisense noncoding RNA in the INK4 locus (ANRIL), was recently shown. INK4b-ARF-INK4a encodes 3 tumor-suppressor proteins, p15INK4b, p14ARF, and p16INK4a, and its transcription is a key requirement for replicative or oncogene-induced senescence and constitutes an important barrier for tumor growth. ANRIL gene is transcribed in the antisense orientation of the INK4b-ARF-INK4a gene cluster, and different single-nucleotide polymorphisms are associated with increased susceptibility to several diseases. Although lncRNA-mediated regulation of INK4b-ARF-INK4a gene is not restricted to ANRIL, both polycomb repressive complex-1 (PRC1) and -2 (PRC2) interact with ANRIL to form heterochromatin surrounding the INK4b-ARF-INK4a locus, leading to its repression. This mechanism would provide an increased advantage for bypassing senescence, sustaining the requirements for the proliferation of stem and/or progenitor cell populations or inappropriately leading to oncogenesis through the aberrant saturation of the INK4b-ARF-INK4a locus by PcG complexes. In this review, we summarize recent findings on the underlying epigenetic mechanisms that link PcG function with ANRIL, which impose gene silencing to control cellular homeostasis as well as cancer development. PMID:21828241

  11. 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

  12. Altered microRNA Expression Profiles and Regulation of INK4A/CDKN2A Tumor Suppressor Genes in Canine Breast Cancer Models.

    PubMed

    Lutful Kabir, Farruk Mohammad; DeInnocentes, Patricia; Bird, Richard Curtis

    2015-12-01

    microRNA (miRNA) expression profiling of cancer versus normal cells may reveal the characteristic regulatory features that can be correlated to altered gene expression in both human and animal models of cancers. In this study, the comprehensive expression profiles of the 277 highly characterized miRNAs from the canine genome were evaluated in spontaneous canine mammary tumor (CMT) models harboring defects in a group of cell cycle regulatory and potent tumor suppressor genes of INK4/CDKN2 family including p16/INK4A, p14ARF, and p15/INK4B. A large number of differentially expressed miRNAs were identified in three CMT cell lines to potentially target oncogenes, tumor suppressor genes and cancer biomarkers. A group of the altered miRNAs were identified by miRNA target prediction tools for regulation of the INK4/CDKN2 family tumor suppressor genes. miRNA-141 was experimentally validated for INK4A 3'-UTR target binding in the CMT cell lines providing an essential mechanism for the post-transcriptional regulation of the INK4A tumor suppressor gene in CMT models. A well-recognized group of miRNAs including miR-21, miR-155, miR-9, miR-34a, miR-143/145, and miR-31 were found to be altered in both CMTs and human breast cancer. These altered miRNAs might serve as potential targets for advancing the development of future therapeutic reagents. These findings further strengthen the validity and use of canine breast cancers as appropriate models for the study of human breast cancers. PMID:26095675

  13. RhoA regulates G1-S progression of gastric cancer cells by modulation of multiple INK4 family tumor suppressors.

    PubMed

    Zhang, Siyuan; Tang, Qiulin; Xu, Feng; Xue, Yan; Zhen, Zipeng; Deng, Yu; Liu, Ming; Chen, Ji; Liu, Surui; Qiu, Meng; Liao, Zhengyin; Li, Zhiping; Luo, Deyun; Shi, Fang; Zheng, Yi; Bi, Feng

    2009-04-01

    RhoA, a member of the Rho GTPase family, has been extensively studied in the regulation of cytoskeletal dynamics, gene transcription, cell cycle progression, and cell transformation. Overexpression of RhoA is found in many malignancies and elevated RhoA activity is associated with proliferation phenotypes of cancer cells. We reported previously that RhoA was hyperactivated in gastric cancer tissues and suppression of RhoA activity could partially reverse the proliferation phenotype of gastric cancer cells, but the underlying mechanism has yet to be elucidated. It has been reported that RhoA activation is crucial for the cell cycle G(1)-S procession through the regulation of Cip/Kip family tumor suppressors in benign cell lines. In this study, we found that selective suppression of RhoA or its effectors mammalian Diaphanous 1 and Rho kinase (ROCK) by small interfering RNA and a pharmacologic inhibitor effectively inhibited proliferation and cell cycle G(1)-S transition in gastric cancer lines. Down-regulation of RhoA-mammalian Diaphanous 1 pathway, but not RhoA-ROCK pathway, caused an increase in the expression of p21(Waf1/Cip1) and p27(Kip1), which are coupled with reduced expression and activity of CDK2 and a cytoplasmic mislocalization of p27(Kip1). Suppression of RhoA-ROCK pathway, on the other hand, resulted in an accumulation of p15(INK4b), p16(INK4a), p18(INK4c), and p19(INK4d), leading to reduced expression and activities of CDK4 and CDK6. Thus, RhoA may use two distinct effector pathways in regulating the G(1)-S progression of gastric cancer cells.

  14. 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. PMID:26180585

  15. 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.

  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 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

  17. 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.

  18. 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

  19. Uncoupling between Phenotypic Senescence and Cell Cycle Arrest in Aging p21-Deficient Fibroblasts

    PubMed Central

    Dulić, Vjekoslav; Beney, Georges-Edouard; Frebourg, Guillaume; Drullinger, Linda F.; Stein, Gretchen H.

    2000-01-01

    Irreversible G1 arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21Cip1/SDI1/WAF1 and p16Ink4A. To determine the physiological and molecular events that specifically require p21, we studied senescence in human diploid fibroblasts expressing the human papillomavirus type 16 E6 oncogene, which confers low p21 levels via enhanced p53 degradation. We show that in late-passage E6 cells, high Cdk activity drives the cell cycle, but population expansion is slowed down by crisis-like events, probably owing to defective cell cycle checkpoints. At the end of lifespan, terminal-passage E6 cells exhibited several aspects of the senescent phenotype and accumulated unphosphorylated pRb and p16. However, both replication and cyclin-Cdk2 kinase activity were still not blocked, demonstrating that phenotypic and replicative senescence are uncoupled in the absence of normal p21 levels. At this stage, E6 cells also failed to upregulate p27 and inactivate cyclin-Cdk complexes in response to serum deprivation. Eventually, irreversible G1 arrest occurred coincident with inactivation of cyclin E-Cdk2 owing to association with p21. Similarly, when p21−/− mouse embryo fibroblasts reached the end of their lifespan, they had the appearance of senescent cells yet, in contrast to their wild-type counterparts, they were deficient in downregulating bromodeoxyuridine incorporation, cyclin E- and cyclin A-Cdk2 activity, and inhibiting pRb hyperphosphorylation. These data support the model that the critical event ensuring G1 arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21. PMID:10958672

  20. Genetic analysis of Raf1, Mdm2, c-Myc, Cdc25a and Cdc25b proto-oncogenes in 2',3'-dideoxycytidine- and 1,3-butadiene-induced lymphomas in B6C3F1 mice.

    PubMed

    Zhuang, S; Söderkvist, P

    2000-07-20

    We have previously identified activation of ras proto-oncogenes and inactivation of tumor suppressor genes including p53, p16(INK4a) and p15(INK4b) in 2',3'-dideoxycytidine (ddC)- and/or 1,3-butadiene (BD)-induced lymphomas derived from B6C3F1 (C57BL/6xC3H/He) mice, indicating that alterations of ras signaling pathway, p53 and pRb growth control pathways are important in the development of these chemically induced lymphomas. However, there is still a subset of tumors that displayed no changes in these genes. Thus, we investigated whether the Raf1, Mdm2, c-Myc, Cdc25a and Cdc25b proto-oncogenes, which are implicated in the ras or p53 or pRb pathways, are alternative oncogenic target genes. Analyses of gross genomic alterations by Southern blots failed to reveal rearrangement or amplification in any of the tumors examined. Frequent point mutations on the substrate binding domain of the Raf1 gene has been reported in 1-ethyl-1-nitrosourea (ENU)-induced murine lymphomas and lung tumors, along with a conspicuous lack of ras mutations [U. Naumann, I. Eisenmann-Tappe, U.R. Rapp, The role of raf kinases in development and growth of tumors, Recent Results Cancer Res., 143 (1997) 237-244]. To investigate whether Raf1 mutation is involved in our set of tumor especially those without ras mutations, the PCR-based single-strand conformation analyses (SSCA) and direct DNA sequencing were employed. No mutations but four genetic polymorphisms between C57BL/6 and C3H/He were found, with two of them reported as point mutations previously (op. cit.). The polymorphisms were utilized for allelic loss study of Raf1 locus. Losses of heterozygosity were found in six of 31 BD-induced lymphomas. These results indicate that genetic alterations of c-Myc, Cdc25, Raf1 and Mdm2 proto-oncogenes may not be involved in the development of ddC- and BD-induced lymphomas and the inactivation of tumor suppressor gene(s) located close to Raf1 gene might be important in the development of a subset of BD

  1. The Ink4/Arf locus is a barrier for iPS reprogramming

    PubMed Central

    Li, Han; Collado, Manuel; Villasante, Aranzazu; Strati, Katerina; Ortega, Sagrario; Cañamero, Marta; Blasco, Maria A.; Serrano, Manuel

    2013-01-01

    The mechanisms involved in the reprogramming of differentiated cells into induced Pluripotent Stem (iPS) cells by Oct4, Klf4 and Sox2 (3F) remain poorly understood 1. The Ink4/Arf tumour suppressor locus encodes three potent inhibitors of proliferation, namely p16Ink4a, p15Ink4b and Arf, which are basally expressed in differentiated cells and upregulated by aberrant mitogenic signals 2-4. We show here that the locus is completely silenced in iPS cells, as well as in embryonic stem (ES) cells, acquiring the epigenetic marks of a bivalent chromatin domain, and retaining the ability to be reactivated upon differentiation. Cell culture conditions during reprogramming enhance the expression of the Ink4/Arf locus, further highlighting the importance of silencing the locus to allow proliferation and reprogramming. Indeed, the 3F together repress the Ink4/Arf locus soon after their expression and concomitant with the appearance of the first molecular markers of stemness. This downregulation also occurs in cells carrying the oncoprotein large-T, which functionally inactivates the pathways regulated by the Ink4/Arf locus, thus implying that the silencing of the locus is intrinsic to reprogramming and not the result of a selective process. Genetic inhibition of the Ink4/Arf locus has a profound positive impact on the efficiency of iPS generation, increasing both the kinetics of reprogramming and the number of emerging iPS colonies. In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming through activation of p53 and p21; whereas, in human fibroblasts, INK4a is more important than ARF. Finally, organismal aging upregulates the Ink4/Arf locus 2,5 and, accordingly, reprogramming is less efficient in cells from old organisms, but this defect can be rescued by inhibiting the locus with an shRNA. All together, we conclude that the silencing of Ink4/Arf locus is rate limiting for reprogramming, and its transient inhibition may significantly improve the

  2. Active ERK2 is sufficient to mediate growth arrest and differentiation signaling

    PubMed Central

    Wu, Pui-Kei; Hong, Seung-Keun; Yoon, Seung-Hee; Park, Jong-In

    2015-01-01

    Although extracellular signal-regulated kinase (ERK) ½ has been shown for its necessity for a variety of the Raf/MEK/ERK pathway signaling, its sufficiency in mediating the pathway signaling has not been firmly established. In an effort to address this, we evaluated previously reported ERK2 mutants that exhibit enhanced activity of autophosphorylation of TEY sites in the activation loop for their ability to induce growth arrest and differentiation in LNCaP and PC12 cells. Here, we demonstrate that expression of ERK2-L73P/S151D, containing Lys73Pro and Ser151Asp replacements that synergistically promote ERK autophosphorylation, is sufficient to induce growth arrest and differentiation whereas ERK2-I84A and ERK2-R65S/D319N are not as effective. When compared to the constitutively active MEK1-ΔN3/S218E/S222D, expression of ERK2-L73P/S151D could only mildly increase ERK kinase activity in cells, as determined by the ERK substrates, p90RSK and ELK1. Nevertheless, ERK2-L73P/S151D expression effectively induced downregulation of androgen receptor, Rb and E2F1, and upregulation of p16INK4A and p21CIP1, which were accompanied by cell cycle arrest and morphological differentiation, in LNCaP cells and neurite-like processing in PC12 cells. These effects and TEY site phosphorylation of ERK2-L73P/S151D were abrogated upon introducing the active site-disabling Lys52Arg mutation, confirming its sufficiency in this signaling. Moreover, introduction of the mutations (producing Asp316/319Ala or Asp319Asn) that impair the common docking site/D-domain-based physical interaction of ERK did not significantly affect the ERK2-L73P/S151D signaling, suggesting that ERK2 can mediate growth arrest and differentiation independently of the conventional ERK-target interaction mechanism. Our study presents a convincing example of ERK sufficiency for Raf/MEK/ERK signaling. PMID:25639353

  3. 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.

  4. [The INK4a-ARF locus: role in the genetic predisposition to familial melanoma and in skin carcinogenesis].

    PubMed

    Soufir, N; Basset-Seguin, N

    2001-11-01

    The INK4a-ARF locus, localized on 9p21, encodes two tumor suppressor proteins, p16INK4a and p14ARF, acting respectively through the CDK4-pRb and the p53 pathways. Familial melanoma (comprising between 8 and 12% of all melanoma cases) is a genodermatosis transmitted as an autosomal dominant trait, often associated with clinically atypical moles (AN). Germline mutations of p16INK4a are found in up to 20-30% of melanoma prone families. Mutated families often contain more than three family members affected and/or comprise at least one relative with multiple melanomas. Most of these mutations have been shown to affect p16INK4a protein function (i.e. CDK4 binding or pRB phosphorylation). Germline mutations of p16INK4a are also found in a lesser extend in sporadic multiple melanoma and in familial pancreatic cancer. The INK4a-ARF locus plays also an important role in skin carcinogenesis. P16INK4a UV induced mutations (CC:GG > TT:AA tandem transition or C:G > T:A transition at dipyrimidic site) are found in 12% of sporadic skin carcinomas, mainly in epidermoid tumors, and seem to occur independently of p53 mutations. Xeroderma pigmentosum (XP) is characterized by an inheritable DNA repair defect (involving the nucleotid excision repair (NER) system) predisposing to skin carcinomas. In skin tumors from (XP) patients, p16INK4a UV induced mutations occur more frequently, are often multiple, and significantly associated with the presence of p53 mutations. Such data, which could be related to the XP genetic instability and indicates a possible cooperative effect of inactivation of these pathways in the tumoral process of XP skin tumors. PMID:11741799

  5. The Loss of p16 Expression Worsens the Prognosis of OSCC.

    PubMed

    Pérez-Sayáns, Mario; Suárez-Peñaranda, José M; Padín-Iruegas, Marí E; Gayoso-Diz, Pilar; Reis-De Almeida, Miguel; Barros-Angueira, Francisco; Gándara-Vila, Pilar; Blanco-Carrión, Andrés; García-García, Abel

    2015-01-01

    Oral squamous cell carcinoma is the most common neoplasia of the mouth. Downregulation of p16(INK4a) (a cyclin-dependent kinase inhibitor) has been reported for mouth cancer and it is believed that its inactivation is an early event in oral carcinogenesis. The goal of this article is to quantitatively report expression of p16(INK4a) and the state of methylation in oral squamous cell carcinoma, and evaluate its relationship with the clinical and prognostic factors, in addition to setting out a multivariate model that predicts survival. The mean expression of p16(INK4a) was 7.70 (SD=14.07) (F=0.894; P=0.449). According to the semiquantitative analysis, there were statistically significant differences, where 19 cases were negative (<2 %), 11 at initial stages, and 8 at advanced stages (χ(2)=6.016; P<0.05). The methylation of p16(INK4a) was not associated with any of the clinical or pathologic variables. Kaplan-Meier curve showed a better survival for patients in initial stages (40.72 mo) compared to those in advanced stages (28.6 mo) (P<0.01). Survival was also reduced in a statistically significant manner in patients with any degree of dysplasia in the adjacent margin (P<0.05). During univariate Cox regression analysis, it was observed that individuals with relapse had a higher risk (almost 9 times higher) [P<0.001; hazard ratio=8.91; 95% confidence interval (CI), 4.18-19.02]. During the Cox multivariate analysis for each unit of decrease in p16(INK4a), the risk increased by 1.06) (P<0.05; hazard ratio=0.94; 95% CI, 0.89-1.00). p16(INK4a) expression is reduced with advancing tumor stage and its gene silencing is associated with an increased risk of death.

  6. [Effect of decitabine combined with Trichostatin A on MDS cell line SKM-1 in vitro].

    PubMed

    Yang, Li; Xu, Rui-Rong; Song, Guo-Qi; Hang, Hong-Ming; Liu, Hong; Jiang, Sheng-Hua; Wang, Xin-Feng; Ding, Xun-Sheng

    2008-08-01

    The study was purposed to explore the effect and mechanisms of decitabine and/or Trichostatin A (TSA) on SKM-1 cells in vitro. The effect of decitabine and/or TSA on proliferation of SKM-1cells was analyzed with trypan blue exclusion; the differentiation of SKM-1 cells was detected by nitro-blue tetrazolium (NBT) reduction and flow cytometry; the apoptosis of cells was measured by Annexin V-FITC; the mRNA expression of Fas, survivin and P15(INK4B) in cells treated with decitabine and/or TSA was evaluated by RT-PCR. The results showed that decitabine and/or TSA were capable of inhibiting SKM-1 cell growth and promoting cell differentiation; they stimulated the expression of CD14 and CD11b and inhibited HLA-DR expression; meanwhile and decitabine or/and TSA could induce cell apoptosis, up-regulate mRNA expression of Fas and P15(INK4B), and down-regulate survivin mRNA expression. It is concluded that decitabine can induce apoptosis/differentiation of SKM-1 cells, whose mechanisms may related to the expression of Fas, survivin and P15(INK4B). Decitabine has the synergistic effect with TSA.

  7. 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.

  8. A Kinase-Independent Function of CDK6 Links the Cell Cycle to Tumor Angiogenesis

    PubMed Central

    Kollmann, Karoline; Heller, Gerwin; Schneckenleithner, Christine; Warsch, Wolfgang; Scheicher, Ruth; Ott, Rene G.; Schäfer, Markus; Fajmann, Sabine; Schlederer, Michaela; Schiefer, Ana-Iris; Reichart, Ursula; Mayerhofer, Matthias; Hoeller, Christoph; Zöchbauer-Müller, Sabine; Kerjaschki, Dontscho; Bock, Christoph; Kenner, Lukas; Hoefler, Gerald; Freissmuth, Michael; Green, Anthony R.; Moriggl, Richard; Busslinger, Meinrad; Malumbres, Marcos; Sexl, Veronika

    2013-01-01

    Summary In contrast to its close homolog CDK4, the cell cycle kinase CDK6 is expressed at high levels in lymphoid malignancies. In a model for p185BCR-ABL+ B-acute lymphoid leukemia, we show that CDK6 is part of a transcription complex that induces the expression of the tumor suppressor p16INK4a and the pro-angiogenic factor VEGF-A. This function is independent of CDK6’s kinase activity. High CDK6 expression thus suppresses proliferation by upregulating p16INK4a, providing an internal safeguard. However, in the absence of p16INK4a, CDK6 can exert its full tumor-promoting function by enhancing proliferation and stimulating angiogenesis. The finding that CDK6 connects cell-cycle progression to angiogenesis confirms CDK6’s central role in hematopoietic malignancies and could underlie the selection pressure to upregulate CDK6 and silence p16INK4a. PMID:23948297

  9. Transcription factor Sp3 represses expression of p21CIP¹ via inhibition of productive elongation by RNA polymerase II.

    PubMed

    Valin, Alvaro; Ouyang, Jian; Gill, Grace

    2013-04-01

    Like that of many protein-coding genes, expression of the p21(CIP1) cell cycle inhibitor is regulated at the level of transcription elongation. While many transcriptional activators have been shown to stimulate elongation, the mechanisms by which promoter-specific repressors regulate pausing and elongation by RNA polymerase II (RNA PolII) are not well described. Here we report that the transcription factor Sp3 inhibits basal p21(CIP1) gene expression by promoter-bound RNA PolII. Knockdown of Sp3 led to increased p21(CIP1) mRNA levels and reduced occupancy of the negative elongation factor (NELF) at the p21(CIP1) promoter, although the level of binding of the positive transcription elongation factor b (P-TEFb) kinase was not increased. Sp3 depletion correlated with increased H3K36me3 and H2Bub1, two histone modifications associated with transcription elongation. Further, Sp3 was shown to promote the binding of protein phosphatase 1 (PP1) to the p21(CIP1) promoter, leading to reduced H3S10 phosphorylation, a finding consistent with Sp3-dependent regulation of the local balance between kinase and phosphatase activities. Analysis of other targets of Sp3-mediated repression suggests that, in addition to previously described SUMO modification-dependent chromatin-silencing mechanisms, inhibition of the transition of paused RNA PolII to productive elongation, described here for p21(CIP1), is a general mechanism by which transcription factor Sp3 fine-tunes gene expression.

  10. Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity.

    PubMed

    Khleif, S N; DeGregori, J; Yee, C L; Otterson, G A; Kaye, F J; Nevins, J R; Howley, P M

    1996-04-30

    Alterations of various components of the cell cycle regulatory machinery that controls the progression of cells from a quiescent to a growing state contribute to the development of many human cancers. Such alterations include the deregulated expression of G1 cyclins, the loss of function of activities such as those of protein p16INK4a that control G1 cyclin-dependent kinase activity, and the loss of function of the retinoblastoma protein (RB), which is normally regulated by the G1 cyclin-dependent kinases. Various studies have revealed an inverse relationship in the expression of p16INK4a protein and the presence of functional RB in many cell lines. In this study we show that p16INK4a is expressed in cervical cancer cell lines in which the RB gene, Rb, is not functional, either as a consequence of Rb mutation or expression of the human papillomavirus E7 protein. We also demonstrate that p16INK4a levels are increased in primary cells in which RB has been inactivated by DNA tumor virus proteins. Given the role of RB in controlling E2F transcription factor activity, we investigated the role of E2F in controlling p16INK4a expression. We found that E2F1 overexpression leads to an inhibition of cyclin D1-dependent kinase activity and induces the expression of a p16-related transcript. We conclude that the accumulation of G1 cyclin-dependent kinase activity during normal G1 progression leads to E2F accumulation through the inactivation of RB, and that this then leads to the induction of cyclin kinase inhibitor activity and a shutdown of G1 kinase activity.

  11. High-risk human papillomavirus in non-melanoma skin lesions from renal allograft recipients and immunocompetent patients

    PubMed Central

    Reuschenbach, M; Tran, T; Faulstich, F; Hartschuh, W; Vinokurova, S; Kloor, M; Krautkrämer, E; Zeier, M; von Knebel Doeberitz, M; Sommerer, C

    2011-01-01

    Background: High-risk human papillomaviruses (HR-HPVs) can be detected in a proportion of non-melanoma skin cancers. Data on prevalence are inconclusive, but are essential to estimate the relevance of HR-HPV, particularly with regard to prophylactic HPV vaccines for skin cancer prevention. Methods: High-risk human papillomavirus DNA was investigated in 140 non-melanoma skin lesions from 54 immunocompetent patients and 33 immunosuppressed renal allograft recipients. Expression of p16INK4a, a marker for HR-HPV oncogene expression in the uterine cervix, and of p53 and pRB was evaluated immunohistochemically. Results: The highest prevalence of HR-HPV was found in squamous cell cancer (SCC) (46.2% (6 out of 13) in immunosuppressed and 23.5% (4 out of 17) in immunocompetent patients). High-risk human papillomavirus positivity was accompanied by diffuse p16INK4a expression in most SCC (P<0.001) and basal cell cancers (P=0.02), while almost all SCC in situ were p16INK4a positive irrespective of HR-HPV presence (P=0.66). Diffuse p16INK4a expression was associated with lack of pRB expression (P=0.001). p53 was strongly expressed in 40.0% (56 out of 140) of the lesions irrespective of HR-HPV presence. Conclusion: High-risk human papillomavirus can be detected in lesions of keratinised squamous epithelia. The association of HR-HPV with diffuse p16INK4a expression might indicate HR-HPV oncogene expression in a proportion of lesions. Overexpression of p53 suggests p53 pathway alterations in HR-HPV-positive and -negative lesions. PMID:21427726

  12. 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...

  13. Stabilization of the Retinoblastoma Protein by A-Type Nuclear Lamins Is Required for INK4A-Mediated Cell Cycle Arrest†

    PubMed Central

    Nitta, Ryan T.; Jameson, Samantha A.; Kudlow, Brian A.; Conlan, Lindus A.; Kennedy, Brian K.

    2006-01-01

    Mutations in the LMNA gene, which encodes all A-type lamins, including lamin A and lamin C, cause a variety of tissue-specific degenerative diseases termed laminopathies. Little is known about the pathogenesis of these disorders. Previous studies have indicated that A-type lamins interact with the retinoblastoma protein (pRB). Here we probe the functional consequences of this association and further examine links between nuclear structure and cell cycle control. Since pRB is required for cell cycle arrest by p16ink4a, we tested the responsiveness of multiple lamin A/C-depleted cell lines to overexpression of this CDK inhibitor and tumor suppressor. We find that the loss of A-type lamin expression results in marked destabilization of pRB. This reduction in pRB renders cells resistant to p16ink4a-mediated G1 arrest. Reintroduction of lamin A, lamin C, or pRB restores p16ink4a-responsiveness to Lmna−/− cells. An array of lamin A mutants, representing a variety of pathologies as well as lamin A processing mutants, was introduced into Lmna−/− cells. Of these, a mutant associated with mandibuloacral dysplasia (MAD R527H), as well as two lamin A processing mutants, but not other disease-associated mutants, failed to restore p16ink4a responsiveness. Although our findings do not rule out links between altered pRB function and laminopathies, they fail to support such an assertion. These findings do link lamin A/C to the functional activation of a critical tumor suppressor pathway and further the possibility that somatic mutations in LMNA contribute to tumor progression. PMID:16809772

  14. Novel and recurrent p14 mutations in Italian familial melanoma.

    PubMed

    Binni, F; Antigoni, I; De Simone, P; Majore, S; Silipo, V; Crisi, A; Amantea, A; Pacchiarini, D; Castori, M; De Bernardo, C; Catricalà, C; Grammatico, P

    2010-06-01

    CDKN2A and CDK4 are the only known high-penetrant genes conferring proneness to cutaneous melanoma. The CDKN2A locus consists of four exons and encodes several alternate transcripts, two of which are p16(INK4a) and p14(ARF), and originate from different open reading frames. Exon 1alpha is specific for p16(INK4a), while exon 1beta characterizes p14(ARF). Most CDKN2A mutations are located in exons 1alpha and 2, while exon 1beta variations have been identified in rare melanoma-prone pedigrees. In a previous study, we investigated 155 Italian melanoma cases, including 94 familial melanomas (FAMs) and 61 sporadic multiple primary melanomas (MPMs), for p16(INK4a)/CDK4 germline alterations and identified 15 p16(INK4a) and 1 CDK4 point mutations. In the present work, we extended our search to p14(ARF) mutations and CDKN2A deletions in the remaining samples. We identified the recurrent g.193+1G> A mutation in two FAM cases, while an additional pedigree displayed the previously undescribed variant g.161G> A. Multiplex ligation-dependent probe amplification (MLPA) screening for copy variations resulted negative in all cases. In Italy, the overall frequency of p14(ARF) mutations is 3.2% in FAM and 0% in sporadic MPM. Re-evaluation of our patients' cohort emphasizes that the chance of identifying CDKN2A/CDK4 mutations in FAM is mainly influenced by the number of affected family members and the presence of one or more MPM cases. Accordingly, mutation rate rises to 61% in selected cases. Further studies are expected in order to investigate CDKN2A rarer mutations, including atypical deletions and inherited epimutations. PMID:20132244

  15. 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.

  16. Telomeres, stem cells, senescence, and cancer

    PubMed Central

    Sharpless, Norman E.; DePinho, Ronald A.

    2004-01-01

    Mammalian aging occurs in part because of a decline in the restorative capacity of tissue stem cells. These self-renewing cells are rendered malignant by a small number of oncogenic mutations, and overlapping tumor suppressor mechanisms (e.g., p16INK4a-Rb, ARF-p53, and the telomere) have evolved to ward against this possibility. These beneficial antitumor pathways, however, appear also to limit the stem cell life span, thereby contributing to aging. PMID:14722605

  17. 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. PMID:26529033

  18. Androgen receptor accelerates premature senescence of human dermal papilla cells in association with DNA damage.

    PubMed

    Yang, Yi-Chien; Fu, Hung-Chun; Wu, Ching-Yuan; Wei, Kuo-Ting; Huang, Ko-En; Kang, Hong-Yo

    2013-01-01

    The dermal papilla, located in the hair follicle, expresses androgen receptor and plays an important role in hair growth. Androgen/Androgen receptor actions have been implicated in the pathogenesis of androgenetic alopecia, but the exact mechanism is not well known. Recent studies suggest that balding dermal papilla cells exhibit premature senescence, upregulation of p16(INK4a), and nuclear expression of DNA damage markers. To investigate whether androgen/AR signaling influences the premature senescence of dermal papilla cells, we first compared frontal scalp dermal papilla cells of androgenetic alopecia patients with matched normal controls and observed that premature senescence is more prominent in the dermal papilla cells of androgenetic alopecia patients. Exposure of androgen induced premature senescence in dermal papilla cells from non-balding frontal and transitional zone of balding scalp follicles but not in beard follicles. Overexpression of the AR promoted androgen-induced premature senescence in association with p16(INK4a) upregulation, whereas knockdown of the androgen receptor diminished the effects of androgen. An analysis of γ-H2AX expression in response to androgen/androgen receptor signaling suggested that DNA damage contributes to androgen/androgen receptor-accelerated premature senescence. These results define androgen/androgen receptor signaling as an accelerator of premature senescence in dermal papilla cells and suggest that the androgen/androgen receptor-mediated DNA damage-p16(INK4a) axis is a potential therapeutic target in the treatment of androgenetic alopecia.

  19. 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%.

  20. 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

  1. Curcumin Down-Regulates DNA Methyltransferase 1 and Plays an Anti-Leukemic Role in Acute Myeloid Leukemia

    PubMed Central

    Yu, Jianhua; Peng, Yong; Wu, Lai-Chu; Xie, Zhiliang; Deng, Youcai; Hughes, Tiffany; He, Shun; Mo, XiaoKui; Chiu, Ming; Wang, Qi-En; He, Xiaoming; Liu, Shujun; Grever, Michael R.; Chan, Kenneth K.; Liu, Zhongfa

    2013-01-01

    Bioactive components from dietary supplements such as curcumin may represent attractive agents for cancer prevention or treatment. DNA methylation plays a critical role in acute myeloid leukemia (AML) development, and presents an excellent target for treatment of this disease. However, it remains largely unknown how curcumin, a component of the popular Indian spice turmeric, plays a role in DNA hypomethylation to reactivate silenced tumor suppressor genes and to present a potential treatment option for AML. Here we show that curcumin down-regulates DNMT1 expression in AML cell lines, both in vitro and in vivo, and in primary AML cells ex vivo. Mechanistically, curcumin reduced the expression of positive regulators of DNMT1, p65 and Sp1, which correlated with a reduction in binding of these transcription factors to the DNMT1 promoter in AML cell lines. This curcumin-mediated down-regulation of DNMT1 expression was concomitant with p15INK4B tumor suppressor gene reactivation, hypomethylation of the p15INK4B promoter, G1 cell cycle arrest, and induction of tumor cell apoptosis in vitro. In mice implanted with the human AML MV4–11 cell line, administration of curcumin resulted in remarkable suppression of AML tumor growth. Collectively, our data indicate that curcumin shows promise as a potential treatment for AML, and our findings provide a basis for future studies to test the clinical efficacy of curcumin – whether used as a single agent or as an adjuvant – for AML treatment. PMID:23457487

  2. 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.

  3. Stratification of HPV-induced cervical pathology using the virally encoded molecular marker E4 in combination with p16 or MCM.

    PubMed

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

    2015-07-01

    High-risk human papillomavirus (HPV) types cause cervical lesions of varying severity, ranging from transient productive infections to high-grade neoplasia. Disease stratification requires the examination of lesional pathology, and possibly also the detection of biomarkers. P16(INK4a) and MCM are established surrogates of high-risk HPV E6/E7 activity, and can be extensively expressed in high-grade lesions. Here we have combined these two cellular biomarkers with detection of the abundant HPV-encoded E4 protein in order to identify both productive and transforming lesions. This approach has allowed us to distinguish true papillomavirus infections from similar pathologies, and has allowed us to divide the heterogeneous CIN2 category into those that are CIN1-like and express E4, and those that more closely resemble nonproductive CIN3. To achieve this, 530 lesional areas were evaluated according to standard pathology criteria and by using a multiple staining approach that allows us to superimpose biomarker patterns either singly or in combination onto an annotated hematoxylin and eosin (H&E) image. Conventional grading of neoplasia was established by review panel, and compared directly with the composite molecular pathology visualized on the same tissue section. The detection of E4 coincided with the onset of vacuolation, becoming abundant in koilocytes as the MCM marker declined and cells lost their defined nuclear margins as visualized by standard H&E staining. Of the dual marker approaches, p16(INK4a) and E4 appeared most promising, with E4 generally identifying areas of low-grade disease even when p16(INK4a) was present. Extensive p16(INK4a) expression usually coincided with an absence of E4 expression or its focal retention in sporadic cells within the lesion. Our results suggest that a straightforward molecular evaluation of HPV life-cycle deregulation in cervical neoplasia may help improve disease stratification, and that this can be achieved using complementary

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

    PubMed Central

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

    2015-01-01

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

  5. 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

  6. 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

  7. Metformin Induced AMPK Activation, G0/G1 Phase Cell Cycle Arrest and the Inhibition of Growth of Esophageal Squamous Cell Carcinomas In Vitro and In Vivo.

    PubMed

    Cai, Xianbin; Hu, Xi; Tan, Xiaojun; Cheng, Weijie; Wang, Qinjia; Chen, Xiaofeng; Guan, Yinghong; Chen, Chong; Jing, Xubin

    2015-01-01

    Esophageal squamous cell carcinomas (ESCC) have become a severe threat to health and the current treatments for ESCC are frequently not effective. Recent epidemiological studies suggest that the anti-hyperglycemic agent metformin may reduce the risk of developing cancer, including ESCC, among diabetic patients. However, the antitumor effects of metformin on ESCC and the mechanisms underlying its cell cycle regulation remain elusive. The findings reported herein show that the anti-proliferative action of metformin on ESCC cell lines is partially mediated by AMPK. Moreover, we observed that metformin induced G0/G1 phase arrest accompanied by the up-regulation of p21CIP1 and p27KIP1. In vivo experiments further showed that metformin inhibited tumor growth in a ESCC xenograft model. Most importantly, the up-regulation of AMPK, p53, p21CIP1, p27KIP1 and the down-regulation of cyclinD1 are involved in the anti-tumor action of metformin in vivo. In conclusion, metformin inhibits the growth of ESCC cells both in cell cultures and in an animal model. AMPK, p53, p21CIP1, p27KIP1 and cyclinD1 are involved in the inhibition of tumor growth that is induced by metformin and cell cycle arrest in ESCC. These findings indicate that metformin has the potential for use in the treatment of ESCC.

  8. CDC20 maintains tumor initiating cells

    PubMed Central

    Xie, Qi; Wu, Qiulian; Mack, Stephen C.; Yang, Kailin; Kim, Leo; Hubert, Christopher G.; Flavahan, William A.; Chu, Chengwei; Bao, Shideng; Rich, Jeremy N.

    2015-01-01

    Glioblastoma is the most prevalent and lethal primary intrinsic brain tumor. Glioblastoma displays hierarchical arrangement with a population of self-renewing and tumorigenic glioma tumor initiating cells (TICs), or cancer stem cells. While non-neoplastic neural stem cells are generally quiescent, glioblastoma TICs are often proliferative with mitotic control offering a potential point of fragility. Here, we interrogate the role of cell-division cycle protein 20 (CDC20), an essential activator of anaphase-promoting complex (APC) E3 ubiquitination ligase, in the maintenance of TICs. By chromatin analysis and immunoblotting, CDC20 was preferentially expressed in TICs relative to matched non-TICs. Targeting CDC20 expression by RNA interference attenuated TIC proliferation, self-renewal and in vivo tumor growth. CDC20 disruption mediated its effects through induction of apoptosis and inhibition of cell cycle progression. CDC20 maintains TICs through degradation of p21CIP1/WAF1, a critical negative regulator of TICs. Inhibiting CDC20 stabilized p21CIP1/WAF1, resulting in repression of several genes critical to tumor growth and survival, including CDC25C, c-Myc and Survivin. Transcriptional control of CDC20 is mediated by FOXM1, a central transcription factor in TICs. These results suggest CDC20 is a critical regulator of TIC proliferation and survival, linking two key TIC nodes – FOXM1 and p21CIP1/WAF1 — elucidating a potential point for therapeutic intervention. PMID:25938542

  9. p19ARF-independent induction of p53 and cell cycle arrest by Raf in murine keratinocytes

    PubMed Central

    Roper, Elizabeth; Weinberg, Wendy; Watt, Fiona M.; Land, Hartmut

    2001-01-01

    In tumorigenesis of the skin, activated Ras co-operates with mutations that inactivate the tumour suppressor p53, but the molecular basis for this co-operation remains unresolved. Here we show that activation of the Raf/MAP kinase pathway in primary mouse keratinocytes leads to a p53 and p21Cip1-dependent cycle arrest and to terminal differentiation. Raf activation in keratinocytes lacking p53 or p21Cip1 genes leads to expression of differentiation markers, but the cells do not cease to proliferate. Thus, loss of p53 or p21Cip1 function is necessary to disable growth-inhibitory Raf/MAP kinase signalling. Activation of oncogenes, including Ras, has been reported to stabilize and activate p53 via induction of the tumour suppressor p19ARF. However, the response to Raf in p19ARF–/– keratinocytes was indistinguishable from wild-type controls. Thus, p19ARF is not essential for Raf-induced p53 induction and cell cycle arrest in keratinocytes, indicating that oncogenes engage p53 activity via multiple mechanisms. PMID:11258707

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

    PubMed

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

    2006-04-01

    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.

  11. Cell cycle gene-specific control of transcription has a critical role in proliferation of primordial germ cells

    PubMed Central

    Okamura, Daiji; Maeda, Ikuma; Taniguchi, Hirofumi; Tokitake, Yuko; Ikeda, Makiko; Ozato, Keiko; Mise, Nathan; Abe, Kuniya; Noce, Toshiaki; Izpisua Belmonte, Juan Carlos; Matsui, Yasuhisa

    2012-01-01

    Transcription elongation is stimulated by positive transcription elongation factor b (P-TEFb), for which activity is repressed in the 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. We show here a critical role of 7SK snRNP in growth control of primordial germ cells (PGCs). The expression of p15INK4b, a cyclin-dependent kinase inhibitor (CDKI) gene, in PGCs is selectively activated by P-TEFb and its recruiting molecule, Brd4, when the amount of active P-TEFb is increased due to reduction of the 7SK snRNP, and PGCs consequently undergo growth arrest. These results indicate that CDKI gene-specific control of transcription by 7SK snRNP plays a pivotal role in the maintenance of PGC proliferation. PMID:23154982

  12. Interferometric detection of early markers for epithelial ovarian cancer and prognostic markers for acute lymphocytic leukemia

    NASA Astrophysics Data System (ADS)

    O'Neil, P.; Zhao, M.; Wang, X.; Nolte, D. D.

    2010-02-01

    We are developing fluorescence-free interferometric biosensors for the early detection of epithelial ovarian cancer (EOC) and prognosis of acute lymphocytic leukemia (ALL). We can detect potential early markers for EOC (CA125, human epididymus protein 4, osteopontin) spiked into serum as well as elevated CA125 in EOC patient serum. For ALL prognosis we are focusing on three intracellular protein markers (p73, p57/Kip2, and p15/Ink4b), the down-regulation of any two being indicative of a more aggressive cancer. We have detected p15 and p57 spiked into buffer and are preparing to test positive and negative control lysates from bone marrow biopsies.

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

    PubMed Central

    Al-Ansari, Mysoon M.; Aboussekhra, Abdelilah

    2015-01-01

    Increasing evidence support the critical roles of active stromal fibroblasts in breast cancer development and spread. However, the mediators and the mechanisms of regulation are still not well defined. We have shown here that the tumor suppressor p16INK4A protein inhibits the pro-carcinogenic effects of breast stromal fibroblasts through repressing the expression/secretion of IL-6. Indeed, p16INK4A suppresses IL-6 at the mRNA and protein levels. This effect is mediated trough miR-146b-5p, which inhibits IL-6 expression through a specific sequence at the IL-6 3′UTR. In addition, we present clear evidence that miR-146b-5p inhibition is sufficient to transactivate breast stromal fibroblasts, which promote epithelial-to-mesenchymal-transition in breast cancer cells in a paracrine manner. By contrast, ectopic expression of miR-146b-5p in active fibroblasts abrogated their pro-carcinogenic effects. The physiological importance of miR-146b-5p inhibition was revealed by showing that the levels of pre-miR-146b-5p as well as its mature form are reduced in cancer-associated fibroblasts as compared with their normal adjacent counterparts from cancer-free tissues isolated from the same patients. Interestingly, treatment of active breast stromal fibroblasts with curcumin increased the level of the p16INK4A coding CDKN2A mRNA and miR-146b-5p and suppressed IL-6, which confirms the repressive effect of these two tumor suppressor molecules on IL-6, and shows the possible “normalization” of cancer-related active fibroblasts. These results show that miR-146b-5p has non-cell-autonomous tumor suppressor function through inhibition of IL-6, suggesting that targeting this microRNA in breast stromal fibroblasts could be of great therapeutic value. PMID:26338965

  14. Disentangling the aneuploidy and senescence paradoxes: a study of triploid breast cancers non-responsive to neoadjuvant therapy.

    PubMed

    Gerashchenko, B I; Salmina, K; Eglitis, J; Huna, A; Grjunberga, V; Erenpreisa, J

    2016-04-01

    Aneuploid cells should have a reduced proliferation rate due to difficulty in proceeding through mitosis. However, contrary to this, high aneuploidy is associated with aggressive tumour growth and poor survival prognosis, in particular in triploid breast cancer. A further paradox revolves around the observation that, while cell senescence should inhibit proliferation, the senescence marker p16INK4a correlates with poor treatment outcome in patients with a very aggressive triple-negative breast carcinoma (TNBC). In this study, we aim to pour light on the possible relationship of these conundrums with polyploidy of tumour cells. We performed detailed analysis of DNA histogram profiles in diagnostic core biopsies of 30 cases of operable breast cancer and found that near triploidy in TNBC and other forms correlated with weak or no response to neoadjuvant chemotherapy (NAC) as scored by Miller-Payne index. Polyploid cells in operation samples from tumours that were non-responsive to NAC treatment were Ki67 and CD44 positive. In addition, polyploid cells were positive for markers of embryonic stemness (OCT4, SOX2, NANOG) and senescence (p16INK4a). The relationship patterns between p16INK4a and NANOG were heterogeneous, with predominantly mutually exclusive expression but also synergistic and intermediate variants in the same samples. We conclude that the aneuploidy and senescence paradoxes can be explained by the mutual platform of polyploidy, conferring genomic and epigenetic instability as a survival advantage. Such cells are able to bypass aneuploidy restrictions of conventional mitosis and overcome the barrier of senescence by a shift to self-renewal, resulting in progression of cancer.

  15. Expression of the MOZ-TIF2 oncoprotein in mice represses senescence

    PubMed Central

    Largeot, Anne; Perez-Campo, Flor Maria; Marinopoulou, Elli; Lie-a-Ling, Michael; Kouskoff, Valerie; Lacaud, Georges

    2016-01-01

    The MOZ-TIF2 translocation, which fuses monocytic leukemia zinc finger protein (MOZ) histone acetyltransferase (HAT) with the nuclear co-activator TIF2, is associated with the development of acute myeloid leukemia. We recently found that in the absence of MOZ HAT activity, p16INK4a transcriptional levels are significantly increased, triggering an early entrance into replicative senescence. Because oncogenic fusion proteins must bypass cellular safeguard mechanisms, such as senescence and apoptosis, to induce leukemia, we hypothesized that this repressive activity of MOZ over p16INK4a transcription could be preserved, or even reinforced, in MOZ leukemogenic fusion proteins, such as MOZ-TIF2. We describe here that, indeed, MOZ-TIF2 silences expression of the CDKN2A locus (p16INK4a and p19ARF), inhibits the triggering of senescence and enhances proliferation, providing conditions favorable to the development of leukemia. Furthermore, we describe that abolishing the MOZ HAT activity of the fusion protein leads to a significant increase in expression of the CDKN2A locus and the number of hematopoietic progenitors undergoing senescence. Finally, we report that inhibition of senescence by MOZ-TIF2 is associated with increased apoptosis, suggesting a role for the fusion protein in p53 apoptosis-versus-senescence balance. Our results underscore the importance of the HAT activity of MOZ, preserved in the fusion protein, for repression of the CDKN2A locus transcription and the subsequent block of senescence, a necessary step for the survival of leukemic cells. PMID:26854485

  16. HPV infection and the alterations of the pRB pathway in oral carcinogenesis.

    PubMed

    Lim, Kue Peng; Hamid, Sharifah; Lau, Shin-Hin; Teo, Soo-Hwang; Cheong, Sok Ching

    2007-06-01

    Inactivation of the retinoblastoma (pRB) pathway is a common event in oral squamous cell carcinoma particularly through the aberrant expression of the components within this pathway. This study examines the alterations of molecules within the pRB pathway by looking at the presence of homozygous deletions in p16(INK4A) and the expression patterns of pRB, cyclin D1 and CDK4, as well as the presence of human papillomavirus (HPV) in our samples. In our study, 5/20 samples demonstrated deletions of p16(INK4A) exon 1alpha. pRB overexpression was found in 20/20 samples, the expression was mainly observed in all layers of the epithelia, particularly in the basal layer where cells are actively dividing and aberrant pRB expression was found in 12/20 samples. Cyclin D1 and CDK4 overexpression was detected in 6/20 and 2/20 samples respectively in comparison to hyperplasias where both proteins were either not expressed or expressed at minimal levels (<10%). Strikingly, HPV was found to be present in all of our samples, suggesting that HPV plays a significant role in driving oral carcinogenesis. Notably, 17/20 of our samples showed more than one alteration in the pRB pathway, however, we did not find any significant relationship between the presence of HPV, homozygous deletion of p16(INK4A) and overexpression of pRB, cyclin D1 and CDK4. Collectively, this data demonstrates that alterations in the pRB pathway are a common event and involve the aberration of more than one molecule within the pathway. Furthermore, the involvement of HPV in all our samples suggests that HPV infection may play an important role in oral carcinogenesis.

  17. Disentangling the aneuploidy and senescence paradoxes: a study of triploid breast cancers non-responsive to neoadjuvant therapy.

    PubMed

    Gerashchenko, B I; Salmina, K; Eglitis, J; Huna, A; Grjunberga, V; Erenpreisa, J

    2016-04-01

    Aneuploid cells should have a reduced proliferation rate due to difficulty in proceeding through mitosis. However, contrary to this, high aneuploidy is associated with aggressive tumour growth and poor survival prognosis, in particular in triploid breast cancer. A further paradox revolves around the observation that, while cell senescence should inhibit proliferation, the senescence marker p16INK4a correlates with poor treatment outcome in patients with a very aggressive triple-negative breast carcinoma (TNBC). In this study, we aim to pour light on the possible relationship of these conundrums with polyploidy of tumour cells. We performed detailed analysis of DNA histogram profiles in diagnostic core biopsies of 30 cases of operable breast cancer and found that near triploidy in TNBC and other forms correlated with weak or no response to neoadjuvant chemotherapy (NAC) as scored by Miller-Payne index. Polyploid cells in operation samples from tumours that were non-responsive to NAC treatment were Ki67 and CD44 positive. In addition, polyploid cells were positive for markers of embryonic stemness (OCT4, SOX2, NANOG) and senescence (p16INK4a). The relationship patterns between p16INK4a and NANOG were heterogeneous, with predominantly mutually exclusive expression but also synergistic and intermediate variants in the same samples. We conclude that the aneuploidy and senescence paradoxes can be explained by the mutual platform of polyploidy, conferring genomic and epigenetic instability as a survival advantage. Such cells are able to bypass aneuploidy restrictions of conventional mitosis and overcome the barrier of senescence by a shift to self-renewal, resulting in progression of cancer. PMID:26860864

  18. 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

  19. High-risk human papillomavirus infection in different histological subtypes of renal cell carcinoma.

    PubMed

    Farhadi, Ali; Behzad-Behbahani, Abbas; Geramizadeh, Bita; Sekawi, Zamberi; Rahsaz, Marjan; Sharifzadeh, Sedigheh

    2014-07-01

    Limited data exist regarding whether a high-risk human papillomavirus (HR-HPV) infection increases the risk of developing renal cell carcinoma. The aim of this study was to investigate whether HPV infection has a role in the pathogenesis or development of a certain histological subtype of renal cell carcinoma. Formalin-fixed paraffin-embedded (FFPE) specimens of 122 patients with histopathologically proven renal cell carcinoma and their respective peritumoral tissues were examined. The presence of HPV-DNA was determined by a combination of MY/GP+ consensus primers and HPV-16/18 type specific nested PCRs followed by direct sequencing. Catalyzed signal-amplified colorimetric in situ hybridization (CSAC-ISH) technique was applied to determine the physical status of viral genome. The expression of p16INK4a and HPV L1 capsid proteins was evaluated using immunohistochemistry. HPV genome was detected in 37 (30.3%) tumor specimens and their four (4.1%) corresponding peritumoral tissues. HPV-18 was the most common viral type identified followed by HPV-16 and 58. Immunoexpression of p16INK4a was detected in 24 (20.3%) cases. Data analysis showed a significant correlation between p16INK4a expression and the presence of HR-HPV DNA (P < 0.001). CSAC-ISH analysis confirmed HR-HPV infection in 45% of tumors, which were previously tested positive for HPV-DNA. Diffuse signal pattern was identified in 15 (83.3%) samples whereas a mixed pattern of diffuse and punctate signals was only detectable in three cases. The results indicate an association of HR-HPV types with renal cell carcinoma. It is proposed that HPV infection in high-grade tumors might precede disease progression in a number of tumors, particularly of the papillary subtype.

  20. Dietary choline deficiency alters global and gene-specific DNA methylation in the developing hippocampus of mouse fetal brains.

    PubMed

    Niculescu, Mihai D; Craciunescu, Corneliu N; Zeisel, Steven H

    2006-01-01

    The availability of choline during critical periods of fetal development alters hippocampal development and affects memory function throughout life. Choline deficiency during fetal development reduces proliferation and migration of neuronal precursor cells in the mouse fetal hippocampus and these changes are associated with modifications in the protein levels of some cell cycle regulators and early differentiation markers. We fed C57 BL/6 mouse dams diets deficient or normal in choline content from days 12 to 17 of pregnancy, and then collected fetal brains on embryonic day 17. Using laser-capture micro-dissection we harvested cells from the ventricular and subventricular zones of Ammon's horn and from the prime germinal zone of the dentate gyrus (hippocampus). In the ventricular and subventricular zones from the choline-deficient group, we observed increased protein levels for kinase-associated phosphatase (Kap) and for p15(INK4b) (two cell cycle inhibitors). In the dentate gyrus, we observed increased levels of calretinin (an early marker of neuronal differentiation). In fetal brain from mothers fed a choline-deficient diet, DNA global methylation was decreased in the ventricular and subventricular zones of Ammon's horn. We also observed decreased gene-specific DNA methylation of the gene (Cdkn3) that encodes for Kap, correlating with increased expression of this protein. This was not the case for p15(INK4b) or calretinin (Cdkn2b and Calb2, respectively). These data suggest that choline deficiency-induced changes in gene methylation could mediate the expression of a cell cycle regulator and thereby alter brain development.

  1. 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.

  2. FOXM1 Induces a Global Methylation Signature That Mimics the Cancer Epigenome in Head and Neck Squamous Cell Carcinoma

    PubMed Central

    Teh, Muy-Teck; Gemenetzidis, Emilios; Patel, Deeviyaben; Tariq, Rameez; Nadir, Ayesha; Bahta, Adiam W.; Waseem, Ahmad; Hutchison, Iain L.

    2012-01-01

    The oncogene FOXM1 has been implicated in all major types of human cancer. We recently showed that aberrant FOXM1 expression causes stem cell compartment expansion resulting in the initiation of hyperplasia. We have previously shown that FOXM1 regulates HELLS, a SNF2/helicase involved in DNA methylation, implicating FOXM1 in epigenetic regulation. Here, we have demonstrated using primary normal human oral keratinocytes (NOK) that upregulation of FOXM1 suppressed the tumour suppressor gene p16INK4A (CDKN2A) through promoter hypermethylation. Knockdown of HELLS using siRNA re-activated the mRNA expression of p16INK4A and concomitant downregulation of two DNA methyltransferases DNMT1 and DNMT3B. The dose-dependent upregulation of endogenous FOXM1 (isoform B) expression during tumour progression across a panel of normal primary NOK strains (n = 8), dysplasias (n = 5) and head and neck squamous cell carcinoma (HNSCC) cell lines (n = 11) correlated positively with endogenous expressions of HELLS, BMI1, DNMT1 and DNMT3B and negatively with p16INK4A and involucrin. Bisulfite modification and methylation-specific promoter analysis using absolute quantitative PCR (MS-qPCR) showed that upregulation of FOXM1 significantly induced p16INK4A promoter hypermethylation (10-fold, P<0.05) in primary NOK cells. Using a non-bias genome-wide promoter methylation microarray profiling method, we revealed that aberrant FOXM1 expression in primary NOK induced a global hypomethylation pattern similar to that found in an HNSCC (SCC15) cell line. Following validation experiments using absolute qPCR, we have identified a set of differentially methylated genes, found to be inversely correlated with in vivo mRNA expression levels of clinical HNSCC tumour biopsy samples. This study provided the first evidence, using primary normal human cells and tumour tissues, that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from

  3. 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.

  4. 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.

  5. 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

  6. Loss of ARF sensitizes transgenic BRAFV600E mice to UV-induced melanoma via suppression of XPC

    PubMed Central

    Luo, Chi; Sheng, Jinghao; Hu, Miaofen G.; Haluska, Frank G.; Cui, Rutao; Xu, Zhengping; Tsichlis, Philip N.; Hu, Guo-fu; Hinds, Philip W.

    2013-01-01

    Both genetic mutations and ultraviolet (UV) irradiation can predispose individuals to melanoma. Although BRAFV600E is the most prevalent oncogene in melanoma, the BRAFV600E mutant is not sufficient to induce tumors in vivo. Mutation at the CDKN2A locus is another melanoma-predisposing event that can disrupt the function of both p16INK4a and ARF. Numerous studies have focused on the role of p16INK4a in melanoma, but the involvement of ARF, a well-known p53 activator, is still controversial. Using a transgenic BRAFV600E mouse model previously generated in our laboratory, we report that loss of ARF is able to enhance spontaneous melanoma formation and cause profound sensitivity to neonatal UVB exposure. Mechanistically, BRAFV600E and ARF deletion synergize to inhibit nucleotide excision repair by epigenetically repressing XPC and inhibiting the E2F4/DP1 complex. We suggest that the deletion of ARF promotes melanomagenesis not by abrogating p53 activation but by acting in concert with BRAFV600E to increase the load of DNA damage caused by UV irradiation. PMID:23650282

  7. Oxindole alkaloids from Uncaria tomentosa induce apoptosis in proliferating, G0/G1-arrested and bcl-2-expressing acute lymphoblastic leukaemia cells.

    PubMed

    Bacher, Nicole; Tiefenthaler, Martin; Sturm, Sonja; Stuppner, Hermann; Ausserlechner, Michael J; Kofler, Reinhard; Konwalinka, Günther

    2006-03-01

    Natural products are still an untapped source of promising lead compounds for the generation of antineoplastic drugs. Here, we investigated for the first time the antiproliferative and apoptotic effects of highly purified oxindole alkaloids, namely isopteropodine (A1), pteropodine (A2), isomitraphylline (A3), uncarine F (A4) and mitraphylline (A5) obtained from Uncaria tomentosa, a South American Rubiaceae, on human lymphoblastic leukaemia T cells (CCRF-CEM-C7H2). Four of the five tested alkaloids inhibited proliferation of acute lymphoblastic leukaemia cells. Furthermore, the antiproliferative effect of the most potent alkaloids pteropodine (A2) and uncarine F (A4) correlated with induction of apoptosis. After 48 h, 100 micromol/l A2 or A4 increased apoptotic cells by 57%. CEM-C7H2 sublines with tetracycline-regulated expression of bcl-2, p16ink4A or constitutively expressing the cowpox virus protein crm-A were used for further studies of the apoptosis-inducing properties of these alkaloids. Neither overexpression of bcl-2 or crm-A nor cell-cycle arrest in G0/G1 phase by tetracycline-regulated expression of p16INK4A could prevent alkaloid-induced apoptosis. Our results show the strong apoptotic effects of pteropodine and uncarine F on acute leukaemic lymphoblasts and recommend the alkaloids for further studies in xenograft models.

  8. 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

  9. Loss of the tyrosine phosphatase PTPRD leads to aberrant STAT3 activation and promotes gliomagenesis.

    PubMed

    Ortiz, Berenice; Fabius, Armida W M; Wu, Wei H; Pedraza, Alicia; Brennan, Cameron W; Schultz, Nikolaus; Pitter, Kenneth L; Bromberg, Jacqueline F; Huse, Jason T; Holland, Eric C; Chan, Timothy A

    2014-06-01

    PTPRD, which encodes the protein tyrosine phosphatase receptor-δ, is one of the most frequently inactivated genes across human cancers, including glioblastoma multiforme (GBM). PTPRD undergoes both deletion and mutation in cancers, with copy number loss comprising the primary mode of inactivation in GBM. However, it is unknown whether loss of PTPRD promotes tumorigenesis in vivo, and the mechanistic basis of PTPRD function in tumors is unclear. Here, using genomic analysis and a glioma mouse model, we demonstrate that loss of Ptprd accelerates tumor formation and define the oncogenic context in which Ptprd loss acts. Specifically, we show that in human GBMs, heterozygous loss of PTPRD is the predominant type of lesion and that loss of PTPRD and the CDKN2A/p16(INK4A) tumor suppressor frequently co-occur. Accordingly, heterozygous loss of Ptprd cooperates with p16 deletion to drive gliomagenesis in mice. Moreover, loss of the Ptprd phosphatase resulted in phospho-Stat3 accumulation and constitutive activation of Stat3-driven genetic programs. Surprisingly, the consequences of Ptprd loss are maximal in the heterozygous state, demonstrating a tight dependence on gene dosage. Ptprd loss did not increase cell proliferation but rather altered pathways governing the macrophage response. In total, we reveal that PTPRD is a bona fide tumor suppressor, pinpoint PTPRD loss as a cause of aberrant STAT3 activation in gliomas, and establish PTPRD loss, in the setting of CDKN2A/p16(INK4A) deletion, as a driver of glioma progression.

  10. [Human papillomavirus in squamous cell cancer of the head and neck. A study at the Ulm Military Hospital, Germany].

    PubMed

    Maier, M; Kraft, K; Steinestel, K; Schramm, A; Lorenz, K J; Tisch, M; Schwerer, M; Maier, H

    2013-07-01

    There is increasing evidence worldwide that human papillomavirus is a major risk factor for head and neck cancer. Only few studies on this association have been performed in Germany to date. For the purposes of the present study, tumor specimens from 223 patients with squamous cell cancer of the oral cavity, oropharynx, hypopharynx and larynx were analyzed for HPV DNA and p16INK4a expression. The prevalence of HPV genotype 16 (HPV16) DNA in the study population was 17.5%. Further high-risk HPV types were not detected. All HPV16-positive tumors showed intense p16INK4a expression. HPV16 prevalence was highest in tonsillar carcinoma (37.5%) and lowest in laryngeal cancer (2.8%). We observed a significantly higher incidence of cervical lymph node metastases in patients with HPV16-positive tonsillar carcinoma in comparison to HPV-negative tumors (p < 0.016). Tobacco and/or alcohol consumption was significantly lower in patients with HPV-positive tumors (p < 0.0001).

  11. The FBXO4 Tumor Suppressor Functions as a Barrier to BrafV600E-Dependent Metastatic Melanoma

    PubMed Central

    Lee, Eric K.; Lian, Zhaorui; D'Andrea, Kurt; Letrero, Richard; Sheng, WeiQi; Liu, Shujing; Diehl, J. Nathaniel; Pytel, Dariusz; Barbash, Olena; Schuchter, Lynn; Amaravaradi, Ravi; Xu, Xiaowei; Herlyn, Meenhard; Nathanson, Katherine L.

    2013-01-01

    Cyclin D1–cyclin-dependent kinase 4/6 (CDK4/6) dysregulation is a major contributor to melanomagenesis. Clinical evidence has revealed that p16INK4A, an allosteric inhibitor of CDK4/6, is inactivated in over half of human melanomas, and numerous animal models have demonstrated that p16INK4A deletion promotes melanoma. FBXO4, a specificity factor for the E3 ligase that directs timely cyclin D1 proteolysis, has not been studied in melanoma. We demonstrate that Fbxo4 deficiency induces Braf-driven melanoma and that this phenotype depends on cyclin D1 accumulation in mice, underscoring the importance of this ubiquitin ligase in tumor suppression. Furthermore, we have identified a substrate-binding mutation, FBXO4 I377M, that selectively disrupts cyclin D1 degradation while preserving proteolysis of the other known FBXO4 substrate, TRF1. The I377M mutation and Fbxo4 deficiency result in nuclear accumulation of cyclin D1, a key transforming neoplastic event. Collectively, these data provide evidence that FBXO4 dysfunction, as a mechanism for cyclin D1 overexpression, is a contributor to human malignancy. PMID:24019069

  12. The FBXO4 tumor suppressor functions as a barrier to BRAFV600E-dependent metastatic melanoma.

    PubMed

    Lee, Eric K; Lian, Zhaorui; D'Andrea, Kurt; Letrero, Richard; Sheng, WeiQi; Liu, Shujing; Diehl, J Nathaniel; Pytel, Dariusz; Barbash, Olena; Schuchter, Lynn; Amaravaradi, Ravi; Xu, Xiaowei; Herlyn, Meenhard; Nathanson, Katherine L; Diehl, J Alan

    2013-11-01

    Cyclin D1-cyclin-dependent kinase 4/6 (CDK4/6) dysregulation is a major contributor to melanomagenesis. Clinical evidence has revealed that p16(INK4A), an allosteric inhibitor of CDK4/6, is inactivated in over half of human melanomas, and numerous animal models have demonstrated that p16(INK4A) deletion promotes melanoma. FBXO4, a specificity factor for the E3 ligase that directs timely cyclin D1 proteolysis, has not been studied in melanoma. We demonstrate that Fbxo4 deficiency induces Braf-driven melanoma and that this phenotype depends on cyclin D1 accumulation in mice, underscoring the importance of this ubiquitin ligase in tumor suppression. Furthermore, we have identified a substrate-binding mutation, FBXO4 I377M, that selectively disrupts cyclin D1 degradation while preserving proteolysis of the other known FBXO4 substrate, TRF1. The I377M mutation and Fbxo4 deficiency result in nuclear accumulation of cyclin D1, a key transforming neoplastic event. Collectively, these data provide evidence that FBXO4 dysfunction, as a mechanism for cyclin D1 overexpression, is a contributor to human malignancy.

  13. 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.

  14. Increased size of solid organs in patients with Chuvash polycythemia and in mice with altered expression of HIF-1alpha and HIF-2alpha.

    PubMed

    Yoon, Donghoon; Okhotin, David V; Kim, Bumjun; Okhotina, Yulia; Okhotin, Daniel J; Miasnikova, Galina Y; Sergueeva, Adelina I; Polyakova, Lydia A; Maslow, Alexei; Lee, Yonggu; Semenza, Gregg L; Prchal, Josef T; Gordeuk, Victor R

    2010-05-01

    Chuvash polycythemia, the first hereditary disease associated with dysregulated oxygen-sensing to be recognized, is characterized by a homozygous germ-line loss-of-function mutation of the VHL gene (VHL(R200W)) resulting in elevated hypoxia inducible factor (HIF)-1alpha and HIF-2alpha levels, increased red cell mass and propensity to thrombosis. Organ volume is determined by the size and number of cells, and the underlying molecular control mechanisms are not fully elucidated. Work from several groups has demonstrated that the proliferation of cells is regulated in opposite directions by HIF-1alpha and HIF-2alpha. HIF-1alpha inhibits cell proliferation by displacing MYC from the promoter of the gene encoding the cyclin-dependent kinase inhibitor, p21(Cip1), thereby inducing its expression. In contrast, HIF-2alpha promotes MYC activity and cell proliferation. Here we report that the volumes of liver, spleen, and kidneys relative to body mass were larger in 30 individuals with Chuvash polycythemia than in 30 matched Chuvash controls. In Hif1a(+/-) mice, which are heterozygous for a null (knockout) allele at the locus encoding HIF-1alpha, hepatic HIF-2alpha mRNA was increased (2-fold) and the mass of the liver was increased, compared with wild-type littermates, without significant difference in cell volume. Hepatic p21(Cip1) mRNA levels were 9.5-fold lower in Hif1a(+/-) mice compared with wild-type littermates. These data suggest that, in addition to increased red cell mass, the sizes of liver, spleen, and kidneys are increased in Chuvash polycythemia. At least in the liver, this phenotype may result from increased HIF-2alpha and decreased p21(Cip1) levels leading to increased hepatocyte proliferation. PMID:20140661

  15. 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.

  16. CDK2 Activation in Mouse Epidermis Induces Keratinocyte Proliferation but Does Not Affect Skin Tumor Development

    PubMed Central

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

    2008-01-01

    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 p21Cip1 and p27Kip1. 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 K5Cdk4D158N mice. The D158N mutation blocks CDK4 kinase activity without interfering with its binding capability. CDK2 activation via overexpression of CDK4D158N, but not of CDK2, resulted in epidermal hyperplasia. We observed elevated levels of p21Cip1 in K5Cdk2, but not in K5Cdk4D158N, epidermis, suggesting that CDK2 overexpression elicits a p21Cip1 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. PMID:18599613

  17. 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

  18. 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.

  19. Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway

    PubMed Central

    Pumiglia, Kevin M.; Decker, Stuart J.

    1997-01-01

    The mitogen-activated protein kinase (MAPK) cascade plays a crucial role in the transduction of extracellular signals into responses governing growth and differentiation. The effects of a specific inhibitor of the MAPK kinase (MEK)/MAPK pathway (PD98059) on nerve growth factor (NGF)-induced growth arrest and inhibition of cell cycle-dependent kinases (CDKs) have been examined. Treatment of NIH 3T3 cells expressing TRKA with PD98059 dramatically reversed the complete inhibition of growth of these cells caused by NGF. PD98059 also blocked the ability of NGF to inhibit the activities of CDK4 and CDK2, while partially preventing NGF induction of p21Cip1/WAF1. To independently evaluate the involvement of the MEK/MAPK pathway in growth arrest, an inducible activated form of the Raf-1 protooncogene (ΔRAF-1:ER) was expressed in these cells. Activation of ΔRAF-1:ER resulted in a prolonged increase in MAPK activity and growth arrest of these cells, with concomitant induction of p21Cip1/WAF1 and inhibition of CDK2 activity. These effects of ΔRAF-1:ER activation were all reversed by treatment of cells with PD98059. These data indicate that in addition to functioning as a positive effector of growth, stimulation of the MEK/MAPK pathway can result in an inhibition of CDK activity and cell cycle arrest. PMID:9012803

  20. Knockdown of Sec8 promotes cell-cycle arrest at G1/S phase by inducing p21 via control of FOXO proteins.

    PubMed

    Tanaka, Toshiaki; Iino, Mituyoshi

    2014-02-01

    p21(Cip1) protein inhibits the activity of cyclins at the G(1) checkpoint and influences transition of cells from the G(1) to the S phase of the cell cycle. Moreover, expression of members of the FOXO family (active form of forkhead transcription factors of the O class) in dividing cells promotes cell-cycle arrest at the G(1)/S boundary via regulation of p21(Cip1). Recently, the exocyst complex, including Sec8, has been implicated in various roles independent of its role in secretion, such as cell migration, invadopodia formation, cytokinesis, glucose uptake and neural development. Given the essential roles of the exocyst complex in cellular and developmental processes, disruption of its function may be involved in various diseases such as cancer, diabetes and neuronal disorders. However, the relationship between Sec8 and the cell cycle remains to be elucidated. In this study, knockdown of Sec8 inhibited cell growth and promoted cell-cycle arrest at the G(1)/S phase by control of p21 expression and retinoblastoma protein phosphorylation. Furthermore, Sec8 regulated FOXO family proteins via ubiquitin-proteasome degradation by regulating the expression of the murine double minute 2 (Mdm2) protein but not S-phase kinase-associated protein 2 (Skp2).

  1. Statin-induced depletion of geranylgeranyl pyrophosphate inhibits cell proliferation by a novel pathway of Skp2 degradation.

    PubMed

    Vosper, Jonathan; Masuccio, Alessia; Kullmann, Michael; Ploner, Christian; Geley, Stephan; Hengst, Ludger

    2015-02-20

    Statins, such as lovastatin, can induce a cell cycle arrest in the G1 phase. This robust antiproliferative activity remains intact in many cancer cells that are deficient in cell cycle checkpoints and leads to an increased expression of CDK inhibitor proteins p27Kip1 and p21Cip1. The molecular details of this statin-induced growth arrest remains unclear. Here we present evidence that lovastatin can induce the degradation of Skp2, a subunit of the SCFSkp2 ubiquitin ligase that targets p27Kip1 and p21Cip1 for proteasomal destruction. The statin-induced degradation of Skp2 is cell cycle phase independent and does not require its well characterised degradation pathway mediated by APC/CCdh1- or Skp2 autoubiquitination. An N-terminal domain preceding the F-box of Skp2 is both necessary and sufficient for its statin mediated degradation. The degradation of Skp2 results from statin induced depletion of geranylgeranyl isoprenoid intermediates of cholesterol biosynthesis. Inhibition of geranylgeranyl-transferase-I also promotes APC/CCdh1- independent degradation of Skp2, indicating that de-modification of a geranylgeranylated protein triggers this novel pathway of Skp2 degradation.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27121132

  3. 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

    2016-05-01

    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. PMID:27121132

  4. The KRAB Zinc Finger Protein Roma/Zfp157 Is a Critical Regulator of Cell-Cycle Progression and Genomic Stability

    PubMed Central

    Ho, Teresa L.F.; Guilbaud, Guillaume; Blow, J. Julian; Sale, Julian E.; Watson, Christine J.

    2016-01-01

    Summary Regulation of DNA replication and cell division is essential for tissue growth and maintenance of genomic integrity and is particularly important in tissues that undergo continuous regeneration such as mammary glands. We have previously shown that disruption of the KRAB-domain zinc finger protein Roma/Zfp157 results in hyperproliferation of mammary epithelial cells (MECs) during pregnancy. Here, we delineate the mechanism by which Roma engenders this phenotype. Ablation of Roma in MECs leads to unscheduled proliferation, replication stress, DNA damage, and genomic instability. Furthermore, mouse embryonic fibroblasts (MEFs) depleted for Roma exhibit downregulation of p21Cip1 and geminin and have accelerated replication fork velocities, which is accompanied by a high rate of mitotic errors and polyploidy. In contrast, overexpression of Roma in MECs halts cell-cycle progression, whereas siRNA-mediated p21Cip1 knockdown ameliorates, in part, this phenotype. Thus, Roma is an essential regulator of the cell cycle and is required to maintain genomic stability. PMID:27149840

  5. No Viral Association Found in a Set of Differentiated Vulvar Intraepithelial Neoplasia Cases by Human Papillomavirus and Pan-Viral Microarray Testing

    PubMed Central

    Saglam, Ozlen; Samayoa, Erik; Somasekar, Sneha; Naccache, Samia; Iwasaki, Akiko; Chiu, Charles Y

    2015-01-01

    Vulvar Intraepithelial Neoplasia (VIN) is the precursor lesion of Vulvar Squamous Cell Carcinoma (VSCC), and the differentiated type (dVIN) is more frequently observed in relation to VSCC. In contrast to usual-type VIN (uVIN), which is related to infection by human papillomavirus (HPV), a germline mutation in the p53 gene is thought to be associated with ~90% of dVIN cases. To date, no infectious agent has been identified in association with dVIN, and studies investigating this possibility have been hindered by the difficulty in accurately diagnosing dVIN from small biopsies. Here, we used immunostaining for p16ink4a, a biomarker for HPV infection, to study 14 uVIN high-grade VIN and 14 dVIN cases, and to select 10 dVIN cases to broadly screen for all known viruses using a pan-viral microarray platform (ViroChip). All of the uVIN tissue samples, including 8 warty and 6 basaloid cases, showed positivity with the p16ink4a immunostain. The staining pattern was full-thickness for all except two cases in which positive staining was localized in the lower 1/3 of the epidermis. In contrast, immunostaining for p16ink4a was negative in all dVIN cases. ViroChip analysis of 10 pure dVIN samples confirmed the absence of human papillomavirus subtypes or any other virus with the exception of a single sample that showed a weak microarray signature to a porcine herpesvirus. Follow-up PCR testing of the sample was negative for herpesvirus, and in-depth metagenomic next-generation sequencing revealed only sequences corresponding to non-pathogenic viral flora and bacterial contamination. In this study, we demonstrated lack of a virus association in 10 dVIN cases. Alternative pathways for carcinogenesis such as the p53 mutation should be considered for investigation of potential treatment options in dVIN. PMID:25894343

  6. Melanoma susceptibility genes and risk assessment.

    PubMed

    Marzuka-Alcalá, Alexander; Gabree, Michele Jacobs; Tsao, Hensin

    2014-01-01

    Familial melanoma accounts for approximately a tenth of all melanoma cases. The most commonly known melanoma susceptibility gene is the highly penetrant CDKN2A (p16INK4a) locus, which is transmitted in an autosomal dominant fashion and accounts for approximately 20-50 % of familial melanoma cases. Mutated p16INK4a shows impaired capacity to inhibit the cyclin D1-CDK4 complex, allowing for unchecked cell cycle progression. Mutations in the second protein coded by CDKN2A, p14ARF, are much less common and result in proteasomal degradation of p53 with subsequent accumulation of DNA damage as the cell progresses through the cell cycle without a functional p53-mediated DNA damage response. Mutations in CDK4 that impair the inhibitory interaction with p16INK4a also increase melanoma risk but these mutations are extremely rare. Genes of the melanin biosynthetic pathway, including MC1R and MITF, have also been implicated in melanomagenesis. MC1R variants were traditionally thought to increase risk for melanoma secondary to intensified UV-mediated DNA damage in the setting of absent photoprotective eumelanin. Accumulation of pheomelanin, which appears to have a carcinogenic effect regardless of UV exposure, may be a more likely mechanism. Impaired SUMOylation of the E318K variant of MITF results in increased transcription of genes that confer melanocytes with a pro-malignant phenotype. Mutations in the tumor suppressor BAP1 enhance the metastatic potential of uveal melanoma and predispose to cutaneous/ocular melanoma, atypical melanocytic tumors, and other internal malignancies (COMMON syndrome). Genome-wide association studies have identified numerous low-risk alleles. Although several melanoma susceptibility genes have been identified, risk assessment tools have been developed only for the most common gene implicated with hereditary melanoma, CDKN2A. MelaPRO, a validated model that relies on Mendelian inheritance and Bayesian probability theories, estimates carrier

  7. The effect of human papillomavirus on DNA repair in head and neck squamous cell carcinoma.

    PubMed

    Low, Garren M; Thylur, David S; N Yamamoto, Vicky; Sinha, Uttam K

    2016-10-01

    Much of the current literature regarding the molecular pathophysiology of human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSCC) has focused on the virus's effect on cell cycle modulation and cell proliferation. A second mechanism of pathogenicity employed by HPV, dysregulation of cellular DNA repair processes, has been more sparsely studied. The purpose of this review is to describe current understanding about the effect of HPV on DNA repair in HNSCC, taking cues from cervical cancer literature. HPV affects DNA-damage response pathways by interacting with many proteins, including ATM, ATR, MRN, γ-H2AX, Chk1, Chk2, p53, BRCA1, BRCA2, RAD51, Rb-related proteins 107 and 130, Tip60, and p16INK4A. Further elucidation of these pathways could lead to development of targeted therapies and improvement of current treatment protocols.

  8. The effect of human papillomavirus on DNA repair in head and neck squamous cell carcinoma.

    PubMed

    Low, Garren M; Thylur, David S; N Yamamoto, Vicky; Sinha, Uttam K

    2016-10-01

    Much of the current literature regarding the molecular pathophysiology of human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSCC) has focused on the virus's effect on cell cycle modulation and cell proliferation. A second mechanism of pathogenicity employed by HPV, dysregulation of cellular DNA repair processes, has been more sparsely studied. The purpose of this review is to describe current understanding about the effect of HPV on DNA repair in HNSCC, taking cues from cervical cancer literature. HPV affects DNA-damage response pathways by interacting with many proteins, including ATM, ATR, MRN, γ-H2AX, Chk1, Chk2, p53, BRCA1, BRCA2, RAD51, Rb-related proteins 107 and 130, Tip60, and p16INK4A. Further elucidation of these pathways could lead to development of targeted therapies and improvement of current treatment protocols. PMID:27688101

  9. 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

  10. Germline Transgenic Methods for Tracking Cells and Testing Gene Function during Regeneration in the Axolotl

    PubMed Central

    Khattak, Shahryar; Schuez, Maritta; Richter, Tobias; Knapp, Dunja; Haigo, Saori L.; Sandoval-Guzmán, Tatiana; Hradlikova, Kristyna; Duemmler, Annett; Kerney, Ryan; Tanaka, Elly M.

    2013-01-01

    The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum (axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly overexpress the cell-cycle inhibitor p16INK4a, which negatively regulates spinal cord regeneration. These tissue-specific germline axolotl lines and tightly inducible Cre drivers and LoxP reporter lines render this classical regeneration model molecularly accessible. PMID:24052945

  11. Germline transgenic methods for tracking cells and testing gene function during regeneration in the axolotl.

    PubMed

    Khattak, Shahryar; Schuez, Maritta; Richter, Tobias; Knapp, Dunja; Haigo, Saori L; Sandoval-Guzmán, Tatiana; Hradlikova, Kristyna; Duemmler, Annett; Kerney, Ryan; Tanaka, Elly M

    2013-01-01

    The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum (axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly overexpress the cell-cycle inhibitor p16 (INK4a) , which negatively regulates spinal cord regeneration. These tissue-specific germline axolotl lines and tightly inducible Cre drivers and LoxP reporter lines render this classical regeneration model molecularly accessible. PMID:24052945

  12. Novel CDKN2A mutation detected in Spanish melanoma pedigree.

    PubMed

    de Torre, Carlos; Martínez-Escribano, Jorge

    2010-08-01

    We have examined alterations in the cyclin-dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent kinase 4 (CDK4), major melanoma predisposing genes, in a Spanish melanoma-prone population comprising 61 patients from 45 families. Using an extensive genetic analysis of these genes, including sequence analysis and multiplex ligation-dependent probe amplification, we have found four different CDKN2A alterations in cases from seven melanoma kindred. Three of them are CDKN2A mutations previously described in the Mediterranean population (p.G101W, p.V59G and c.358delG) in addition to an undescribed deletion (p. M54del) which has been detected in a melanoma kindred. This codon deletion affects an essential residue in the interaction of p16INK4A with cdk6 and has not been reported in melanoma patients and other cancers. PMID:20653773

  13. Genetic and epigenetic changes in nasopharyngeal carcinoma.

    PubMed

    Lo, Kwok-Wai; Huang, Dolly P

    2002-12-01

    Nasopharyngeal carcinoma (NPC) is a malignancy with remarkable racial and geographic distribution. The development of this EBV-associated cancer likely involves cumulative genetic and epigenetic changes in a background of predisposed genetic and environmental factors. Genome-wide studies have unravelled multiple chromosomal abnormalities with involvement of specific oncogenes and tumour suppressor genes. Alterations of genes such as Ras association domain family 1A (RASSF1A), p16/INK4A, p14/ARF suggest that multiple cellular pathways were dysregulated in the NPC cells. Studies on the precancerous lesions revealed early genetic changes and a critical role of EBV latent infection in the development of this cancer. Based on the existing findings, a pathogenetic model for NPC is proposed.

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

    PubMed

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

    2016-04-26

    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.

  15. [HPV-associated carcinomas of the female genital tract. Molecular mechanisms of development].

    PubMed

    Reuschenbach, M; Vinokurova, S; von Knebel Doeberitz, M

    2011-11-01

    Infections with human papillomaviruses (HPV) are a common occurrence in both men and women. In contrast HPV-associated neoplasias are relatively rare and occur only in certain areas of the body. The virus has obviously developed efficient mechanisms for its persistence without inducing too much damage to the host. The formation of neoplasia seems to be more an exception. Epigenetic mechanisms play an important role in the regulation of viral gene expression. Investigations have indicated that exactly the transition from the permissive infection stage to a transformation stage, where neoplastic alterations can occur due to expression of the viral oncogenes, is associated with certain methylation patterns of the viral genome which promote the expression of the oncogenes E6 and E7. The transforming stage is seen as the actual carcinogenic event and can be immunohistochemically detected by the biomarker p16(INK4a).

  16. Promoter methylation of TIMP3 and CDH1 predicts better outcome in head and neck squamous cell carcinoma treated by radiotherapy only.

    PubMed

    De Schutter, H; Geeraerts, H; Verbeken, E; Nuyts, S

    2009-02-01

    As with other solid tumor types, head and neck squamous cell carcinoma (HNSCC) has been identified as an epigenetic, as well as genetic, disease. Consequently, promoter hypermethylation, being the most important aberrant epigenetic characteristic, has been intensively investigated for its biomarker potential in this cancer type. As many of these evaluations are obscured by a heterogeneity of treatments, the current study aimed to evaluate the incidence and prognostic value of the promoter hypermethylation of TIMP3, CDH1, DAPK, RASSF1A, p16INK4A and MGMT in HNSCC treated solely by radiotherapy. In 46 patients with advanced HNSCC treated with a hybrid accelerated fractionation radiotherapy schedule, DNA extracted from pretreatment paraffin-embedded tumor biopsies was used to determine the methylation status of the genes of interest by methylation-specific PCR (MSP). The detected epigenetic silencing was related with outcome in terms of locoregional control (LRC), and overall (OS), disease-free (DFS) and disease-specific survival (DSS). Tumor biopsies revealed the epigenetic silencing of MGMT in 42.5% (17 of 40) of patients and of TIMP3 in 40.5% (17 of 42) of cases. For the remaining investigated genes, a lower methylation percentage was detected: 13.2% (5 of 38) for CDH1, 11.4% (4 of 44) for DAPK, 4.8% (2 of 42) for p16INK4A and 2.4% (1 of 41) for RASSF1A. The promoter hypermethylation of TIMP3 and CDH1 was significantly related with better LRC (p=0.009 and p=0.02, respectively), OS (p=0.005 and p=0.002, respectively), DFS (p=0.02 and p=0.004, respectively) and DSS (p=0.12 and p=0.007, respectively). In conclusion, in this representative group of 46 patients with advanced HNSCC treated by radiotherapy only, the epigenetic silencing of TIMP3 and CDH1 predicted a better outcome.

  17. Dynamic assembly of chromatin complexes during cellular senescence: implications for the growth arrest of human melanocytic nevi

    PubMed Central

    Bandyopadhyay, Debdutta; Curry, Jonathan L; Lin, Qiushi; Richards, Hunter W; Chen, Dahu; Hornsby, Peter J; Timchenko, Nikolai A; Medrano, Estela E

    2007-01-01

    The retinoblastoma (RB)/p16INK4a pathway regulates senescence of human melanocytes in culture and oncogene-induced senescence of melanocytic nevi in vivo. This senescence response is likely due to chromatin modifications because RB complexes from senescent melanocytes contain increased levels of histone deacetylase (HDAC) activity and tethered HDAC1. Here we show that HDAC1 is prominently detected in p16INK4a-positive, senescent intradermal melanocytic nevi but not in proliferating, recurrent nevus cells that localize to the epidermal/dermal junction. To assess the role of HDAC1 in the senescence of melanocytes and nevi, we used tetracycline-based inducible expression systems in cultured melanocytic cells. We found that HDAC1 drives a sequential and cooperative activity of chromatin remodeling effectors, including transient recruitment of Brahma (Brm1) into RB/HDAC1 mega-complexes, formation of heterochromatin protein 1β (HP1β)/SUV39H1 foci, methylation of H3-K9, stable association of RB with chromatin and significant global heterochromatinization. These chromatin changes coincide with expression of typical markers of senescence, including the senescent-associated β-galactosidase marker. Notably, formation of RB/HP1β foci and early tethering of RB to chromatin depends on intact Brm1 ATPase activity. As cells reached senescence, ejection of Brm1 from chromatin coincided with its dissociation from HP1β/RB and relocalization to protein complexes of lower molecular weight. These results provide new insights into the role of the RB pathway in regulating cellular senescence and implicate HDAC1 as a likely mediator of early chromatin remodeling events. PMID:17578512

  18. Antagonizing pathways leading to differential dynamics in colon carcinogenesis in Shugoshin1 (Sgo1)-haploinsufficient chromosome instability model.

    PubMed

    Rao, Chinthalapally V; Sanghera, Saira; Zhang, Yuting; Biddick, Laura; Reddy, Arun; Lightfoot, Stan; Dai, Wei; Yamada, Hiroshi Y

    2016-05-01

    Colon cancer is the second most lethal cancer. It is predicted to claim 50,310 lives in 2014. Chromosome Instability (CIN) is observed in 80-90% of colon cancers, and is thought to contribute to colon cancer progression and recurrence. However, there are no animal models of CIN that have been validated for studies of colon cancer development or drug testing. In this study, we sought to validate a mitotic error-induced CIN model mouse, the Shugoshin1 (Sgo1) haploinsufficient mouse, as a colon cancer study model. Wild-type and Sgo1(-/+) mice were treated with the colonic carcinogen, azoxymethane (AOM). We tracked colon tumor development 12, 24, and 36 wk after treatment to assess progression of colon tumorigenesis. Initially, more precancerous lesions, Aberrant Crypt Foci (ACF), developed in Sgo1(-/+) mice. However, the ACF did not develop straightforwardly into larger tumors. At the 36-wk endpoint, the number of gross tumors in Sgo1(-/+) mice was no different from that in wild-type controls. However, Copy Number Variation (CNV) analysis indicated that fully developed colon tumor in Sgo1(-/+) mice carried 13.75 times more CNV. Immunohistological analyses indicated that Sgo1(-/+) mice differentially expressed IL-6, Bcl2, and p16(INK4A) . We propose that formation of ACF in Sgo1(-/+) mice is facilitated by the IL6-STAT3-SOCS3 oncogenic pathway and by the Bcl2-anti-apoptotic pathway, yet further development of the ACF to tumors is inhibited by the p16(INK4A) tumor suppressor pathway. Manipulating these pathways would be beneficial for inhibiting development of colon cancer with CIN. PMID:25773652

  19. 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. PMID:27203461

  20. No role for human papillomavirus in esophageal squamous cell carcinoma in China.

    PubMed

    Koshiol, Jill; Wei, Wen-Qiang; Kreimer, Aimee R; Chen, Wen; Gravitt, Patti; Ren, Jian-Song; Abnet, Christian C; Wang, Jian-Bing; Kamangar, Farin; Lin, Dong-Mei; von Knebel-Doeberitz, Magnus; Zhang, Yu; Viscidi, Raphael; Wang, Guo-Qing; Gillison, Maura L; Roth, Mark J; Dong, Zhi-Wei; Kim, Esther; Taylor, Philip R; Qiao, You-Lin; Dawsey, Sanford M

    2010-07-01

    Certain regions of China have high rates of esophageal squamous cell carcinoma (ESCC). Previous studies of human papillomavirus (HPV), a proposed causal factor, have produced highly variable results. We attempted to evaluate HPV and ESCC more definitively using extreme care to prevent DNA contamination. We collected tissue and serum in China from 272 histopathologically-confirmed ESCC cases with rigorous attention to good molecular biology technique. We tested for HPV DNA in fresh-frozen tumor tissue using PCR with PGMY L1 consensus primers and HPV16 and 18 type-specific E6 and E7 primers, and in formalin-fixed paraffin-embedded tumor tissue using SPF(10) L1 primers. In HPV-positive cases, we evaluated p16(INK4a) overexpression and HPV E6/E7 seropositivity as evidence of carcinogenic HPV activity. beta-globin, and thus DNA, was adequate in 98.2% of the frozen tumor tissues (267/272). Of these, 99.6% (95% confidence interval (CI) = 97.9-100.0%) were negative for HPV DNA by PGMY, and 100% (95% CI = 98.6-100%) were negative by HPV16/18 E6/E7 PCR. In the corresponding formalin-fixed paraffin-embedded tumor specimens, 99.3% (95% CI = 97.3-99.9%) were HPV negative by SPF(10). By PGMY, 1 case tested weakly positive for HPV89, a noncancer causing HPV type. By SPF(10), 2 cases tested weakly positive: 1 for HPV16 and 1 for HPV31. No HPV DNA-positive case had evidence of HPV oncogene activity as measured by p16(INK4a) overexpression or E6/E7 seropositivity. This study provides the most definitive evidence to date that HPV is not involved in ESCC carcinogenesis in China. HPV DNA contamination cannot be ruled out as an explanation for high HPV prevalence in ESCC tissue studies with less stringent tissue procurement and processing protocols.

  1. Biological evidence for a causal role of HPV16 in a small fraction of laryngeal squamous cell carcinoma

    PubMed Central

    Halec, G; Holzinger, D; Schmitt, M; Flechtenmacher, C; Dyckhoff, G; Lloveras, B; Höfler, D; Bosch, F X; Pawlita, M

    2013-01-01

    Background: Human papillomavirus (HPV) is a causal factor in virtually all cervical and a subset of oropharyngeal squamous cell carcinoma (OP-SCC), whereas its role in laryngeal squamous cell carcinoma (L-SCC) is unclear. Methods: Formalin-fixed paraffin-embedded (N=154) and deep-frozen tissues (N=55) of 102 L-SCC patients were analysed for the presence of 51 mucosal HPV types. HPV DNA-positive (HPV DNA+) cases were analysed for E6*I mRNA transcripts of all high risk (HR)/probably/possibly (p)HR-HPV identified, and for HPV type 16 (HPV16) viral load. Expression of p16INK4a, pRb, cyclin D1 and p53 was analysed by immunohistochemistry. Results: Ninety-two patients were valid in DNA analysis, of which 32 (35%) had at least one HPV DNA+ sample. Among the 29 single infections, 22 (76%) were HPV16, 2 (7%) HPV56 and 1 each (4%) HPV45, HPV53, HPV70, HPV11 and HPV42. Three cases harboured HPV16 with HPV33 (twice) or HPV45. Only 32% of HPV DNA+ findings were reproducible. Among HPV16 DNA+ L-SCC, 2 out of 23 (9%) had high viral loads, 5 out of 25 (21%) expressed E6*I mRNA and 3 out of 21 (14%) showed high p16INK4a and low pRb expression (all three HPV16 RNA-positive), immunohistochemical marker combination not identified in any other HPV DNA+ or HPV DNA-negative (HPV DNA−) L-SCC, respectively. Conclusion: HPV type 16 has a causative role in a small subgroup of L-SCC (<5% in this German hospital series). PMID:23778529

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

    PubMed Central

    Kagawa, Shingo; Natsuizaka, Mitsuteru; Whelan, Kelly A.; Facompre, Nicole; Naganuma, Seiji; Ohashi, Shinya; Kinugasa, Hideaki; Egloff, Ann Marie; Basu, Devraj; Gimotty, Phyllis A.; Klein-Szanto, Andres J; Bass, Adam; Wong, Kwok-Kin; Diehl, J. Alan; Rustgi, Anil K.; Nakagawa, Hiroshi

    2014-01-01

    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 (RNAi) 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 p16INK4A-Rb pathway. Loss of p16INK4A 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 TGF-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. PMID:24931169

  3. 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

  4. Polyomavirus-associated Trichodysplasia spinulosa involves hyperproliferation, pRB phosphorylation and upregulation of p16 and p21.

    PubMed

    Kazem, Siamaque; van der Meijden, Els; Wang, Richard C; Rosenberg, Arlene S; Pope, Elena; Benoit, Taylor; Fleckman, Philip; Feltkamp, Mariet C W

    2014-01-01

    Trichodysplasia spinulosa (TS) is a proliferative skin disease observed in severely immunocompromized patients. It is characterized by papule and trichohyalin-rich spicule formation, epidermal acanthosis and distention of dysmorphic hair follicles overpopulated by inner root sheath cells (IRS). TS probably results from active infection with the TS-associated polyomavirus (TSPyV), as indicated by high viral-load, virus protein expression and particle formation. The underlying pathogenic mechanism imposed by TSPyV infection has not been solved yet. By analogy with other polyomaviruses, such as the Merkel cell polyomavirus associated with Merkel cell carcinoma, we hypothesized that TSPyV T-antigen promotes proliferation of infected IRS cells. Therefore, we analyzed TS biopsy sections for markers of cell proliferation (Ki-67) and cell cycle regulation (p16ink4a, p21waf, pRB, phosphorylated pRB), and the putatively transforming TSPyV early large tumor (LT) antigen. Intense Ki-67 staining was detected especially in the margins of TS hair follicles, which colocalized with TSPyV LT-antigen detection. In this area, staining was also noted for pRB and particularly phosphorylated pRB, as well as p16ink4a and p21waf. Healthy control hair follicles did not or hardly stained for these markers. Trichohyalin was particularly detected in the center of TS follicles that stained negative for Ki-67 and TSPyV LT-antigen. In summary, we provide evidence for clustering of TSPyV LT-antigen-expressing and proliferating cells in the follicle margins that overproduce negative cell cycle regulatory proteins. These data are compatible with a scenario of TSPyV T-antigen-mediated cell cycle progression, potentially creating a pool of proliferating cells that enable viral DNA replication and drive papule and spicule formation.

  5. Functional inactivation of Rb sensitizes cancer cells to TSC2 inactivation induced cell Death

    PubMed Central

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

    2012-01-01

    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 p16ink4a 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 p16ink4a 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. PMID:23022476

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

    PubMed Central

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

    2009-01-01

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

  7. Structure-Guided Discovery of Selective Antagonists for the Chromodomain of Polycomb Repressive Protein CBX7.

    PubMed

    Ren, Chunyan; Smith, Steven G; Yap, Kyoko; Li, SiDe; Li, Jiaojie; Mezei, Mihaly; Rodriguez, Yoel; Vincek, Adam; Aguilo, Francesca; Walsh, Martin J; Zhou, Ming-Ming

    2016-06-01

    The chromobox 7 (CBX7) protein of the polycomb repressive complex 1 (PRC1) functions to repress transcription of tumor suppressor p16 (INK4a) through long noncoding RNA, ANRIL (antisense noncoding RNA in the INK4 locus) directed chromodomain (ChD) binding to trimethylated lysine 27 of histone H3 (H3K27me3), resulting in chromatin compaction at the INK4a/ARF locus. In this study, we report structure-guided discovery of two distinct classes of small-molecule antagonists for the CBX7ChD. Our Class A compounds, a series including analogues of the previously reported MS452, inhibit CBX7ChD/methyl-lysine binding by occupying the H3K27me3 peptide binding site, whereas our Class B compound, the newly discovered MS351, appears to inhibit H3K27me3 binding when CBX7ChD is bound to RNA. Our crystal structure of the CBX7ChD/MS351 complex reveals the molecular details of ligand recognition by the aromatic cage residues that typically engage in methyl-lysine binding. We further demonstrate that MS351 effectively induces transcriptional derepression of CBX7 target genes, including p16 (INK4a) in mouse embryonic stem cells and human prostate cancer PC3 cells. Thus, MS351 represents a new class of ChD antagonists that selectively targets the biologically active form of CBX7 of the PRC1 in long noncoding RNA- and H3K27me3-directed gene transcriptional repression. PMID:27326334

  8. 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.

  9. Potential role of human papilloma virus in the pathogenesis of gastric cancer

    PubMed Central

    Snietura, Miroslaw; Waniczek, Dariusz; Piglowski, Wojciech; Kopec, Agnieszka; Nowakowska-Zajdel, Ewa; Lorenc, Zbigniew; Muc-Wierzgon, Malgorzata

    2014-01-01

    AIM: To demonstrate the presence and biological activity of human papilloma virus (HPV) in gastric cancer (GAC) tissues. METHODS: The study involved 84 surgically treated patients with gastric adenocarcinoma, regardless of the clinical stage of the disease. The presence of HPV DNA of high oncogenic risk types in formalin-fixed, paraffin-embedded tumor samples was determined using quantitative polymerase chain reaction analysis. A stringent protocol of prevention of cross- and environmental contamination was applied during DNA isolation, and amplification, as well as confirmation of the biological activity of the virus in tumor cells, was implemented. The study utilized the Real-time High Risk HPV test, which detects the DNA of 14 HPV subtypes that are considered to have high oncogenic potential. The overexpression of the p16INK4a protein assessed immunohistochemically was considered confirmation of the HPV infection. RESULTS: Among the 89 patients initially included in the study group, diagnostic results were obtained for 84 individuals. In five cases, either the histopathological material was too scant to isolate the necessary amount of DNA, or the isolated DNA was significantly degraded, resulting in the failure of internal control amplification within the predefined number of 35 cycles. Those patients were excluded from further analysis. The amplification of HPV DNA was demonstrated in none of the 84 tissue samples; thus, all cases were considered to have a negative DNA status of highly oncogenic HPV subtypes. Immunohistochemical staining provided diagnostic results for all of the examined tissue samples, and excluded the accumulation of the p16INK4a protein in tumor cells, thus confirming the lack of active HPV infection in all of the individuals. CONCLUSION: The study does not confirm the presence or biological activity of HPV in tumor tissues. Thus, the relationship between GAC and HPV infection, in the Central European population seems doubtful. PMID

  10. 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.

  11. The role of human papillomavirus in head and neck cancer in Senegal

    PubMed Central

    2013-01-01

    Background Exploring the presence and role of human papillomavirus (HPV) in head and neck cancer (HNC) is a necessary step to evaluate the potential impact of HPV prophylactic vaccines. Objective To assess the prevalence and oncogenic role of HPV in HNC in Senegal. Methods This is a multicenter cross-sectional study. Paraffin-embedded blocks of cases diagnosed with invasive HNC between 2002 and 2010 were collected from 4 pathology laboratories in Senegal. Presence of HPV DNA was determined by PCR and DEIA, and genotyping performed with LiPA25. Tubulin analysis was performed to assess DNA quality. HPV DNA-positive cases were tested for p16INK4a expression. Findings A total of 117 cases were included in the analysis: 71% were men, mean age was 52 years old (SD ±18.3), and 96% of cases were squamous cell carcinoma. Analysis was performed on 41 oral cavity tumors, 64 laryngeal tumors, 5 oropharyngeal tumors and 7 pharyngeal tumors. Only four cases (3.4%; 95% CI = 0.9%-8.5%) harbored HPV DNA. HPV types detected were HPV16, HPV35 and HPV45. However, among HPV-positive cases, none showed p16INK4a overexpression. Conclusion Our findings indicate that HPV DNA prevalence in HNC in Senegal is very low, suggesting that HPV is not a strong risk factor for these cancers. Additional larger studies are needed to confirm these findings and explore other potential risk factors specific to the region. PMID:23594504

  12. 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.

  13. Epstein-Barr virus nuclear antigen 3A partially coincides with EBNA3C genome-wide and is tethered to DNA through BATF complexes.

    PubMed

    Schmidt, Stefanie C S; Jiang, Sizun; Zhou, Hufeng; Willox, Bradford; Holthaus, Amy M; Kharchenko, Peter V; Johannsen, Eric C; Kieff, Elliott; Zhao, Bo

    2015-01-13

    Epstein-Barr Virus (EBV) conversion of B-lymphocytes to Lymphoblastoid Cell Lines (LCLs) requires four EBV nuclear antigen (EBNA) oncoproteins: EBNA2, EBNALP, EBNA3A, and EBNA3C. EBNA2 and EBNALP associate with EBV and cell enhancers, up-regulate the EBNA promoter, MYC, and EBV Latent infection Membrane Proteins (LMPs), which up-regulate BCL2 to protect EBV-infected B-cells from MYC proliferation-induced cell death. LCL proliferation induces p16(INK4A) and p14(ARF)-mediated cell senescence. EBNA3A and EBNA3C jointly suppress p16(INK4A) and p14(ARF), enabling continuous cell proliferation. Analyses of the EBNA3A human genome-wide ChIP-seq landscape revealed 37% of 10,000 EBNA3A sites to be at strong enhancers; 28% to be at weak enhancers; 4.4% to be at active promoters; and 6.9% to be at weak and poised promoters. EBNA3A colocalized with BATF-IRF4, ETS-IRF4, RUNX3, and other B-cell Transcription Factors (TFs). EBNA3A sites clustered into seven unique groups, with differing B-cell TFs and epigenetic marks. EBNA3A coincidence with BATF-IRF4 or RUNX3 was associated with stronger EBNA3A ChIP-Seq signals. EBNA3A was at MYC, CDKN2A/B, CCND2, CXCL9/10, and BCL2, together with RUNX3, BATF, IRF4, and SPI1. ChIP-re-ChIP revealed complexes of EBNA3A on DNA with BATF. These data strongly support a model in which EBNA3A is tethered to DNA through a BATF-containing protein complexes to enable continuous cell proliferation.

  14. 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.

  15. 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.

  16. Clinicopathological significance of p15 promoter hypermethylation in multiple myeloma: a meta-analysis.

    PubMed

    Wei, Bing; Yang, Shuhua; Zhang, Bo; Feng, Yong

    2016-01-01

    Published studies reported that loss of function of the p15(INK4B) gene is caused by hypermethylation; however, whether or not the inactivation is associated with the incidence and clinical significance of multiple myeloma (MM) remains unclear. In this study, we performed a meta-analysis to quantitatively determine the effects of p15 hypermethylation on the incidence of MM. The related research articles in English and Chinese languages were evaluated. The data were extracted and assessed independently. The pooled data were analyzed and odds ratios were calculated and summarized. Sixteen eligible studies were selected for final analysis. We demonstrated that p15 hypermethylation is significantly higher in MM than that in normal bone marrow, as well as monoclonal gammopathy of undetermined significance. However, aberrant p15 hypermethylation was not significantly higher in advanced MM than that in early-stage MM. The results of this study reveal that p15 hypermethylation is correlated with an increased risk in the progression of monoclonal gammopathy of undetermined significance to MM. p15 hypermethylation, which induces the loss of function of the p15 gene, plays a critical role in the early tumorigenesis of MM and serves as a reputable diagnostic marker and potential drug target. PMID:27445492

  17. Clinicopathological significance of p15 promoter hypermethylation in multiple myeloma: a meta-analysis

    PubMed Central

    Wei, Bing; Yang, Shuhua; Zhang, Bo; Feng, Yong

    2016-01-01

    Published studies reported that loss of function of the p15INK4B gene is caused by hypermethylation; however, whether or not the inactivation is associated with the incidence and clinical significance of multiple myeloma (MM) remains unclear. In this study, we performed a meta-analysis to quantitatively determine the effects of p15 hypermethylation on the incidence of MM. The related research articles in English and Chinese languages were evaluated. The data were extracted and assessed independently. The pooled data were analyzed and odds ratios were calculated and summarized. Sixteen eligible studies were selected for final analysis. We demonstrated that p15 hypermethylation is significantly higher in MM than that in normal bone marrow, as well as monoclonal gammopathy of undetermined significance. However, aberrant p15 hypermethylation was not significantly higher in advanced MM than that in early-stage MM. The results of this study reveal that p15 hypermethylation is correlated with an increased risk in the progression of monoclonal gammopathy of undetermined significance to MM. p15 hypermethylation, which induces the loss of function of the p15 gene, plays a critical role in the early tumorigenesis of MM and serves as a reputable diagnostic marker and potential drug target. PMID:27445492

  18. Trisomy of the Dscr1 gene suppresses early progression of pancreatic intraepithelial neoplasia driven by oncogenic Kras.

    PubMed

    Lee, Jang Choon; Shin, Jimin; Baek, Kwan-Hyuck

    2013-10-11

    Individuals with Down syndrome exhibit remarkably reduced incidence of most solid tumors including pancreatic cancer. Multiple mechanisms arising from the genetic complexity underlying Down syndrome has been suggested to contribute to such a broad cancer protection. In this study, utilizing a genetically engineered mouse model of pancreatic cancer, we demonstrate that trisomy of the Down syndrome critical region-1 (Dscr1), an endogenous calcineurin inhibitor localized on chromosome 21, suppresses the progression of pancreatic intraepithelial neoplasia-1A (PanIN-1A) to PanIN-1B lesions without affecting the initiation of PanIN lesions mediated by oncogenic Kras(G12D). In addition, we show that Dscr1 trisomy attenuates nuclear localization of nuclear factor of activated T-cells (NFAT) accompanied by upregulation of the p15(Ink4b) tumor suppressor and reduction of cell proliferation in early PanIN lesions. Our data suggest that attenuation of calcineurin-NFAT signaling in neoplastic pancreatic ductal epithelium by a single extra copy of Dscr1 is sufficient to inhibit the progression of early PanIN lesions driven by oncogenic Kras, and thus may be a potential mechanism underlying reduced incidence of pancreatic cancer in Down syndrome individuals. PMID:24041692

  19. Polycomb repressive complex PRC2 regulates Xenopus retina development downstream of Wnt/β-catenin signaling

    PubMed Central

    Aldiri, Issam; Moore, Kathryn B.; Hutcheson, David A.; Zhang, Jianmin; Vetter, Monica L.

    2013-01-01

    The histone methyltransferase complex PRC2 controls key steps in developmental transitions and cell fate choices; however, its roles in vertebrate eye development remain unknown. Here, we report that in Xenopus, PRC2 regulates the progression of retinal progenitors from proliferation to differentiation. We show that the PRC2 core components are enriched in retinal progenitors and downregulated in differentiated cells. Knockdown of the PRC2 core component Ezh2 leads to reduced retinal progenitor proliferation, in part due to upregulation of the Cdk inhibitor p15Ink4b. In addition, although PRC2 knockdown does not alter eye patterning, retinal progenitor gene expression or expression of the neural competence factor Sox2, it does cause suppression of proneural bHLH gene expression, indicating that PRC2 is crucial for the initiation of neural differentiation in the retina. Consistent with this, knocking down or blocking PRC2 function constrains the generation of most retinal neural cell types and promotes a Müller glial cell fate decision. We also show that Wnt/β-catenin signaling acting through the receptor Frizzled 5, but independent of Sox2, regulates expression of key PRC2 subunits in the developing retina. This is consistent with a role for this pathway in coordinating proliferation and the transition to neurogenesis in the Xenopus retina. Our data establish PRC2 as a regulator of proliferation and differentiation during eye development. PMID:23739135

  20. p38α MAPK-mediated induction and interaction of FOXO3a and p53 contribute to the inhibited-growth and induced-apoptosis of human lung adenocarcinoma cells by berberine

    PubMed Central

    2014-01-01

    Background Berberine (BBR), a component from traditional Chinese medicine, has been shown to possess anti-tumor activity against a wide spectrum of cancer cells including human lung cancer, but the detailed mechanism underlining this has not been well elucidated. Methods In this study, the effect of berberine on cell growth and apoptosis were assessed by MTT, flow cytometry and Hoechst 33258 staining assays. The phosphorylation of p38 MAPK and ERK1/2, and expressions of p38 MAPK isoforms α and β, total ERK1/2, p53, FOXO3a and p21 protein were evaluated by Western Blot analysis. Silencing of p38 MAPK isoform α and β, p53, FOXO3a and p21 were performed by siRNA methods. Exogenous expression of FOXO3a was carried out by electroporated transfection assays. Results We showed that BBR significantly inhibited growth and induced cell cycle arrest of non small cell lung cancer (NSCLC) cells in the G0/G1 phase in a dose-dependent manner. Furthermore, we found that BBR increased phosphorylation of p38 MAPK and ERK1/2 in a time-dependent and induced protein expression of tumor suppressor p53 and transcription factor FOXO3a in a dose-dependent fashion. The specific inhibitor of p38 MAPK (SB203580), and silencing of p38α MAPK by small interfering RNAs (siRNAs), but not ERK1/2 inhibitor (PD98059) blocked the stimulatory effects of BBR on protein expression of p53 and FOXO3a. Interestingly, inhibition of p53 using one specific inhibitor (Pifithrin-α) and silencing of p53 using siRNAs overcome the inhibitory effect of BBR on cell growth. Silencing of FOXO3a appeared to attenuate the effect of BBR on p53 expression, cell proliferation and apoptosis. Furthermore, BBR induces the protein expression of cell cycle inhibitor p21 (CIP1/WAF1), which was not observed in cells silencing of p53 or FOXO3α gene. Intriguingly, exogenous expression of FOXO3a enhanced the expression of p21 (CIP1/WAF1) and strengthened BBR-induced apoptosis. Conclusion Our results show that BBR inhibits

  1. The beta-isoform of the BRCA2 and CDKN1A(p21)-interacting protein (BCCIP) stabilizes nuclear RPL23/uL14.

    PubMed

    Wyler, Emanuel; Wandrey, Franziska; Badertscher, Lukas; Montellese, Christian; Alper, Daniel; Kutay, Ulrike

    2014-10-16

    BRCA2 and CDKN1A(p21,CIP1)-interacting protein (BCCIP) is an evolutionary conserved protein implicated in maintenance of genome stability and cell cycle progression. Two isoforms of BCCIP with distinct C-terminal domains exist in humans. We show that mammalian BCCIPβ, but not BCCIPα, forms a ternary complex with the ribosomal protein RPL23/uL14 and the pre-60S trans-acting factor eIF6. Complex formation is dependent on an intact C-terminal domain of BCCIPβ. Depletion of BCCIPβ reduces the pool of free RPL23, and decreases eIF6 levels in nucleoli. Overexpression of BCCIPβ leads to nucleoplasmic accumulation of extra-ribosomal RPL23 and stabilizes overexpressed RPL23, suggesting that BCCIPβ functions as nuclear chaperone for RPL23.

  2. p53 as Batman: using a movie plot to understand control of the cell cycle.

    PubMed

    Gadi, Nikhita; Foley, Sage E; Nowey, Mark; Plopper, George E

    2013-04-16

    This Teaching Resource provides and describes a two-part classroom exercise to help students understand control of the cell cycle, with a focus on the transcription factor p53, the E3 ubiquitin ligase Mdm2, the Mdm2 inhibitor ARF, the kinases ATM and ATR, the kinase Chk2, and the cell cycle inhibitor p21(Cip1). Students use characters and scenes from the movie The Dark Knight to represent elements of the cell cycle control machinery, then they apply these characters and scenes to translate a primary research article on p53 function into a new movie scene in the "Batman universe." This exercise is appropriate for college-level courses in cell biology and cancer biology and requires students to have a background in introductory cell biology. Explicit learning outcomes and associated assessment methods are provided, as well as slides, student assignments, the primary research article, and an instructor's guide for the exercise.

  3. Nac1 interacts with the POZ-domain transcription factor, Miz1.

    PubMed

    Stead, Mark A; Wright, Stephanie C

    2014-06-05

    Nac1 (nucleus accumbens 1) is a POZ (poxvirus and zinc finger)-domain transcriptional repressor that is expressed at high levels in ovarian serous carcinoma. Here we identify Nac1 as a novel interacting partner of the POZ-domain transcriptional activator, Miz1 (Myc-interacting zinc-finger protein 1), and using chemical crosslinking we show that this association is mediated by a heterodimeric interaction of the Nac1 and Miz1 POZ domains. Nac1 is found in discrete bodies within the nucleus of mammalian cells, and we demonstrate the relocalization of Miz1 to these structures in transfected HeLa cells. We show that siRNA (small interfering RNA)-mediated knockdown of Nac1 in ovarian cancer cells results in increased levels of the Miz1 target gene product, p21Cip1. The interaction of Nac1 with Miz1 may thus be relevant to its mechanism of tumourigenesis in ovarian cancer.

  4. 6-Gingerol Inhibits Growth of Colon Cancer Cell LoVo via Induction of G2/M Arrest.

    PubMed

    Lin, Ching-Bin; Lin, Chun-Che; Tsay, Gregory J

    2012-01-01

    6-Gingerol, a natural component of ginger, has been widely reported to possess antiinflammatory and antitumorigenic activities. Despite its potential efficacy against cancer, the anti-tumor mechanisms of 6-gingerol are complicated and remain sketchy. In the present study, we aimed to investigate the anti-tumor effects of 6-gingerol on colon cancer cells. Our results revealed that 6-gingerol treatment significantly reduced the cell viability of human colon cancer cell, LoVo, in a dose-dependent manner. Further flow cytometric analysis showed that 6-gingerol induced significant G2/M phase arrest and had slight influence on sub-G1 phase in LoVo cells. Therefore, levels of cyclins, cyclin-dependent kinases (CDKs), and their regulatory proteins involved in S-G2/M transition were investigated. Our findings revealed that levels of cyclin A, cyclin B1, and CDK1 were diminished; in contrast, levels of the negative cell cycle regulators p27(Kip1) and p21(Cip1) were increased in response to 6-gingerol treatment. In addition, 6-gingerol treatment elevated intracellular reactive oxygen species (ROS) and phosphorylation level of p53. These findings indicate that exposure of 6-gingerol may induce intracellular ROS and upregulate p53, p27(Kip1), and p21(Cip1) levels leading to consequent decrease of CDK1, cyclin A, and cyclin B1 as result of cell cycle arrest in LoVo cells. It would be suggested that 6-gingerol should be beneficial to treatment of colon cancer.

  5. 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

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

    PubMed

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

    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

  7. Acute activation of AMP-activated protein kinase prevents H2O2-induced premature senescence in primary human keratinocytes.

    PubMed

    Ido, Yasuo; Duranton, Albert; Lan, Fan; Cacicedo, Jose M; Chen, Tai C; Breton, Lionel; Ruderman, Neil B

    2012-01-01

    We investigated the effects of AMPK on H(2)O(2)-induced premature senescence in primary human keratinocytes. Incubation with 50 µM H(2)O(2) for 2 h resulted in premature senescence with characteristic increases in senescence-associated ß-galactosidase (SA-gal) staining 3 days later and no changes in AMPK or p38 MAPK activity. The increase in SA-gal staining was preceded by increases in both p53 phosphorylation (S15) (1 h) and transactivation (6 h) and the abundance of the cyclin inhibitor p21(CIP1) (16 h). Incubation with AICAR or resveratrol, both of which activated AMPK, prevented the H(2)O(2)-induced increases in both SA-Gal staining and p21 abundance. In addition, AICAR diminished the increase in p53 transactivation. The decreases in SA-Gal expression induced by resveratrol and AICAR were prevented by the pharmacological AMPK inhibitor Compound C, expression of a DN-AMPK or AMPK knock-down with shRNA. Likewise, both knockdown of AMPK and expression of DN-AMPK were sufficient to induce senescence, even in the absence of exogenous H(2)O(2). As reported by others, we found that AMPK activation by itself increased p53 phosphorylation at S15 in embryonic fibroblasts (MEF), whereas under the same conditions it decreased p53 phosphorylation in the keratinocytes, human aortic endothelial cells, and human HT1080 fibrosarcoma cells. In conclusion, the results indicate that H(2)O(2) at low concentrations causes premature senescence in human keratinocytes by activating p53-p21(CIP1) signaling and that these effects can be prevented by acute AMPK activation and enhanced by AMPK downregulation. They also suggest that this action of AMPK may be cell or context-specific. PMID:22514710

  8. Dietary feeding of silibinin prevents early biomarkers of UVB radiation-induced carcinogenesis in SKH-1 hairless mouse epidermis.

    PubMed

    Gu, Mallikarjuna; Dhanalakshmi, Sivanandhan; Singh, Rana P; Agarwal, Rajesh

    2005-05-01

    Solar radiation is the causal etiologic factor in the development of nonmelanoma skin cancer (NMSC). Depletion of the stratospheric ozone layer leads to an increase in ambient UV radiation loads, which are expected to further raise skin cancer incidence in many temperate parts of the world, including the United States, suggesting that skin cancer chemopreventive approaches via biomarker efficacy studies or vice versa are highly warranted. Based on our recent study reporting strong efficacy of silibinin against photocarcinogenesis, we assessed here the protective effects of its dietary feeding on UVB-induced biomarkers involved in NMSC providing a mechanistic rationale for an early-on silibinin efficacy in skin cancer prevention. Dietary feeding of silibinin at 1% dose (w/w) to SKH-1 hairless mice for 2 weeks before a single UVB irradiation at 180 mJ/cm(2) dose resulted in a strong and significant (P < 0.001) decrease in UVB-induced thymine dimer-positive cells and proliferating cell nuclear antigen, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and apoptotic sunburn cells together with an increase (P < 0.001) in p53 and p21/cip1-positive cell population in epidermis. These findings suggest that dietary feeding of silibinin affords strong protection against UVB-induced damages in skin epidermis by (a) either preventing DNA damage or enhancing repair, (b) reducing UVB-induced hyperproliferative response, and (c) inhibiting UVB-caused apoptosis and sunburn cell formation, possibly via silibinin-caused up-regulation of p53 and p21/cip1 as major UVB-damage control sensors.

  9. 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

  10. Prevention of carcinogenesis and inhibition of breast cancer tumor burden by dietary stearate

    PubMed Central

    Li, Chuanyu; Zhao, Xiangmin; Toline, Eric C.; Siegal, Gene P.; Evans, Lynda M.; Ibrahim-Hashim, Arig; Desmond, Renee A.; Hardy, Robert W.

    2011-01-01

    Previous studies have shown that stearate (C18:0), a dietary long-chain saturated fatty acid, inhibits breast cancer cell neoplastic progression; however, little is known about the mechanism modulating these processes. We demonstrate that stearate, at physiological concentrations, inhibits cell cycle progression in human breast cancer cells at both the G1 and G2 phases. Stearate also increases cell cycle inhibitor p21CIP1/WAF1 and p27KIP1 levels and concomitantly decreases cyclin-dependent kinase 2 (Cdk2) phosphorylation. Our data also show that stearate induces Ras– guanosine triphosphate formation and causes increased phosphorylation of extracellular signal-regulated kinase (pERK). The MEK1 inhibitor, PD98059, reversed stearate-induced p21CIP1/WAF1 upregulation, but only partially restored stearate-induced dephosphorylation of Cdk2. The Ras/mitogen-activated protein kinase/ERK pathway has been linked to cell cycle regulation but generally in a positive way. Interestingly, we found that stearate inhibits both Rho activation and expression in vitro. In addition, constitutively active RhoC reversed stearate-induced upregulation of p27KIP1, providing further evidence of Rho involvement. To test the effect of stearate in vivo, we used the N-Nitroso-N-methylurea rat breast cancer carcinogen model. We found that dietary stearate reduces the incidence of carcinogen-induced mammary cancer and reduces tumor burden. Importantly, mammary tumor cells from rats on a stearate diet had reduced expression of RhoA and B as well as total Rho compared with a low-fat diet. Overall, these data indicate that stearate inhibits breast cancer cell proliferation by inhibiting key check points in the cell cycle as well as Rho expression in vitro and in vivo and inhibits tumor burden and carcinogen-induced mammary cancer in vivo. PMID:21586513

  11. Mortalin (GRP75/HSPA9) upregulation promotes survival and proliferation of medullary thyroid carcinoma cells

    PubMed Central

    Starenki, Dmytro; Hong, Seung-Keun; Lloyd, Ricardo V.; Park, Jong-In

    2014-01-01

    Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the RET proto-oncogene. For therapy of advanced MTC, the Food and Drug Administration recently approved vandetanib and cabozantinib, the tyrosine kinase inhibitors targeting RET, vascular endothelial growth factor receptor, epidermal growth factor receptor, and/or c-MET. Nevertheless, not all patients respond to these drugs, demanding additional therapeutic strategies. We found that mortalin (HSPA9/GRP75), a member of HSP70 family, is upregulated in human MTC tissues and that its depletion robustly induces cell death and growth arrest in MTC cell lines in culture and in mouse xenografts. These effects were accompanied by substantial downregulation of RET, induction of the tumor suppressor TP53, and altered expression of cell cycle regulatory machinery and apoptosis markers including E2F-1, p21CIP1, p27KIP1, and Bcl-2 family proteins. Our investigation of the molecular mechanisms underlying these effects revealed that mortalin depletion induces transient MEK/ERK activation and altered mitochondrial bioenergetics in MTC cells, as indicated by depolarized mitochondrial membrane, decreased oxygen consumption and extracellular acidification, and increased oxidative stress. Intriguingly, mortalin depletion induced growth arrest partly via the MEK/ERK pathway whereas it induced cell death by causing mitochondrial dysfunction in a Bcl-2 dependent manner. However, TP53 was not necessary for these effects except for p21CIP1 induction. Moreover, mortalin depletion downregulated RET expression independently of MEK/ERK and TP53. These data demonstrate that mortalin is a key regulator of multiple signaling and metabolic pathways pivotal to MTC cell survival and proliferation, proposing mortalin as a novel therapeutic target for MTC. PMID:25435367

  12. 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.

  13. EVI1, a target gene for amplification at 3q26, antagonizes transforming growth factor-β-mediated growth inhibition in hepatocellular carcinoma.

    PubMed

    Yasui, Kohichiroh; Konishi, Chika; Gen, Yasuyuki; Endo, Mio; Dohi, Osamu; Tomie, Akira; Kitaichi, Tomoko; Yamada, Nobuhisa; Iwai, Naoto; Nishikawa, Taichiro; Yamaguchi, Kanji; Moriguchi, Michihisa; Sumida, Yoshio; Mitsuyoshi, Hironori; Tanaka, Shinji; Arii, Shigeki; Itoh, Yoshito

    2015-07-01

    EVI1 (ecotropic viral integration site 1) is one of the most aggressive oncogenes associated with myeloid leukemia. We investigated DNA copy number aberrations in human hepatocellular carcinoma (HCC) cell lines using a high-density oligonucleotide microarray. We found that a novel amplification at the chromosomal region 3q26 occurs in the HCC cell line JHH-1, and that MECOM (MDS1 and EVI1 complex locus), which lies within the 3q26 region, was amplified. Quantitative PCR analysis of the three transcripts transcribed from MECOM indicated that only EVI1, but not the fusion transcript MDS1-EVI1 or MDS1, was overexpressed in JHH-1 cells and was significantly upregulated in 22 (61%) of 36 primary HCC tumors when compared with their non-tumorous counterparts. A copy number gain of EVI1 was observed in 24 (36%) of 66 primary HCC tumors. High EVI1 expression was significantly associated with larger tumor size and higher level of des-γ-carboxy prothrombin, a tumor marker for HCC. Knockdown of EVI1 resulted in increased induction of the cyclin-dependent kinase inhibitor p15(INK) (4B) by transforming growth factor (TGF)-β and decreased expression of c-Myc, cyclin D1, and phosphorylated Rb in TGF-β-treated cells. Consequently, knockdown of EVI1 led to reduced DNA synthesis and cell viability. Collectively, our results suggest that EVI1 is a probable target gene that acts as a driving force for the amplification at 3q26 in HCC and that the oncoprotein EVI1 antagonizes TGF-β-mediated growth inhibition of HCC cells.

  14. Opposing Growth Regulatory Roles of Protein Kinase D Isoforms in Human Keratinocytes*

    PubMed Central

    Ryvkin, Vladislav; Rashel, Mohammad; Gaddapara, Trivikram; Ghazizadeh, Soosan

    2015-01-01

    PKD is a family of three serine/threonine kinases (PKD-1, -2, and -3) involved in the regulation of diverse biological processes including proliferation, migration, secretion, and cell survival. We have previously shown that despite expression of all three isoforms in mouse epidermis, PKD1 plays a unique and critical role in wound healing, phorbol ester-induced hyperplasia, and tumor development. In translating our findings to the human, we discovered that PKD1 is not expressed in human keratinocytes (KCs) and there is a divergence in the expression and function of other PKD isoforms. Contrary to mouse KCs, treatment of cultured human KCs with pharmacological inhibitors of PKDs resulted in growth arrest. We found that PKD2 and PKD3 are expressed differentially in proliferating and differentiating human KCs, with the former uniformly present in both compartments whereas the latter is predominantly expressed in the proliferating compartment. Knockdown of individual PKD isoforms in human KCs revealed contrasting growth regulatory roles for PKD2 and PKD3. Loss of PKD2 enhanced KC proliferative potential while loss of PKD3 resulted in a progressive proliferation defect, loss of clonogenicity and diminished tissue regenerative ability. This proliferation defect was correlated with up-regulation of CDK4/6 inhibitor p15INK4B and induction of a p53-independent G1 cell cycle arrest. Simultaneous silencing of PKD isoforms resulted in a more pronounced proliferation defect consistent with a predominant role for PKD3 in proliferating KCs. These data underline the importance and complexity of PKD signaling in human epidermis and suggest a central role for PKD3 signaling in maintaining human epidermal homeostasis. PMID:25802335

  15. 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

  16. Anti-breast cancer activity of Fine Black ginseng (Panax ginseng Meyer) and ginsenoside Rg5

    PubMed Central

    Kim, Shin-Jung; Kim, An Keun

    2014-01-01

    Background Black ginseng (Ginseng Radix nigra, BG) refers to the ginseng steamed for nine times and fine roots (hairy roots) of that is called fine black ginseng (FBG). It is known that the content of saponin of FBG is higher than that of BG. Therefore, in this study, we examined antitumor effects against MCF-7 breast cancer cells to target the FBG extract and its main component, ginsenoside Rg5 (Rg5). Methods Action mechanism was determined by MTT assay, cell cycle assay and western blot analysis. Results The results from MTT assay showed that MCF-7 cell proliferation was inhibited by Rg5 treatment for 24, 48 and 72 h in a dose-dependent manner. Rg5 at different concentrations (0, 25, 50 and 100 μM), induced cell cycle arrest in G0/G1 phase through regulation of cell cycle-related proteins in MCF-7 cells. As shown in the results from western blot analysis, Rg5 increased expression of p53, p21WAF1/CIP1 and p15INK4B and decreased expression of Cyclin D1, Cyclin E2 and CDK4. Expression of apoptosis–related proteins including Bax, PARP and Cytochrome c was also regulated by Rg5. These results indicate that Rg5 stimulated cell apoptosis and cell cycle arrest at G0/G1 phase via regulation of cell cycle-associated proteins in MCF-7 cells. Conclusion Rg5 promotes breast cancer cell apoptosis in a multi-path manner with higher potency compared to 20(S)-ginsenoside Rg3 (Rg3) in MCF-7 (HER2−/ER+) and MDA-MB-453 (HER2+/ER−) human breast cancer cell lines, and this suggests that Rg5 might be an effective natural new material in improving breast cancer. PMID:26045685

  17. Aberrant cell cycle progression contributes to the early-stage accelerated carcinogenesis in transgenic epidermis expressing the dominant negative TGFbetaRII.

    PubMed

    Go, C; He, W; Zhong, L; Li, P; Huang, J; Brinkley, B R; Wang, X J

    2000-07-27

    Mutations in the transforming growth factor beta type II receptor (TGFbetaRII) have been found in various malignant tumors, suggesting that loss of TGFbeta signaling plays a causal role in late-stage cancer development. To test whether loss of TGFbetaRII is involved in early-stage carcinogenesis, we have generated transgenic mice expressing a dominant negative TGFbetaRII (deltabetaRII) in the epidermis. These mice exhibited an increased susceptibility to chemical carcinogenesis protocols at both early and late stages. In the current study, parameters for cell cycle progression and chromosome instability were analysed in deltabetaRII tumors. DeltabetaRII papillomas showed an increased S phase in flow cytometry. Bromodeoxyuridine (BrdU) labeling and mitotic indices in deltabetaRII papillomas also showed a threefold increase compared to papillomas developing in non-transgenic mice. When papillomas further progressed to squamous cell carcinomas (SCC), both control and deltabetaRII SCC showed similar BrdU labeling indices and percentages of S phase cells. However, deltabetaRII SCC cells showed a sixfold increase in the G2/M population. Mitotic indices in deltabetaRII SCC also showed a threefold increase compared to non-transgenic SCC. Consistent with a perturbed cell cycle, deltabetaRII papillomas and SCC showed reduced expression of the TGFbeta target genes p15 (INK4b), p21 (WAF-1) and p27 (Kip1), inhibitors of cyclin-dependent kinases (cdks). However, most deltabetaRII papilloma cells exhibited normal centrosome numbers, and deltabetaRII SCC exhibited a similar extent of centrosome abnormalities compared to control SCC (35-40% cells). Most of deltabetaRII SCC exhibited diploid chromosome profiles. These data indicate that inactivation of TGFbetaRII accelerates skin tumorigenesis at early stages by the acceleration of loss of cell cycle control, but not by increased chromosome instability.

  18. 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

  19. 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

  20. 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

    2015-12-01

    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

  1. Epstein-Barr virus nuclear antigen 3C binds to BATF/IRF4 or SPI1/IRF4 composite sites and recruits Sin3A to repress CDKN2A.

    PubMed

    Jiang, Sizun; Willox, Bradford; Zhou, Hufeng; Holthaus, Amy M; Wang, Anqi; Shi, Tommy T; Maruo, Seiji; Kharchenko, Peter V; Johannsen, Eric C; Kieff, Elliott; Zhao, Bo

    2014-01-01

    Epstein-Barr virus nuclear antigen 3C (EBNA3C) repression of CDKN2A p14(ARF) and p16(INK4A) is essential for immortal human B-lymphoblastoid cell line (LCL) growth. EBNA3C ChIP-sequencing identified >13,000 EBNA3C sites in LCL DNA. Most EBNA3C sites were associated with active transcription; 64% were strong H3K4me1- and H3K27ac-marked enhancers and 16% were active promoters marked by H3K4me3 and H3K9ac. Using ENCODE LCL transcription factor ChIP-sequencing data, EBNA3C sites coincided (±250 bp) with RUNX3 (64%), BATF (55%), ATF2 (51%), IRF4 (41%), MEF2A (35%), PAX5 (34%), SPI1 (29%), BCL11a (28%), SP1 (26%), TCF12 (23%), NF-κB (23%), POU2F2 (23%), and RBPJ (16%). EBNA3C sites separated into five distinct clusters: (i) Sin3A, (ii) EBNA2/RBPJ, (iii) SPI1, and (iv) strong or (v) weak BATF/IRF4. EBNA3C signals were positively affected by RUNX3, BATF/IRF4 (AICE) and SPI1/IRF4 (EICE) cooccupancy. Gene set enrichment analyses correlated EBNA3C/Sin3A promoter sites with transcription down-regulation (P < 1.6 × 10(-4)). EBNA3C signals were strongest at BATF/IRF4 and SPI1/IRF4 composite sites. EBNA3C bound strongly to the p14(ARF) promoter through SPI1/IRF4/BATF/RUNX3, establishing RBPJ-, Sin3A-, and REST-mediated repression. EBNA3C immune precipitated with Sin3A and conditional EBNA3C inactivation significantly decreased Sin3A binding at the p14(ARF) promoter (P < 0.05). These data support a model in which EBNA3C binds strongly to BATF/IRF4/SPI1/RUNX3 sites to enhance transcription and recruits RBPJ/Sin3A- and REST/NRSF-repressive complexes to repress p14(ARF) and p16(INK4A) expression. PMID:24344258

  2. Hypermethylation of gene promoters in peripheral blood leukocytes in humans long term after radiation exposure.

    PubMed

    Kuzmina, Nina S; Lapteva, Nellya Sh; Rubanovich, Alexander V

    2016-04-01

    Some human genes known to undergo age-related promoter hypermethylation. These epigenetic modifications are similar to those occurring in the course of certain diseases, e.g. some types of cancer, which in turn may also associate with age. Given external genotoxic factors may additionally contribute to hypermethylation, this study was designed to analyzes, using methylation-sensitive polymerase chain reaction (PCR), the CpG island hypermethylation in RASSF1A, CDKN2A (including p16/INK4A and p14/ARF) and GSTP1 promoters in peripheral blood leukocytes of individuals exposed to ionizing radiation long time ago. One hundred and twenty-four irradiated subjects (24-77 years old at sampling: 83 Chernobyl Nuclear Power Plant clean-up workers, 21 nuclear workers, 20 residents of territories with radioactive contamination) and 208 unirradiated volunteers (19-77 years old at sampling) were enrolled. In addition, 74 non-exposed offspring (2-51 years old at sampling) born to irradiated parents were examined. The frequency of individuals displaying promoter methylation of at least one gene in exposed group was significantly higher as compared to the control group (OR=5.44, 95% CI=2.62-11.76, p=3.9×10(-7)). No significant difference was found between the frequency of subjects with the revealed promoter methylation in the group of offspring born to irradiated parents and in the control group. The increase in the number of methylated loci of RASSF1A and p14/ARF was associated with age (β=0.242; p=1.7×10(-5)). In contrast, hypermethylation of p16/INK4A and GSTP1 genes correlated with the fact of radiation exposure only (β=0.290; p=1.7×10(-7)). The latter finding demonstrates that methylation changes in blood leukocytes of healthy subjects exposed to radiation resemble those reported in human malignancies. Additional studies are required to identify the dose-response of epigenetic markers specifically associating with radiation-induced premature aging and/or with the development

  3. 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

  4. Hypermethylation of gene promoters in peripheral blood leukocytes in humans long term after radiation exposure.

    PubMed

    Kuzmina, Nina S; Lapteva, Nellya Sh; Rubanovich, Alexander V

    2016-04-01

    Some human genes known to undergo age-related promoter hypermethylation. These epigenetic modifications are similar to those occurring in the course of certain diseases, e.g. some types of cancer, which in turn may also associate with age. Given external genotoxic factors may additionally contribute to hypermethylation, this study was designed to analyzes, using methylation-sensitive polymerase chain reaction (PCR), the CpG island hypermethylation in RASSF1A, CDKN2A (including p16/INK4A and p14/ARF) and GSTP1 promoters in peripheral blood leukocytes of individuals exposed to ionizing radiation long time ago. One hundred and twenty-four irradiated subjects (24-77 years old at sampling: 83 Chernobyl Nuclear Power Plant clean-up workers, 21 nuclear workers, 20 residents of territories with radioactive contamination) and 208 unirradiated volunteers (19-77 years old at sampling) were enrolled. In addition, 74 non-exposed offspring (2-51 years old at sampling) born to irradiated parents were examined. The frequency of individuals displaying promoter methylation of at least one gene in exposed group was significantly higher as compared to the control group (OR=5.44, 95% CI=2.62-11.76, p=3.9×10(-7)). No significant difference was found between the frequency of subjects with the revealed promoter methylation in the group of offspring born to irradiated parents and in the control group. The increase in the number of methylated loci of RASSF1A and p14/ARF was associated with age (β=0.242; p=1.7×10(-5)). In contrast, hypermethylation of p16/INK4A and GSTP1 genes correlated with the fact of radiation exposure only (β=0.290; p=1.7×10(-7)). The latter finding demonstrates that methylation changes in blood leukocytes of healthy subjects exposed to radiation resemble those reported in human malignancies. Additional studies are required to identify the dose-response of epigenetic markers specifically associating with radiation-induced premature aging and/or with the development

  5. 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

  6. Pathfinder cells provide a novel therapeutic intervention for acute kidney injury.

    PubMed

    McGlynn, Liane M; Eller, Kathrin; MacDonald, Alasdair I; Macintyre, Alan; Russell, David; Koppelstaetter, Christian; Davies, R W; Shiels, Paul G

    2013-02-01

    Pathfinder cells (PCs) are a novel class of adult-derived cells that facilitate functional repair of host tissue. We used rat PCs to demonstrate that they enable the functional mitigation of ischemia reperfusion (I/R) injury in a mouse model of renal damage. Female C57BL/6 mice were subjected to 30 min of renal ischemia and treated with intravenous (i.v.) injection of saline (control) or male rat pancreas-derived PCs in blinded experimentation. Kidney function was assessed 14 days after treatment by measuring serum creatinine (SC) levels. Kidney tissue was assessed by immunohistochemistry (IHC) for markers of cellular damage, proliferation, and senescence (TUNEL, Ki67, p16(ink4a), p21). Fluorescence in situ hybridization (FISH) was performed to determine the presence of any rat (i.e., pathfinder) cells in the mouse tissue. PC-treated animals demonstrated superior renal function at day 14 post-I/R, in comparison to saline-treated controls, as measured by SC levels (0.13 mg/dL vs. 0.23 mg/dL, p<0.001). PC-treated kidney tissue expressed significantly lower levels of p16(ink4a) in comparison to the control group (p=0.009). FISH analysis demonstrated that the overwhelming majority of repaired kidney tissue was mouse in origin. Rat PCs were only detected at a frequency of 0.02%. These data confirm that PCs have the ability to mitigate functional damage to kidney tissue following I/R injury. Kidneys of PC-treated animals showed evidence of improved function and reduced expression of damage markers. The PCs appear to act in a paracrine fashion, stimulating the host tissue to recover functionally, rather than by differentiating into renal cells. This study demonstrates that pancreatic-derived PCs from the adult rat can enable functional repair of renal damage in mice. It validates the use of PCs to regenerate damaged tissues and also offers a novel therapeutic intervention for repair of solid organ damage in situ.

  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

    2016-01-01

    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. 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.

  9. 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

  10. No role for human papillomavirus in esophageal squamous cell carcinoma in China.

    PubMed

    Koshiol, Jill; Wei, Wen-Qiang; Kreimer, Aimee R; Chen, Wen; Gravitt, Patti; Ren, Jian-Song; Abnet, Christian C; Wang, Jian-Bing; Kamangar, Farin; Lin, Dong-Mei; von Knebel-Doeberitz, Magnus; Zhang, Yu; Viscidi, Raphael; Wang, Guo-Qing; Gillison, Maura L; Roth, Mark J; Dong, Zhi-Wei; Kim, Esther; Taylor, Philip R; Qiao, You-Lin; Dawsey, Sanford M

    2010-07-01

    Certain regions of China have high rates of esophageal squamous cell carcinoma (ESCC). Previous studies of human papillomavirus (HPV), a proposed causal factor, have produced highly variable results. We attempted to evaluate HPV and ESCC more definitively using extreme care to prevent DNA contamination. We collected tissue and serum in China from 272 histopathologically-confirmed ESCC cases with rigorous attention to good molecular biology technique. We tested for HPV DNA in fresh-frozen tumor tissue using PCR with PGMY L1 consensus primers and HPV16 and 18 type-specific E6 and E7 primers, and in formalin-fixed paraffin-embedded tumor tissue using SPF(10) L1 primers. In HPV-positive cases, we evaluated p16(INK4a) overexpression and HPV E6/E7 seropositivity as evidence of carcinogenic HPV activity. beta-globin, and thus DNA, was adequate in 98.2% of the frozen tumor tissues (267/272). Of these, 99.6% (95% confidence interval (CI) = 97.9-100.0%) were negative for HPV DNA by PGMY, and 100% (95% CI = 98.6-100%) were negative by HPV16/18 E6/E7 PCR. In the corresponding formalin-fixed paraffin-embedded tumor specimens, 99.3% (95% CI = 97.3-99.9%) were HPV negative by SPF(10). By PGMY, 1 case tested weakly positive for HPV89, a noncancer causing HPV type. By SPF(10), 2 cases tested weakly positive: 1 for HPV16 and 1 for HPV31. No HPV DNA-positive case had evidence of HPV oncogene activity as measured by p16(INK4a) overexpression or E6/E7 seropositivity. This study provides the most definitive evidence to date that HPV is not involved in ESCC carcinogenesis in China. HPV DNA contamination cannot be ruled out as an explanation for high HPV prevalence in ESCC tissue studies with less stringent tissue procurement and processing protocols. PMID:19918949

  11. Epigenetic Alterations in Ultraviolet Radiation-Induced Skin Carcinogenesis: Interaction of Bioactive Dietary Components on Epigenetic Targets†

    PubMed Central

    Katiyar, Santosh K.; Singh, Tripti; Prasad, Ram; Sun, Qian; Vaid, Mudit

    2011-01-01

    The importance of epigenetic alterations in the development of various diseases including the cancers has been realized. As epigenetic changes are reversible heritable changes, these can be utilized as an effective strategy for the prevention of cancers. DNA methylation is the most characterized epigenetic mechanism that can be inherited without changing the DNA sequence. Although limited, but available data suggest that silencing of tumor suppressor genes in ultraviolet (UV) radiation-exposed epidermis leads to photocarcinogenesis and is associated with a network of epigenetic modifications including alterations in DNA methylation, DNA methyltransferases and histone acetylations. Various bioactive dietary components have been shown to protect skin from UV radiation-induced skin tumors in animal models. The role of bioactive dietary components, such as, (−)-epicatechins from green tea and proanthocyanidins from grape seeds, has been assessed in chemoprevention of UV-induced skin carcinogenesis and underlying epigenetic mechanism in vitro and in vivo animal models. These bioactive components have the ability to block UV-induced DNA hypermethylation and histone modifications in the skin required for the silencing of tumor suppressor genes (e.g., Cip1/p21, p16INK4a). These information are of importance for understanding the role of epigenetic modulation in UV-induced skin tumor and the chemopreventive mechanism of bioactive dietary components. PMID:22017262

  12. Genomic and molecular aberrations in malignant peripheral nerve sheath tumor and their roles in personalized target therapy.

    PubMed

    Yang, Jilong; Du, Xiaoling

    2013-09-01

    Malignant peripheral nerve sheath tumors (MPNSTs) are malignant tumors with a high rate of local recurrence and a significant tendency to metastasize. Its dismal outcome points to the urgent need to establish better therapeutic strategies for patients harboring MPNSTs. The investigations of genomic and molecular aberrations in MPNSTs which detect many chromosomal aberrations, pathway abnormalities, and specific molecular aberrant events would supply multiple potential therapy targets and contribute to achievement of personalized medicine. The involved genes in the significant gains aberrations include BIRC5, CCNE2, DAB2, DDX15, EGFR, DAB2, MSH2, CDK6, HGF, ITGB4, KCNK12, LAMA3, LOXL2, MET, and PDGFRA. The involved genes in the significant deletion aberrations include CDH1, GLTSCR2, EGR1, CTSB, GATA3, SULT2A1, GLTSCR2, HMMR/RHAMM, LICAM2, MMP13, p16/INK4a, RASSF2, NM-23H1, and TP53. These genetic aberrations involve in several important signaling pathways such as TFF, EGFR, ARF, IGF1R signaling pathways. The genomic and molecular aberrations of EGFR, IGF1R, SOX9, EYA4, TOP2A, ETV4, and BIRC5 exhibit great promise as personalized therapeutic targets for MPNST patients. PMID:23830351

  13. Chromosome 7p11.2 (EGFR) variation influences glioma risk

    PubMed Central

    Sanson, Marc; Hosking, Fay J.; Shete, Sanjay; Zelenika, Diana; Dobbins, Sara E.; Ma, Yussanne; Enciso-Mora, Victor; Idbaih, Ahmed; Delattre, Jean-Yves; Hoang-Xuan, Khe; Marie, Yannick; Boisselier, Blandine; Carpentier, Catherine; Wang, Xiao-Wei; Di Stefano, Anna Luisa; Labussière, Marianne; Gousias, Konstantinos; Schramm, Johannes; Boland, Anne; Lechner, Doris; Gut, Ivo; Armstrong, Georgina; Liu, Yanhong; Yu, Robert; Lau, Ching; Di Bernardo, Maria Chiara; Robertson, Lindsay B.; Muir, Kenneth; Hepworth, Sarah; Swerdlow, Anthony; Schoemaker, Minouk J; Wichmann, H.-Erich; Müller, Martina; Schreiber, Stefan; Franke, Andre; Moebus, Susanne; Eisele, Lewin; Försti, Asta; Hemminki, Kari; Lathrop, Mark; Bondy, Melissa; Houlston, Richard S.; Simon, Matthias

    2011-01-01

    While gliomas are the most common primary brain tumors, their etiology is largely unknown. To identify novel risk loci for glioma, we conducted genome-wide association (GWA) analysis of two case–control series from France and Germany (2269 cases and 2500 controls). Pooling these data with previously reported UK and US GWA studies provided data on 4147 glioma cases and 7435 controls genotyped for 424 460 common tagging single-nucleotide polymorphisms. Using these data, we demonstrate two statistically independent associations between glioma and rs11979158 and rs2252586, at 7p11.2 which encompasses the EGFR gene (population-corrected statistics, Pc = 7.72 × 10−8 and 2.09 × 10−8, respectively). Both associations were independent of tumor subtype, and were independent of EGFR amplification, p16INK4a deletion and IDH1 mutation status in tumors; compatible with driver effects of the variants on glioma development. These findings show that variation in 7p11.2 is a determinant of inherited glioma risk. PMID:21531791

  14. 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

  15. p19ARF is a critical mediator of both cellular senescence and an innate immune response associated with MYC inactivation in mouse model of acute leukemia

    PubMed Central

    Yetil, Alper; Anchang, Benedict; Gouw, Arvin M.; Adam, Stacey J.; Zabuawala, Tahera; Parameswaran, Ramya; van Riggelen, Jan; Plevritis, Sylvia; Felsher, Dean W.

    2015-01-01

    MYC-induced T-ALL exhibit oncogene addiction. Addiction to MYC is a consequence of both cell-autonomous mechanisms, such as proliferative arrest, cellular senescence, and apoptosis, as well as non-cell autonomous mechanisms, such as shutdown of angiogenesis, and recruitment of immune effectors. Here, we show, using transgenic mouse models of MYC-induced T-ALL, that the loss of either p19ARF or p53 abrogates the ability of MYC inactivation to induce sustained tumor regression. Loss of p53 or p19ARF, influenced the ability of MYC inactivation to elicit the shutdown of angiogenesis; however the loss of p19ARF, but not p53, impeded cellular senescence, as measured by SA-beta-galactosidase staining, increased expression of p16INK4A, and specific histone modifications. Moreover, comparative gene expression analysis suggested that a multitude of genes involved in the innate immune response were expressed in p19ARF wild-type, but not null, tumors upon MYC inactivation. Indeed, the loss of p19ARF, but not p53, impeded the in situ recruitment of macrophages to the tumor microenvironment. Finally, p19ARF null-associated gene signature prognosticated relapse-free survival in human patients with ALL. Therefore, p19ARF appears to be important to regulating cellular senescence and innate immune response that may contribute to the therapeutic response of ALL. PMID:25784651

  16. A rapid and scalable system for studying gene function in mice using conditional RNA interference

    PubMed Central

    Premsrirut, Prem K.; Dow, Lukas E.; Kim, Sang Yong; Camiolo, Matthew; Malone, Colin D.; Miething, Cornelius; Scuoppo, Claudio; Zuber, Johannes; Dickins, Ross A.; Kogan, Scott C.; Shroyer, Kenneth R.; Sordella, Raffaella; Hannon, Gregory J.; Lowe, Scott W.

    2011-01-01

    Summary RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16INK4a, p19ARF and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19ARF as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PMID:21458673

  17. 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. PMID:25746661

  18. An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice

    PubMed Central

    Sindrilaru, Anca; Peters, Thorsten; Wieschalka, Stefan; Baican, Corina; Baican, Adrian; Peter, Henriette; Hainzl, Adelheid; Schatz, Susanne; Qi, Yu; Schlecht, Andrea; Weiss, Johannes M.; Wlaschek, Meinhard; Sunderkötter, Cord; Scharffetter-Kochanek, Karin

    2011-01-01

    Uncontrolled macrophage activation is now considered to be a critical event in the pathogenesis of chronic inflammatory diseases such as atherosclerosis, multiple sclerosis, and chronic venous leg ulcers. However, it is still unclear which environmental cues induce persistent activation of macrophages in vivo and how macrophage-derived effector molecules maintain chronic inflammation and affect resident fibroblasts essential for tissue homeostasis and repair. We used a complementary approach studying human subjects with chronic venous leg ulcers, a model disease for macrophage-driven chronic inflammation, while establishing a mouse model closely reflecting its pathogenesis. Here, we have shown that iron overloading of macrophages — as was found to occur in human chronic venous leg ulcers and the mouse model — induced a macrophage population in situ with an unrestrained proinflammatory M1 activation state. Via enhanced TNF-α and hydroxyl radical release, this macrophage population perpetuated inflammation and induced a p16INK4a-dependent senescence program in resident fibroblasts, eventually leading to impaired wound healing. This study provides insight into the role of what we believe to be a previously undescribed iron-induced macrophage population in vivo. Targeting this population may hold promise for the development of novel therapies for chronic inflammatory diseases such as chronic venous leg ulcers. PMID:21317534

  19. Increased expression of senescence markers in cystic fibrosis airways.

    PubMed

    Fischer, Bernard M; Wong, Jessica K; Degan, Simone; Kummarapurugu, Apparao B; Zheng, Shuo; Haridass, Prashamsha; Voynow, Judith A

    2013-03-15

    Cystic Fibrosis (CF) is a chronic lung disease characterized by chronic neutrophilic airway inflammation and increased levels of neutrophil elastase (NE) in the airways. We have previously reported that NE treatment triggers cell cycle arrest. Cell cycle arrest can lead to senescence, a complete loss of replicative capacity. Importantly, senescent cells can be proinflammatory and would perpetuate CF chronic inflammation. By immunohistochemistry, we evaluated whether airway sections from CF and control subjects expressed markers of senescence, including p16(INK4a) (p16), a cyclin-dependent kinase inhibitor, phospho-Histone H2A.X (γH2A.X), and phospho-checkpoint 2 kinase (phospho-Chk2), which are also DNA damage response markers. Compared with airway epithelium from control subjects, CF airway epithelium had increased levels of expression of all three senescence markers. We hypothesized that the high load of NE in the CF airway triggers epithelial senescence by upregulating expression of p16, which inhibits cyclin-dependent kinase 4 (CDK4). Normal human bronchial epithelial (NHBE) cells, cultured in air-liquid interface were treated with NE (0, 200, and 500 nM) to induce visible injury. Total cell lysates were collected and evaluated by Western analysis for p16 protein expression and CDK4 kinase activity. NE significantly increased p16 expression and decreased CDK4 kinase activity in NHBE cells. These results support the concept that NE triggers expression of senescence markers in CF airway epithelial cells. PMID:23316069

  20. Increased RIPK4 expression is associated with progression and poor prognosis in cervical squamous cell carcinoma patients.

    PubMed

    Liu, De-Qing; Li, Fang-Fang; Zhang, Jiang-Bo; Zhou, Tie-Jun; Xue, Wen-Qiong; Zheng, Xiao-Hui; Chen, Yuan-Bin; Liao, Xiao-Yu; Zhang, Lan; Zhang, Shao-Dan; Hu, Ye-Zhu; Jia, Wei-Hua

    2015-07-07

    Aberrant expression of receptor interacting protein kinase 4 (RIPK4), a crucial regulatory protein of Wnt/β-catenin signaling, has recently been reported to be involved in several cancers. Here, we report the potential clinical implication and biological functions of RIPK4 in cervical squamous cell carcinoma (CSCC). One hundred and ninety-eight CSCC cases, 109 low-grade squamous intraepithelial lesions (LSILs), 141 high-grade squamous intraepithelial lesions (HSILs) and 63 chronic cervicitis were collected. The expression of RIPK4 was detected by immunohistochemistry (IHC), and its clinical value and oncogenic functions were further assessed. RIPK4 expression increased significantly with disease progression from 3.2% in chronic cervicitis, 19.3% in LSILs and 85.1% in HSILs to 94.4% in CSCCs (P < 0.001). Moreover, RIPK4 may serve as a useful biomarker to distinguish HSIL from chronic cervicitis/LSIL, which are two different clinical types for therapeutic procedures, with a high sensitivity and specificity (85.1% and 86.6%, respectively) and the performance improved when combined with p16(INK4a). Further, RIPK4 overexpression was associated with overall (HR = 2.085, P = 0.038) and disease-free survival (HR = 1.742, P = 0.037). Knockdown of RIPK4 reduced cell migration and invasion via inhibition of Vimentin, MMP2 and Fibronectin expression in cervical cancer cells. RIPK4 might act as a potential diagnostic and independent prognostic biomarker for CSCC patients.

  1. Epstein-Barr Virus and Gastric Carcinoma – Viral Carcinogenesis through Epigenetic Mechanisms

    PubMed Central

    Uozaki, Hiroshi; Fukayama, Masashi

    2008-01-01

    Epstein-Barr virus (EBV)-associated gastric carcinoma (GC) is the monoclonal growth of EBV-infected epithelial cells, and the entity was recognized only recently. EBV-associated GC is distributed worldwide and more than 90,000 patients are estimated to develop GC annually in association with EBV (10% of total GC). EBV-associated GC occurs in two forms in terms of the histological features, i.e., lymphoepithelioma-like GC and ordinary type of GC. Both share characteristic clinicopathological features, such as the preferential occurrence as multiple cancer and remnant stomach cancer. While the expression of EBV-latent genes is restricted to several in the infected cells (Latency I), EBV-associated GC shows gastric cell phenotype, resistance to apoptosis, and the production of immunomodulator molecules. Recently, global and non-random CpG island methylation of the promoter region of many cancer-related genes has been demonstrated with their decreased expression, such as p16 INK4A, p73 and E-cadherin. This abnormality is accompanied by methylation of the EBV genome itself, suggesting a process of virus-driven hypermethylation in the development of neoplastic cells. Further studies are necessary to determine the precise sequence of EBV infection, methylation, transformation and selection of the predominant clone within the stomach mucosa. Future studies are also desirable for the target and strategy of therapy, such as initiating viral replication or reversing the DNA methylation of cellular genes. PMID:18784828

  2. Activated Factor X Induces Endothelial Cell Senescence Through IGFBP-5

    PubMed Central

    Sanada, Fumihiro; Taniyama, Yoshiaki; Muratsu, Jun; Otsu, Rei; Iwabayashi, Masaaki; Carracedo, Miguel; Rakugi, Hiromi; Morishita, Ryuichi

    2016-01-01

    Uncontrolled coagulation contributes to the pathophysiology of several chronic inflammatory diseases. In these conditions, senescent cells are often observed and is involved in the generation of inflammation. The coincidence of hyper-coagulation, cell senescence, and inflammation suggests the existence of a common underlying mechanism. Recent evidence indicates that activated coagulation factor X (FXa) plays a role in the processes beyond blood coagulation. This non-hematologic function entails the mediation of inflammation and tissue remodeling. We therefore tested the hypothesis that FXa induces cell senescence resulting in tissue inflammation and impaired tissue regeneration. Human umbilical vein endothelial cells were stimulated with FXa for 14 days. The proliferation of cells treated with FXa was significantly smaller, and the fraction of senescence-associated β-galactosidase-positive cells was increased as compared to the control group. RT-qPCR array revealed that FXa increased the expression of IGFBP-5, EGR-1, p53, and p16INK4a. Inhibition of FXa by a direct FXa inhibitor, rivaroxaban, or IGFBP-5 by siRNA decreased FXa-induced cell senescence, restoring cell proliferation. Moreover, in an ischemic hind limb mouse model, FXa inhibited neovascularization by endothelial progenitor cell. However, rivaroxaban significantly restored FXa-induced impaired angiogenesis. In summary, FXa induced endothelial cell senescence through IGFBP-5, resulting in impaired angiogenesis. PMID:27752126

  3. Colorectal Cancer “Methylator Phenotype”: Fact or Artifact?1

    PubMed Central

    Anacleto, Charles; Leopoldino, Andréia M; Rossi, Benedito; Soares, Fernando A; Lopes, Ademar; Rocha, José Cláudio C; Caballero, Otávia; Camargo, Anamaria A; Simpson, Andrew J G; Pena, Sérgio D J

    2005-01-01

    Abstract It has been proposed that human colorectal tumors can be classified into two groups: one in which methylation is rare, and another with methylation of several loci associated with a “CpG island methylated phenotype (CIMP),” characterized by preferential proximal location in the colon, but otherwise poorly defined. There is considerable overlap between this putative methylator phenotype and the well-known mutator phenotype associated with microsatellite instability (MSI). We have examined hypermethylation of the promoter region of five genes (DAPK, MGMT, hMLH1, p16INK4a, and p14ARF) in 106 primary colorectal cancers. A graph depicting the frequency of methylated loci in the series of tumors showed a continuous, monotonically decreasing distribution quite different from the previously claimed discontinuity. We observed a significant association between the presence of three or more methylated loci and the proximal location of the tumors. However, if we remove from analysis the tumors with hMLH1 methylation or those with MSI, the significance vanishes, suggesting that the association between multiple methylations and proximal location was indirect due to the correlation with MSI. Thus, our data do not support the independent existence of the so-called methylator phenotype and suggest that it rather may represent a statistical artifact caused by confounding of associations. PMID:15967110

  4. Investigating the effects of the presence of foreign DNA on DNA methylation and DNA repair events in cultured eukaryotic cells.

    PubMed

    Du Toit, J; van der Westhuizen, F H; Pretorius, P J

    2013-01-01

    Methylation of DNA in eukaryotic cells, global as well as gene-specific, is affected by endogenous and endogenous factors. In this paper, it is reported that deviations in DNA methylation and expression of genes involved in DNA repair and the cell cycle are affected in 143B cultured cells containing an expression vector. Global DNA methylation analysis with cytosine-extension assay revealed a decreased global DNA methylation in the presence of the expression vector. Less promoter-specific methylation, as measured by bisulfite-MS PCR, was observed for MGMT and p16INK4a in vector-containing cells. Comet assay investigations revealed a negative effect on the DNA repair capacity of both BER and NER in Complex III compromised cells. This was reflected in the down-regulation of hOGG1 and ERCC1 expression. The results presented in this paper support the existence of a strong relationship between impaired mitochondrial function and deviations in DNA methylation and extend this relationship to impaired DNA repair.

  5. [Aberrant promoter methylation as biomarker for molecular cytological diagnosis of lung cancer].

    PubMed

    Grote, H J

    2006-01-01

    Aberrant promoter methylation represents a main mechanism of tumor suppressor gene inactivation and may serve as a new source for biomarker discovery. This study investigated its applicability as a molecular tool for lung cancer diagnostics on bronchial aspirates. A methylation assay was developed applying a quantitative methylation specific real-time PCR (QMSP). A total of 552 patients with the differential diagnosis of lung cancer were investigated. The QMSP findings on bronchial aspirates were compared with the methylation status of respective genes investigated in microdissected tumor tissues (QMSP, cloning and sequencing of promoter regions after bisulfite conversion). Among the genes tested a marker panel consisting of APC, p16(INK4a) and RASSF1A proved to be the best suited for lung cancer diagnostics. This panel allowed for a correct diagnosis of lung cancer in cases with an ambiguous or false negative conventional cytology. In a cohort study on 247 patients, the combination of histology (sensitivity 59 %), cytology (sensitivity 44 %) and QMSP-assay (sensitivity 53 %) raised the sensitivity of a single bronchoscopy for the diagnosis of lung cancer up to 81%. The methylation assay yielded its major diagnostic surplus with respect to peripheral tumors representing 59 % of all primaries detected. In patients without antecedent lung cancer its specificity considering malignancy was >99 %. Therefore, the QMSP-assay is a promising technique which could enhance the sensitivity and diagnostic impact of conventional cytology. The assay is applicable to residual material of regular diagnostic cytology even in retrospect.

  6. 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

  7. Premature skin aging features rescued by inhibition of NADPH oxidase activity in XPC-deficient mice.

    PubMed

    Hosseini, Mohsen; Mahfouf, Walid; Serrano-Sanchez, Martin; Raad, Houssam; Harfouche, Ghida; Bonneu, Marc; Claverol, Stephane; Mazurier, Frederic; Rossignol, Rodrigue; Taieb, Alain; Rezvani, Hamid Reza

    2015-04-01

    Xeroderma pigmentosum type C (XP-C) is characterized mostly by a predisposition to skin cancers and accelerated photoaging, but little is known about premature skin aging in this disease. By comparing young and old mice, we found that the level of progerin and p16(INK4a) expression, β-galactosidase activity, and reactive oxygen species, which increase with age, were higher in young Xpc(-/-) mice than in young Xpc(+/+) ones. The expression level of mitochondrial complexes and mitochondrial functions in the skin of young Xpc(-/-) was as low as in control aged Xpc(+/+)animals. Furthermore, the metabolic profile in young Xpc(-/-) mice resembled that found in aged Xpc(+/+) mice. Furthermore, premature skin aging features in young Xpc(-/-) mice were mostly rescued by inhibition of nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) activity by using a NOX1 peptide inhibitor, suggesting that the continuous oxidative stress due to overactivation of NOX1 has a causative role in the underlying pathophysiology. PMID:25437426

  8. miR-494-3p Induces Cellular Senescence and Enhances Radiosensitivity in Human Oral Squamous Carcinoma Cells

    PubMed Central

    Weng, Jui-Hung; Yu, Cheng-Chia; Lee, Yueh-Chun; Lin, Cheng-Wei; Chang, Wen-Wei; Kuo, Yu-Liang

    2016-01-01

    Oral squamous cell carcinoma (OSCC) is the most common malignancy of head and neck. Although radiotherapy is used for OSCC treatment, the occurrence of radioresistant cancer cells limits its efficiency. MicroRNAs (miRNAs) are non-coding RNAs with lengths of 18–25 base pairs and known to be involved in carcinogenesis. We previously demonstrated that by targeting B lymphoma Mo-MLV insertion region 1 homolog (Bmi1), miR-494-3p functions as a putative tumor suppressor miRNA in OSCC. In this study, we further discovered that miR-494-3p could enhance the radiosensitivity of SAS OSCC cells and induce cellular senescence. The overexpression of miR-494-3p in SAS cells increased the population of senescence-associated β-galactosidase positive cells, the expression of p16INK4a and retinoblastoma 1 (RB1), as well as downregulated Bmi1. The knockdown of Bmi1 by lentiviral-mediated delivery of specific short hairpin RNAs (shRNAs) also enhanced the radiosensitivity of SAS cells and the activation of the senescence pathway. Furthermore, the inverse correlation between Bmi1 and miR-494-3p expression was observed among OSCC tissues. Results suggest that miR-494-3p could increase the radiosensitivity of OSCC cells through the induction of cellular senescence caused by the downregulation of Bmi1. PMID:27399693

  9. Senescence induced by RECQL4 dysfunction contributes to Rothmund-Thomson syndrome features in mice.

    PubMed

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

    2014-05-15

    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 p16(INK4a) or/and p21(WAF1) 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 (Recql4(HD)). Tail fibroblasts from Recql4(HD) showed increased SA-β-gal staining and increased DNA damage foci. We also identified sparser tail hair and fewer blood cells in Recql4(HD) 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.

  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. 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.

  12. Senescence-Induced Oxidative Stress Causes Endothelial Dysfunction.

    PubMed

    Bhayadia, Raj; Schmidt, Bernhard M W; Melk, Anette; Hömme, Meike

    2016-02-01

    Age is a risk factor for cardiovascular disease, suggesting a causal relationship between age-related changes and vascular damage. Endothelial dysfunction is an early pathophysiological hallmark in the development of cardiovascular disease. Senescence, the cellular equivalent of aging, was proposed to be involved in endothelial dysfunction, but functional data showing a causal relationship are missing.Endothelium-dependent vasodilation was measured in aortic rings ex vivo. We investigated aortas from aged C57Bl/6 mice (24-28 months), in which p16 (INK4a) and p19 (ARF) expression, markers of stress-induced senescence, were significantly induced compared to young controls (4-6 months). To reflect telomere shortening in human aging, we investigated aortas from telomerase deficient (Terc(-/-)) mice of generation 3 (G3). Endothelium-dependent vasodilation in aged wildtype and in Terc(-/-) G3 mice was impaired. A combination of the superoxide dismutase mimetic 1-Oxyl-2,2,6, 6-tetramethyl-4-hydroxypiperidine (TEMPOL) and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin significantly improved endothelium-dependent vasodilation in aged wildtype and Terc(-/-) G3 mice compared to untreated controls. We show that both, aging and senescence induced by telomere shortening, cause endothelial dysfunction that can be restored by antioxidants, indicating a role for oxidative stress. The observation that cellular senescence is a direct signalling event leading to endothelial dysfunction holds the potential to develop new targets for the prevention of cardiovascular disease.

  13. Emerging roles of hypoxia-inducible factors and reactive oxygen species in cancer and pluripotent stem cells.

    PubMed

    Saito, Shigeo; Lin, Ying-Chu; Tsai, Ming-Ho; Lin, Chang-Shen; Murayama, Yoshinobu; Sato, Ryuji; Yokoyama, Kazunari K

    2015-06-01

    Eukaryotic organisms require oxygen homeostasis to maintain proper cellular function for survival. During conditions of low oxygen tension (hypoxia), cells activate the transcription of genes that induce an adaptive response, which supplies oxygen to tissues. Hypoxia and hypoxia-inducible factors (HIFs) may contribute to the maintenance of putative cancer stem cells, which can continue self-renewal indefinitely and express stemness genes in hypoxic stress environments (stem cell niches). Reactive oxygen species (ROS) have long been recognized as toxic by-products of aerobic metabolism that are harmful to living cells, leading to DNA damage, senescence, or cell death. HIFs may promote a cancer stem cell state, whereas the loss of HIFs induces the production of cellular ROS and activation of proteins p53 and p16(Ink4a), which lead to tumor cell death and senescence. ROS seem to inhibit HIF regulation in cancer cells. By contrast, controversial data have suggested that hypoxia increases the generation of ROS, which prevents hydroxylation of HIF proteins by inducing their transcription as negative feedback. Moreover, hypoxic conditions enhance the generation of induced pluripotent stem cells (iPSCs). During reprogramming of somatic cells into a PSC state, cells attain a metabolic state typically observed in embryonic stem cells (ESCs). ESCs and iPSCs share similar bioenergetic metabolisms, including decreased mitochondrial number and activity, and induced anaerobic glycolysis. This review discusses the current knowledge regarding the emerging roles of ROS homeostasis in cellular reprogramming and the implications of hypoxic regulation in cancer development. PMID:26043406

  14. 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.

  15. 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.

  16. Topical hypochlorite ameliorates NF-κB–mediated skin diseases in mice

    PubMed Central

    Leung, Thomas H.; Zhang, Lillian F.; Wang, Jing; Ning, Shoucheng; Knox, Susan J.; Kim, Seung K.

    2013-01-01

    Nuclear factor-κB (NF-κB) regulates cellular responses to inflammation and aging, and alterations in NF-κB signaling underlie the pathogenesis of multiple human diseases. Effective clinical therapeutics targeting this pathway remain unavailable. In primary human keratinocytes, we found that hypochlorite (HOCl) reversibly inhibited the expression of CCL2 and SOD2, two NF-κB–dependent genes. In cultured cells, HOCl inhibited the activity of inhibitor of NF-κB kinase (IKK), a key regulator of NF-κB activation, by oxidizing cysteine residues Cys114 and Cys115. In NF-κB reporter mice, topical HOCl reduced LPS-induced NF-κB signaling in skin. We further evaluated topical HOCl use in two mouse models of NF-κB–driven epidermal disease. For mice with acute radiation dermatitis, topical HOCl inhibited the expression of NF-κB–dependent genes, decreased disease severity, and prevented skin ulceration. In aged mice, topical HOCl attenuated age-dependent production of p16INK4a and expression of the DNA repair gene Rad50. Additionally, skin of aged HOCl-treated mice acquired enhanced epidermal thickness and proliferation, comparable to skin in juvenile animals. These data suggest that topical HOCl reduces NF-κB–mediated epidermal pathology in radiation dermatitis and skin aging through IKK modulation and motivate the exploration of HOCl use for clinical aims. PMID:24231355

  17. Interfering EZH2 Expression Reverses the Cisplatin Resistance in Human Ovarian Cancer by Inhibiting Autophagy.

    PubMed

    Sun, Yang; Jin, Long; Liu, Jia-Hua; Sui, Yu-Xia; Han, Li-Li; Shen, Xiao-Li

    2016-09-01

    We aimed to determine the effects of the inhibition of enhancer of zeste homolog 2 (EZH2) gene expression on the cisplatin resistance of the human ovarian cancer cell line, SKOV3/DDP, and to identify the underlying mechanisms. SKOV3/DDP cells were stably transfected with pSUPER-EZH2 (EZH2 RNA interference plasmid) or pcDNA3.1-EZH2 (EZH2 gene overexpression plasmid) using the lipofection method. Real-time fluorescence quantitative reverse transcription polymerase chain reaction and western blotting confirmed that EZH2 expression was downregulated in pSUPER-EZH2-transfected cells. Flow cytometry revealed that EZH2 inhibition did not induce apoptosis, but significantly inhibited autophagy. In addition, it significantly increased the expression of the cellular senescence-signaling proteins p14(ARF), p16(INK4a), p53, pRb, and p21, and significantly decreased the expression of cyclin-dependent kinase (CDK)1, CDK2, and H3K27me3. Cellular senescence was characterized by a significant increase in the G0/G1 ratio and the restoration of sensitivity to cisplatin in the drug-resistant cells. These findings suggest that interfering with EZH2 expression can inhibit SKOV3/DDP cell autophagy and reverse resistance to cisplatin. The underlying mechanisms could be associated with the regulation of the cellular senescence-signaling pathway. PMID:27610467

  18. Jun Dimerization Protein 2 Controls Senescence and Differentiation via Regulating Histone Modification

    PubMed Central

    Huang, Yu-Chang; Hasegawa, Hitomi; Wang, Shin-Wei; Ku, Chia-Chen; Lin, Ying-Chu; Chiou, Shyh-Shin; Hou, Ming-Feng; Wu, Deng-Chyang; Tsai, Eing-Mei; Saito, Shigeo; Yamaguchi, Naoto; Yokoyama, Kazunari K.

    2011-01-01

    Transcription factor, Jun dimerization protein 2 (JDP2), binds directly to histones and DNAs and then inhibits the p300-mediated acetylation both of core histones and of reconstituted nucleosomes that contain JDP2 recognition DNA sequences. JDP2 plays a key role as a repressor of adipocyte differentiation by regulation of the expression of the gene C/EBPδ via inhibition of histone acetylation. Moreover, JDP2-deficient mouse embryonic fibroblasts (JDP2−/− MEFs) are resistant to replicative senescence. JDP2 inhibits the recruitment of polycomb repressive complexes (PRC1 and PRC2) to the promoter of the gene encoding p16Ink4a, resulting from the inhibition of methylation of lysine 27 of histone H3 (H3K27). Therefore, it seems that chromatin-remodeling factors, including the PRC complex controlled by JDP2, may be important players in the senescence program. The novel mechanisms that underline the action of JDP2 in inducing cellular senescence and suppressing adipocyte differentiation are reviewed. PMID:21197464

  19. 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

  20. 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.

  1. miR-494-3p Induces Cellular Senescence and Enhances Radiosensitivity in Human Oral Squamous Carcinoma Cells.

    PubMed

    Weng, Jui-Hung; Yu, Cheng-Chia; Lee, Yueh-Chun; Lin, Cheng-Wei; Chang, Wen-Wei; Kuo, Yu-Liang

    2016-01-01

    Oral squamous cell carcinoma (OSCC) is the most common malignancy of head and neck. Although radiotherapy is used for OSCC treatment, the occurrence of radioresistant cancer cells limits its efficiency. MicroRNAs (miRNAs) are non-coding RNAs with lengths of 18-25 base pairs and known to be involved in carcinogenesis. We previously demonstrated that by targeting B lymphoma Mo-MLV insertion region 1 homolog (Bmi1), miR-494-3p functions as a putative tumor suppressor miRNA in OSCC. In this study, we further discovered that miR-494-3p could enhance the radiosensitivity of SAS OSCC cells and induce cellular senescence. The overexpression of miR-494-3p in SAS cells increased the population of senescence-associated β-galactosidase positive cells, the expression of p16(INK4a) and retinoblastoma 1 (RB1), as well as downregulated Bmi1. The knockdown of Bmi1 by lentiviral-mediated delivery of specific short hairpin RNAs (shRNAs) also enhanced the radiosensitivity of SAS cells and the activation of the senescence pathway. Furthermore, the inverse correlation between Bmi1 and miR-494-3p expression was observed among OSCC tissues. Results suggest that miR-494-3p could increase the radiosensitivity of OSCC cells through the induction of cellular senescence caused by the downregulation of Bmi1. PMID:27399693

  2. 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. PMID:27067784

  3. Inhibition of TGF-β Signaling Promotes Human Pancreatic β-Cell Replication.

    PubMed

    Dhawan, Sangeeta; Dirice, Ercument; Kulkarni, Rohit N; Bhushan, Anil

    2016-05-01

    Diabetes is associated with loss of functional pancreatic β-cells, and restoration of β-cells is a major goal for regenerative therapies. Endogenous regeneration of β-cells via β-cell replication has the potential to restore cellular mass; however, pharmacological agents that promote regeneration or expansion of endogenous β-cells have been elusive. The regenerative capacity of β-cells declines rapidly with age, due to accumulation of p16(INK4a), resulting in limited capacity for adult endocrine pancreas regeneration. Here, we show that transforming growth factor-β (TGF-β) signaling via Smad3 integrates with the trithorax complex to activate and maintain Ink4a expression to prevent β-cell replication. Importantly, inhibition of TGF-β signaling can result in repression of the Ink4a/Arf locus, resulting in increased β-cell replication in adult mice. Furthermore, small molecule inhibitors of the TGF-β pathway promote β-cell replication in human islets transplanted into NOD-scid IL-2Rg(null) mice. These data reveal a novel role for TGF-β signaling in the regulation of the Ink4a/Arf locus and highlight the potential of using small molecule inhibitors of TGF-β signaling to promote human β-cell replication. PMID:26936960

  4. Cold-Inducible RNA-Binding Protein Bypasses Replicative Senescence in Primary Cells through Extracellular Signal-Regulated Kinase 1 and 2 Activation▿ †

    PubMed Central

    Artero-Castro, Ana; Callejas, Francisco B.; Castellvi, Josep; Kondoh, Hiroshi; Carnero, Amancio; Fernández-Marcos, Pablo J.; Serrano, Manuel; Ramón y Cajal, Santiago; Lleonart, Matilde E.

    2009-01-01

    Embryonic stem cells are immortalized cells whose proliferation rate is comparable to that of carcinogenic cells. To study the expression of embryonic stem cell genes in primary cells, genetic screening was performed by infecting mouse embryonic fibroblasts (MEFs) with a cDNA library from embryonic stem cells. Cold-inducible RNA-binding protein (CIRP) was identified due to its ability to bypass replicative senescence in primary cells. CIRP enhanced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, and treatment with an MEK inhibitor decreased the proliferation caused by CIRP. In contrast to CIRP upregulation, CIRP downregulation decreased cell proliferation and resulted in inhibition of phosphorylated ERK1/2 inhibition. This is the first evidence that ERK1/2 activation, through the same mechanism as that described for a Val12 mutant K-ras to induce premature senescence, is able to bypass senescence in the absence of p16INK4a, p21WAF1, and p19ARF upregulation. Moreover, these results show that CIRP functions by stimulating general protein synthesis with the involvement of the S6 and 4E-BP1 proteins. The overall effect is an increase in kinase activity of the cyclin D1-CDK4 complex, which is in accordance with the proliferative capacity of CIRP MEFs. Interestingly, CIRP mRNA and protein were upregulated in a subgroup of cancer patients, a finding that may be of relevance for cancer research. PMID:19158277

  5. LIF-JAK1-STAT3 signaling delays contact inhibition of human corneal endothelial cells.

    PubMed

    Liu, Xin; Tseng, Scheffer C G; Zhang, Ming-Chang; Chen, Szu-Yu; Tighe, Sean; Lu, Wen-Juan; Zhu, Ying-Ting

    2015-01-01

    Human corneal endothelial cells (HCECs) responsible for corneal transparency have limited proliferative capacity in vivo because of "contact-inhibition." This feature has hampered the ability to engineer HCECs for transplantation. Previously we have reported an in vitro model of HCECs in which contact inhibition was re-established at Day 21, even though cell junction and cell matrix interaction were not perturbed during isolation. Herein, we observe that such HCEC monolayers continue to expand and retain a normal phenotype for 2 more weeks if cultured in a leukemia inhibitory factor (LIF)-containing serum-free medium. Such expansion is accompanied initially by upregulation of Cyclin E2 colocalized with nuclear translocation of phosphorylated retinoblastoma tumor suppressor (p-Rb) at Day 21 followed by a delay in contact inhibition through activation of LIF-Janus kinase1 (JAK1)-signal transducer and activator of transcription 3 (STAT3) signaling at Day 35. The LIF-JAK1-STAT3 signaling is coupled with upregulation of E2F2 colocalized with nuclear p-Rb and with concomitant downregulation of p16(INK4a), of which upregulation is linked to senescence. Hence, activation of LIF-JAK1-STAT3 signaling to delay contact inhibition can be used as another strategy to facilitate engineering of HCEC grafts to solve the unmet global shortage of corneal grafts. PMID:25695744

  6. Effects of ageing and senescence on pancreatic β-cell function.

    PubMed

    Helman, A; Avrahami, D; Klochendler, A; Glaser, B; Kaestner, K H; Ben-Porath, I; Dor, Y

    2016-09-01

    Ageing is generally associated with deterioration of organ function and regenerative potential. In the case of pancreatic β-cells, an age-related decline in proliferative potential is well documented, and was proposed to contribute to the increased prevalence of type 2 diabetes in the elderly. The effects of ageing on β-cell function, namely glucose-stimulated insulin secretion (GSIS), have not been studied as extensively. Recent work revealed that, surprisingly, β-cells of mature mice and humans secrete more insulin than young β-cells in response to high glucose concentrations, potentially serving to counteract age-related peripheral insulin resistance. This functional change appears to be orchestrated by p16(Ink4A) -driven cellular senescence and downstream remodelling of chromatin structure and DNA methylation, enhancing the expression of genes controlling β-cell function. We propose that activation of the cellular senescence program drives life-long functional maturation of β-cells, due to β-cell hypertrophy, enhanced glucose uptake and more efficient mitochondrial metabolism, in parallel to locking these cells in a non-replicative state. We speculate that the beneficial aspects of this process can be harnessed to enhance GSIS. Other age-related mechanisms, which are currently poorly understood, act to increase basal insulin secretion levels also in low glucose conditions. This leads to an overall reduction in the amplitude of insulin secretion between low and high glucose at old age, which may contribute to a deterioration in metabolic control. PMID:27615132

  7. A high-content cellular senescence screen identifies candidate tumor suppressors, including EPHA3.

    PubMed

    Lahtela, Jenni; Corson, Laura B; Hemmes, Annabrita; Brauer, Matthew J; Koopal, Sonja; Lee, James; Hunsaker, Thomas L; Jackson, Peter K; Verschuren, Emmy W

    2013-02-15

    Activation of a cellular senescence program is a common response to prolonged oncogene activation or tumor suppressor loss, providing a physiological mechanism for tumor suppression in premalignant cells. The link between senescence and tumor suppression supports the hypothesis that a loss-of-function screen measuring bona fide senescence marker activation should identify candidate tumor suppressors. Using a high-content siRNA screening assay for cell morphology and proliferation measures, we identify 12 senescence-regulating kinases and determine their senescence marker signatures, including elevation of senescence-associated β-galactosidase, DNA damage and p53 or p16 (INK4a) expression. Consistent with our hypothesis, SNP array CGH data supports loss of gene copy number of five senescence-suppressing genes across multiple tumor samples. One such candidate is the EPHA3 receptor tyrosine kinase, a gene commonly mutated in human cancer. We demonstrate that selected intracellular EPHA3 tumor-associated point mutations decrease receptor expression level and/or receptor tyrosine kinase (RTK) activity. Our study therefore describes a new strategy to mine for novel candidate tumor suppressors and provides compelling evidence that EPHA3 mutations may promote tumorigenesis only when key senescence-inducing pathways have been inactivated. PMID:23324396

  8. [Inactivation of failsafe programs by Twist oncoproteins and tumor progression].

    PubMed

    Puisieux, A

    2008-01-01

    Multicellular organisms have developed innate defense mechanisms to prevent the expansion of abnormal cells with significant proliferative potential. The two major safeguard mechanisms are premature senescence, which is characterized by definitive cell cycle arrest, and apoptosis, the most common form of programmed cell death. In normal and premalignant cells, the control of these processes is coupled to the regulation of cell proliferation, mainly through the p16 (Ink4A) -Rb and ARF-p53 intracellular signaling pathways. Hence, in benign tumors, aberrant mitogenic activity is counterbalanced by the induction of these oncosuppressive pathways, leading to either apoptosis or senescence which both limit tumor outgrowth. Progression towards malignant and potentially metastatic tumors requires the inhibition of these failsafe programs. Based on our work on Twist oncoproteins, we propose a presentation of recent data on cellular mechanisms by which cancer cells override the surveillance machinery and escape senescence and apoptosis, and we will describe the biological impact of this process on tumor metastasis. PMID:19061727

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Selective impact of CDK4/6 suppression on patient-derived models of pancreatic cancer

    PubMed Central

    Witkiewicz, Agnieszka K.; Borja, Nicholas A.; Franco, Jorge; Brody, Jonathan R.; Yeo, Charles J.; Mansour, John; Choti, Michael A.; McCue, Peter; Knudsen, Erik S.

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDA) harbors an exceedingly poor prognosis, and is generally considered a therapy-recalcitrant disease due to poor response to conventional chemotherapy coupled with non-actionable genetic drivers (e.g. KRAS mutations). However, PDA frequently loses p16ink4a, thereby leading to deregulation of CDK4/6. Surprisingly, in established cell models and xenografts, CDK4/6 inhibition has a modest effect on proliferation and resistance develops rapidly. To determine if such weak response was an intrinsic feature of PDA, we developed primary tumor explants that maintain the tumor environment and recapitulate feuture of primary PDA. The CDK4/6 inhibitor PD-0332991 was highly efficient at suppressing proliferation in 14 of the 15 explants. In the single resistant explant, we identified the rare loss of the RB tumor suppressor as the basis for resistance. Patient-derived xenografts (PDXs) were developed in parallel, and unlike the xenografts emerging from established cell lines, the PDXs maintained the histoarchitecture of the primary tumor. These PDXs were highly sensitive to CDK4/6 inhibition, yielding a complete suppression of PDA proliferation. Together, these data indicate that primary PDA is sensitive to CDK4/6 inhibition, that specific biomarkers can delineate intrinsic resistance, and that established cell line models may not represent an adequate means for evaluating therapeutic sensitivities. PMID:26158861

  15. Selective impact of CDK4/6 suppression on patient-derived models of pancreatic cancer.

    PubMed

    Witkiewicz, Agnieszka K; Borja, Nicholas A; Franco, Jorge; Brody, Jonathan R; Yeo, Charles J; Mansour, John; Choti, Michael A; McCue, Peter; Knudsen, Erik S

    2015-06-30

    Pancreatic ductal adenocarcinoma (PDA) harbors an exceedingly poor prognosis, and is generally considered a therapy-recalcitrant disease due to poor response to conventional chemotherapy coupled with non-actionable genetic drivers (e.g. KRAS mutations). However, PDA frequently loses p16ink4a, thereby leading to deregulation of CDK4/6. Surprisingly, in established cell models and xenografts, CDK4/6 inhibition has a modest effect on proliferation and resistance develops rapidly. To determine if such weak response was an intrinsic feature of PDA, we developed primary tumor explants that maintain the tumor environment and recapitulate feuture of primary PDA. The CDK4/6 inhibitor PD-0332991 was highly efficient at suppressing proliferation in 14 of the 15 explants. In the single resistant explant, we identified the rare loss of the RB tumor suppressor as the basis for resistance. Patient-derived xenografts (PDXs) were developed in parallel, and unlike the xenografts emerging from established cell lines, the PDXs maintained the histoarchitecture of the primary tumor. These PDXs were highly sensitive to CDK4/6 inhibition, yielding a complete suppression of PDA proliferation. Together, these data indicate that primary PDA is sensitive to CDK4/6 inhibition, that specific biomarkers can delineate intrinsic resistance, and that established cell line models may not represent an adequate means for evaluating therapeutic sensitivities.

  16. Metabolic re-programming of pancreatic cancer mediated by CDK4/6 inhibition elicits unique vulnerabilities

    PubMed Central

    Franco, Jorge; Balaji, Uthra; Freinkman, Elizaveta; Witkiewicz, Agnieszka K.; Knudsen, Erik S.

    2016-01-01

    Due to loss of p16ink4a in pancreatic ductal adenocarcinoma (PDA), pharmacological suppression of CDK4/6 could represent a potent target for treatment. In PDA models CDK4/6 inhibition had variable effect on cell cycle, but yielded accumulation of ATP and mitochondria. Pharmacological CDK4/6 inhibitors induce cyclin D1 protein levels; however, RB activation was required and sufficient for mitochondrial accumulation. CDK4/6 inhibition stimulated glycolytic and oxidative metabolism and was associated with an increase in mTORC1 activity. MTOR and MEK inhibitors potently cooperate with CDK4/6 inhibition in eliciting cell cycle exit. However, MTOR inhibition fully suppressed metabolism and yielded apoptosis and suppression of tumor growth. The metabolic state mediated by CDK4/6 inhibition increases mitochondrial number and ROS. Concordantly, the suppression of ROS scavenging or BCL2-antagonists cooperated with CDK4/6 inhibition. Together, these data define the impact of therapeutics on PDA metabolism and provide strategies for converting cytostatic response to tumor cell killing. PMID:26804906

  17. The Matricellular Protein CCN1/CYR61 Induces Fibroblast Senescence and Restricts Fibrosis in Cutaneous Wound Healing

    PubMed Central

    Jun, Joon-Il; Lau, Lester F.

    2010-01-01

    Cellular senescence is a recognised mechanism of tumor suppression; however, its contribution to other pathologies is not well understood. We show that the matricellular protein CCN1/CYR61, which is dynamically expressed at sites of wound repair, can induce fibroblast senescence through its cell adhesion receptors, integrin α6β1 and heparan sulfate proteoglycans. CCN1 induces DNA damage response and p53 activation, and activates the RAC1-NOX1 complex to induce reactive oxygen species (ROS) generation and ROS-dependent activation of the p16INK4a/pRb pathway, leading to senescence and concomitant expression of antifibrotic genes. Senescent fibroblasts accumulate in granulation tissues of healing cutaneous wounds and express antifibrotic genes in wild type mice. These processes are obliterated in knockin mice that express a senescence-defective CCN1 mutant, resulting in exacerbated fibrosis. Topical application of CCN1 protein to wounds reverses these defects. Thus, fibroblast senescence is a CCN1-dependent wound healing response in cutaneous injury, functioning to curb fibrosis during tissue repair. PMID:20526329

  18. 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. PMID:23334329

  19. Familial cutaneous melanoma.

    PubMed

    Hansson, Johan

    2010-01-01

    Approximately 5-10 % of all cutaneous melanomas occur in families with hereditary melanoma predisposition. Worldwide, approximately 20-40% of kindreds with familial elanoma harbor germline mutations in the CDKN2A gene, located on chromosome 9p21, which encodes two different proteins, p16INK4 and p14ARF, both involved in regulation of cell cycle progression and induction of senescence. In different populations several recurring CDKN2A founder mutations have been described. The risk of melanoma in CDKN2A mutations carriers varies between populations and is higher in regions with high sun exposure and high incidence of melanoma in the general population. Some CDKN2A mutations have been associated not only with melanoma but also with increased risk of other malignancies--most notably pancreatic carcinoma. A much smaller number of families have germline mutations in the CDK4 gene on chromosome 12q14, encoding a cyclin dependent kinase which normally interacts with p16INK4A. The management of families with hereditary melanoma is discussed. PMID:20687502

  20. MicroRNAs in the pathogenesis of malignant melanoma.

    PubMed

    Glud, M; Gniadecki, R

    2013-02-01

    Cutaneous malignant melanoma is the most aggressive and lethal form of skin cancer. Over the past decades, its incidence has been increasing by 3-8% per year in western countries while mortality has stabilized. Melanoma is a heterogenous disease and can be subclassified based on distinct clinical characteristics, histopathological features and mutation patterns within NRAS and BRAF genes. Recent data indicate that microRNAs (miRNAs) are involved in the pathogenesis of malignant melanoma. MiRNAs are small, non-coding, regulatory RNA molecules expressed in a tissue and cell specific manner and are known to play a crucial role in cell homeostasis and carcinogenesis. MiRNAs might prove to be powerful cancer biomarkers and future therapeutic targets. In this review, we focused on the miRNA involvement in four molecular pathways known to be deregulated in malignant melanoma, including the RAS-RAF-MEK-ERK pathway, the p16(INK4A) -CDK4-RB pathway, the PIK3-AKT pathway and the MITF pathway. PMID:22621697

  1. 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. PMID:24479804

  2. 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

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

    PubMed

    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

  4. 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

  5. Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice.

    PubMed

    Lund, Anders H; Turner, Geoffrey; Trubetskoy, Alla; Verhoeven, Els; Wientjens, Ellen; Hulsman, Danielle; Russell, Robert; DePinho, Ronald A; Lenz, Jack; van Lohuizen, Maarten

    2002-09-01

    We have used large-scale insertional mutagenesis to identify functional landmarks relevant to cancer in the recently completed mouse genome sequence. We infected Cdkn2a(-/-) mice with Moloney murine leukemia virus (MoMuLV) to screen for loci that can participate in tumorigenesis in collaboration with loss of the Cdkn2a-encoded tumor suppressors p16INK4a and p19ARF. Insertional mutagenesis by the latent retrovirus was synergistic with loss of Cdkn2a expression, as indicated by a marked acceleration in the development of both myeloid and lymphoid tumors. We isolated 747 unique sequences flanking retroviral integration sites and mapped them against the mouse genome sequence databases from Celera and Ensembl. In addition to 17 insertions targeting gene loci known to be cancer-related, we identified a total of 37 new common insertion sites (CISs), of which 8 encode components of signaling pathways that are involved in cancer. The effectiveness of large-scale insertional mutagenesis in a sensitized genetic background is demonstrated by the preference for activation of MAP kinase signaling, collaborating with Cdkn2a loss in generating the lymphoid and myeloid tumors. Collectively, our results show that large-scale retroviral insertional mutagenesis in genetically predisposed mice is useful both as a system for identifying genes underlying cancer and as a genetic framework for the assignment of such genes to specific oncogenic pathways.

  6. Induction of p38δ Expression Plays an Essential Role in Oncogenic ras-Induced Senescence

    PubMed Central

    Kwong, Jinny; Chen, Michelle; Lv, Dan; Luo, Na; Su, Weijun; Xiang, Rong

    2013-01-01

    Oncogene-induced senescence is a stable proliferative arrest that serves as a tumor-suppressing defense mechanism. p38 mitogen-activated protein kinase (MAPK) has been implicated in oncogene-induced senescence and tumor suppression. However, the specific role of each of the four p38 isoforms in oncogene-induced senescence is not fully understood. Here, we demonstrate that p38δ mediates oncogene-induced senescence through a p53- and p16INK4A-independent mechanism. Instead, evidence suggests a link between p38δ and the DNA damage pathways. Moreover, we have discovered a novel mechanism that enhances the expression of p38δ during senescence. In this mechanism, oncogenic ras induces the Raf-1–MEK–extracellular signal-regulated kinase (ERK) pathway, which, in turn, activates the AP-1 and Ets transcription factors that are bound to the p38δ promoter, leading to increased transcription of p38δ. These findings indicate that induction of the prosenescent function of p38δ by oncogenic ras is achieved through 2 mechanisms, transcriptional activation by the Raf-1–MEK–ERK–AP-1/Ets pathway, which increases the cellular concentration of the p38δ protein, and posttranslational modification by MKK3/6, which stimulates the enzymatic activity of p38δ. In addition, these studies identify the AP-1 and Ets transcription factors as novel signaling components in the senescence-inducing pathway. PMID:23878395

  7. Novel therapeutic targets for pancreatic cancer

    PubMed Central

    Tang, Shing-Chun; Chen, Yang-Chao

    2014-01-01

    Pancreatic cancer has become the fourth leading cause of cancer death in the last two decades. Only 3%-15% of patients diagnosed with pancreatic cancer had 5 year survival rate. Drug resistance, high metastasis, poor prognosis and tumour relapse contributed to the malignancies and difficulties in treating pancreatic cancer. The current standard chemotherapy for pancreatic cancer is gemcitabine, however its efficacy is far from satisfactory, one of the reasons is due to the complex tumour microenvironment which decreases effective drug delivery to target cancer cell. Studies of the molecular pathology of pancreatic cancer have revealed that activation of KRAS, overexpression of cyclooxygenase-2, inactivation of p16INK4A and loss of p53 activities occurred in pancreatic cancer. Co-administration of gemcitabine and targeting the molecular pathological events happened in pancreatic cancer has brought an enhanced therapeutic effectiveness of gemcitabine. Therefore, studies looking for novel targets in hindering pancreatic tumour growth are emerging rapidly. In order to give a better understanding of the current findings and to seek the direction in future pancreatic cancer research; in this review we will focus on targets suppressing tumour metastatsis and progression, KRAS activated downstream effectors, the relationship of Notch signaling and Nodal/Activin signaling with pancreatic cancer cells, the current findings of non-coding RNAs in inhibiting pancreatic cancer cell proliferation, brief discussion in transcription remodeling by epigenetic modifiers (e.g., HDAC, BMI1, EZH2) and the plausible therapeutic applications of cancer stem cell and hyaluronan in tumour environment. PMID:25152585

  8. Accelerated aging phenotype in mice with conditional deficiency for mitochondrial superoxide dismutase in the connective tissue.

    PubMed

    Treiber, Nicolai; Maity, Pallab; Singh, Karmveer; Kohn, Matthias; Keist, Alexander F; Ferchiu, Florentina; Sante, Lea; Frese, Sebastian; Bloch, Wilhelm; Kreppel, Florian; Kochanek, Stefan; Sindrilaru, Anca; Iben, Sebastian; Högel, Josef; Ohnmacht, Michael; Claes, Lutz E; Ignatius, Anita; Chung, Jin H; Lee, Min J; Kamenisch, York; Berneburg, Mark; Nikolaus, Thorsten; Braunstein, Kerstin; Sperfeld, Anne-Dorte; Ludolph, Albert C; Briviba, Karlis; Wlaschek, Meinhard; Florin, Lore; Angel, Peter; Scharffetter-Kochanek, Karin

    2011-04-01

    The free radical theory of aging postulates that the production of mitochondrial reactive oxygen species is the major determinant of aging and lifespan. Its role in aging of the connective tissue has not yet been established, even though the incidence of aging-related disorders in connective tissue-rich organs is high, causing major disability in the elderly. We have now addressed this question experimentally by creating mice with conditional deficiency of the mitochondrial manganese superoxide dismutase in fibroblasts and other mesenchyme-derived cells of connective tissues in all organs. Here, we have shown for the first time that the connective tissue-specific lack of superoxide anion detoxification in the mitochondria results in reduced lifespan and premature onset of aging-related phenotypes such as weight loss, skin atrophy, kyphosis (curvature of the spine), osteoporosis and muscle degeneration in mutant mice. Increase in p16(INK4a) , a robust in vivo marker for fibroblast aging, may contribute to the observed phenotype. This novel model is particularly suited to decipher the underlying mechanisms and to develop hopefully novel connective tissue-specific anti-aging strategies.

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

    PubMed

    Mimeault, Murielle; Batra, Surinder K

    2009-04-01

    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 p16(INK4A), p19(ARF), ataxia-telangiectasia mutated (ATM) kinase, p53 and/or the forkhead 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.

  10. 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

  11. 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

  12. Viral and Cellular Biomarkers in the Diagnosis of Cervical Intraepithelial Neoplasia and Cancer

    PubMed Central

    Buonaguro, Luigi; Giorgi-Rossi, Paolo; Buonaguro, Franco M.

    2013-01-01

    Cervical cancer arises from cells localized in the ectoendocervical squamocolumnar junction of the cervix persistently infected with one of about 13 human papillomavirus (HPV) genotypes. The majority of HPV infections induces low grade squamous epithelial lesions that in more than 90% of cases spontaneously regress and in about 10% eventually progress to high grade lesions and even less frequently evolve to invasive cancer. Tumor progression is characterized by (1) increased expression of E6 and E7 genes of high risk HPVs, known to bind to and inactivate p53 and pRb oncosuppressors, respectively; (2) integration of viral DNA into host genome, with disruption of E2 viral genes and host chromosomal loci; and (3) molecular alterations of key regulators of cell cycle. Molecular markers with high sensitivity and specificity in differentiating viral infections associated with cellular abnormalities with high risk of progression are strongly needed for cervical cancer screening and triage. This review will focus on the analysis of clinical validated or candidate biomarkers, such as HPV DNA, HPV E6/E7 mRNA, HPV proteins, p16(INK4a) and Ki67, TOP2A and MCM2 cellular factors, and DNA methylation profiles, which will likely improve the identification of premalignant lesions that have a high risk to evolve into invasive cervical cancer. PMID:24383054

  13. Exercise Prevents Diet-Induced Cellular Senescence in Adipose Tissue.

    PubMed

    Schafer, Marissa J; White, Thomas A; Evans, Glenda; Tonne, Jason M; Verzosa, Grace C; Stout, Michael B; Mazula, Daniel L; Palmer, Allyson K; Baker, Darren J; Jensen, Michael D; Torbenson, Michael S; Miller, Jordan D; Ikeda, Yasuhiro; Tchkonia, Tamara; van Deursen, Jan M; Kirkland, James L; LeBrasseur, Nathan K

    2016-06-01

    Considerable evidence implicates cellular senescence in the biology of aging and chronic disease. Diet and exercise are determinants of healthy aging; however, the extent to which they affect the behavior and accretion of senescent cells within distinct tissues is not clear. Here we tested the hypothesis that exercise prevents premature senescent cell accumulation and systemic metabolic dysfunction induced by a fast-food diet (FFD). Using transgenic mice that express EGFP in response to activation of the senescence-associated p16(INK4a) promoter, we demonstrate that FFD consumption causes deleterious changes in body weight and composition as well as in measures of physical, cardiac, and metabolic health. The harmful effects of the FFD were associated with dramatic increases in several markers of senescence, including p16, EGFP, senescence-associated β-galactosidase, and the senescence-associated secretory phenotype (SASP) specifically in visceral adipose tissue. We show that exercise prevents the accumulation of senescent cells and the expression of the SASP while nullifying the damaging effects of the FFD on parameters of health. We also demonstrate that exercise initiated after long-term FFD feeding reduces senescent phenotype markers in visceral adipose tissue while attenuating physical impairments, suggesting that exercise may provide restorative benefit by mitigating accrued senescent burden. These findings highlight a novel mechanism by which exercise mediates its beneficial effects and reinforces the effect of modifiable lifestyle choices on health span. PMID:26983960

  14. Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation

    PubMed Central

    2014-01-01

    Background Human adipose tissue is an ideal autologous source of mesenchymal stem cells (MSCs) for various regenerative medicine and tissue engineering strategies. Aged patients are one of the primary target populations for many promising applications. It has long been known that advanced age is negatively correlated with an organism’s reparative and regenerative potential, but little and conflicting information is available about the effects of age on the quality of human adipose tissue derived MSCs (hAT-MSCs). Methods To study the influence of age, the expansion and in vitro differentiation potential of hAT-MSCs from young (<30 years), adult (35-50 years) and aged (>60 years) individuals were investigated. MSCs were characterized for expression of the genes p16INK4a and p21 along with measurements of population doublings (PD), superoxide dismutase (SOD) activity, cellular senescence and differentiation potential. Results Aged MSCs displayed senescent features when compared with cells isolated from young donors, concomitant with reduced viability and proliferation. These features were also associated with significantly reduced differentiation potential in aged MSCs compared to young MSCs. Conclusions In conclusion, advancing age negatively impacts stem cell function and such age related alterations may be detrimental for successful stem cell therapies. PMID:24397850

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

    PubMed Central

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

    2004-01-01

    Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. JunBΔ/Δ 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, junBΔ/Δ osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16INK4a 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. PMID:14769860

  16. Avian reovirus nonstructural protein p17-induced G(2)/M cell cycle arrest and host cellular protein translation shutoff involve activation of p53-dependent pathways.

    PubMed

    Chulu, Julius L C; Huang, Wei R; Wang, L; Shih, Wen L; Liu, Hung J

    2010-08-01

    The effects of avian reovirus (ARV) p17 protein on cell cycle progression and host cellular protein translation were studied. ARV infection and ARV p17 transfection resulted in the accumulation of infected and/or transfected cells in the G(2)/M phase of the cell cycle. The accumulation of cells in the G(2)/M phase was accompanied by upregulation and phosphorylation of the G(2)/M-phase proteins ATM, p53, p21(cip1/waf1), Cdc2, cyclin B1, Chk1, Chk2, and Cdc25C, suggesting that p17 induces a G(2)/M cell cycle arrest through activation of the ATM/p53/p21(cip1/waf1)/Cdc2/cyclin B1 and ATM/Chk1/Chk2/Cdc25C pathways. The G(2)/M cell cycle arrest resulted in increased virus replication. In the present study, we also provide evidence demonstrating that p17 protein is responsible for ARV-induced host cellular protein translation shutoff. Increased phosphorylation levels of the eukaryotic translation elongation factor 2 (eEF2) and initiation factor eIF2alpha and reduced phosphorylation levels of the eukaryotic translation initiation factors eIF4E, eIF4B, and eIF4G, as well as 4E-BP1 and Mnk-1 in p17-transfected cells, demonstrated that ARV p17 suppresses translation initiation factors and translation elongation factors to induce host cellular protein translation shutoff. Inhibition of mTOR by rapamycin resulted in a decrease in the levels of phosphorylated 4E-BP1, eIF4B, and eIF4G and an increase in the levels eEF2 but did not affect ARV replication, suggesting that ARV replication was not hindered by inhibition of cap-dependent translation. Taken together, our data indicate that ARV p17-induced G(2)/M arrest and host cellular translation shutoff resulted in increased ARV replication.

  17. Effects of an Indolocarbazole-Derived CDK4 Inhibitor on Breast Cancer Cells

    PubMed Central

    Sun, Yuan; Li, Ying-xia; Wu, Hai-jun; Wu, Si-hung; Wang, Y. Alan; Luo, Dian-zhong; Liao, D. Joshua

    2011-01-01

    Introduction: Cyclin D1 (D1) binds to cyclin-dependent kinases (CDK) 4 or 6 to form a holoenzyme that phosphorylates the Rb protein to promote cell cycle progression from G1 to S phase. Therefore, targeting CDK4/6 may be a good strategy for chemotherapy of cancer. We performed a proof-of-principle study to determine the effect of Naphtho [2, 1-α] pyrrolo [3, 4-c] carbazole-5, 7 (6H, 12H)-dione (NPCD), a novel CDK4 inhibitor, on breast cancer cell lines. Methods: NPCD was synthesized and purified to over 99% purity verified by HPLC. MCF7, MB231, MCF15, T47D and GI101Ap human breast cancer cells were analyzed for the efficacy of NPCD with MTT and clonogenic assays, with FACS and staining for ethidium bromide and acridine orange for cell death and cell cycle profile. Western blot, reverse transcription and PCR were used for studies of gene expression, and co-immunoprecipitation for protein-complex formation. Results: MTT assay showed that NPCD caused growth arrest and apoptosis of MCF7, MDA-MB231, T47D, MCF15 and GI101Ap cells with an IC50 ranging between 3 to 8 µM given as a single dose. The growth arrest persisted for many days after cessation of the treatment, as shown in a clonogenic assay. NPCD could induce or reduce the D1 and CDK4 protein levels, depending on the cell line, but this effect was not correlated with its efficacy. Phosphorylation of D1 at Thr286 was decreased but it unexpectedly did not correlate with the change in D1 level in the cell lines studied. Phosphorylation of the Rb protein was decreased as expected whereas the p27kip1 protein level was decreased unexpectedly. Protein levels of p21cip1, CDK2 and cyclin E were also decreased in some, but not all, of the cell lines, whereas the mRNA levels of D1, CDK4, cyclin E, CDK2, p27kip1 and p21cip1 were increased in different cell lines. Conclusions: NPCD can cause long-lasting growth arrest and cell death of breast cancer cell lines at an IC50 of 3-8 µM. Decreased phosphorylation of Rb by D1-CDK4

  18. 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

  19. CC3/TIP30 affects DNA damage repair

    PubMed Central

    2010-01-01

    Background The pro-apoptotic protein CC3/TIP30 has an unusual cellular function as an inhibitor of nucleocytoplasmic transport. This function is likely to be activated under conditions of stress. A number of studies support the notion that CC3 acts as a tumor and metastasis suppressor in various types of cancer. The yeast homolog of CC3 is likely to be involved in responses to DNA damage. Here we examined the potential role of CC3 in regulation of cellular responses to genotoxic stress. Results We found that forced expression of CC3 in CC3-negative cells strongly delays the repair of UV-induced DNA damage. Exogenously introduced CC3 negatively affects expression levels of DDB2/XPE and p21CIP1, and inhibits induction of c-FOS after UV exposure. In addition, exogenous CC3 prevents the nuclear accumulation of P21CIP in response to UV. These changes in the levels/localization of relevant proteins resulting from the enforced expression of CC3 are likely to contribute to the observed delay in DNA damage repair. Silencing of CC3 in CC3-positive cells has a modest delaying effect on repair of the UV induced damage, but has a much more significant negative affect on the translesion DNA synthesis after UV exposure. This could be related to the higher expression levels and increased nuclear localization of p21CIP1 in cells where expression of CC3 is silenced. Expression of CC3 also inhibits repair of oxidative DNA damage and leads to a decrease in levels of nucleoredoxin, that could contribute to the reduced viability of CC3 expressing cells after oxidative insult. Conclusions Manipulation of the cellular levels of CC3 alters expression levels and/or subcellular localization of proteins that exhibit nucleocytoplasmic shuttling. This results in altered responses to genotoxic stress and adversely affects DNA damage repair by affecting the recruitment of adequate amounts of required proteins to proper cellular compartments. Excess of cellular CC3 has a significant negative

  20. Critical roles of DMP1 in human epidermal growth factor receptor 2/neu-Arf-p53 signaling and breast cancer development.

    PubMed

    Taneja, Pankaj; Maglic, Dejan; Kai, Fumitake; Sugiyama, Takayuki; Kendig, Robert D; Frazier, Donna P; Willingham, Mark C; Inoue, Kazushi

    2010-11-15

    Human epidermal growth factor receptor 2 (HER2) overexpression stimulates cell growth in p53-mutated cells while it inhibits cell proliferation in those with wild-type p53, but the molecular mechanism is unknown. The Dmp1 promoter was activated by HER2/neu through the phosphatidylinositol-3'-kinase-Akt-NF-κB pathway, which in turn stimulated Arf transcription. Binding of p65 and p52 subunits of NF-κB was shown to the Dmp1 promoter and that of Dmp1 to the Arf promoter on HER2/neu overexpression. Both Dmp1 and p53 were induced in premalignant lesions from mouse mammary tumor virus-neu mice, and mammary tumorigenesis was significantly accelerated in both Dmp1+/- and Dmp1-/- mice. Selective deletion of Dmp1 and/or overexpression of Tbx2/Pokemon was found in >50% of wild-type HER2/neu carcinomas, although the involvement of Arf, Mdm2, or p53 was rare. Tumors from Dmp1+/-, Dmp1-/-, and wild-type neu mice with hemizygous Dmp1 deletion showed significant downregulation of Arf and p21Cip1/WAF1, showing p53 inactivity and more aggressive phenotypes than tumors without Dmp1 deletion. Notably, endogenous hDMP1 mRNA decreased when HER2 was depleted in human breast cancer cells. Our study shows the pivotal roles of Dmp1 in HER2/neu-p53 signaling and breast carcinogenesis. PMID:21062982

  1. Dmp1α Inhibits HER2/neu-Induced Mammary Tumorigenesis

    PubMed Central

    Fry, Elizabeth A.; Taneja, Pankaj; Maglic, Dejan; Zhu, Sinan; Sui, Guangchao; Inoue, Kazushi

    2013-01-01

    Our recent study shows a pivotal role of Dmp1 in quenching hyperproliferative signals from HER2 to the Arf-p53 pathway as a safety mechanism to prevent breast carcinogenesis. To directly demonstrate the role of Dmp1 in preventing HER2/neu-driven oncogenic transformation, we established Flag-Dmp1α transgenic mice (MDTG) under the control of the mouse mammary tumor virus (MMTV) promoter. The mice were viable but exhibited poorly developed mammary glands with markedly reduced milk production; thus more than half of parous females were unable to support the lives of new born pups. The mammary glands of the MDTG mice had very low Ki-67 expression but high levels of Arf, Ink4a, p53, and p21Cip1, markers of senescence and accelerated aging. In all strains of generated MDTG;neu mice, tumor development was significantly delayed with decreased tumor weight. Tumors from MDTG;neu mice expressed Flag-Dmp1α and Ki-67 in a mutually exclusive fashion indicating that transgenic Dmp1α prevented tumor growth in vivo. Genomic DNA analyses showed that the Dmp1α transgene was partially lost in half of the MDTG;neu tumors, and Western blot analyses showed Dmp1α protein downregulation in 80% of the cases. Our data demonstrate critical roles of Dmp1 in preventing mammary tumorigenesis and raise the possibility of treating breast cancer by restoring Dmp1α expression. PMID:24205004

  2. DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories.

    PubMed

    Montecucco, A; Rossi, R; Levin, D S; Gary, R; Park, M S; Motycka, T A; Ciarrocchi, G; Villa, A; Biamonti, G; Tomkinson, A E

    1998-07-01

    In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.

  3. 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. PMID:25813896

  4. Inhibition of liver trans-sulphuration pathway by propargylglycine mimics gene expression changes found in the mammary gland of weaned lactating rats: role of glutathione.

    PubMed Central

    Zaragozá, Rosa; García, Concha; Rus, A Diana; Pallardó, Federico V; Barber, Teresa; Torres, Luis; Miralles, Vicente J; Viña, Juan R

    2003-01-01

    In the lactating mammary gland, weaning produces mitochondrial cytochrome c release and nuclear DNA fragmentation, as determined by gel electrophoresis. This is followed by a significant decrease in lactation. Weaning for 2 h produces an early induction of the tumour suppressor/transcription factor p53, whereas the oncoprotein c-Jun and c-Jun N-terminal kinase are elevated after 24 h of weaning when compared with controls. The expression of p21(cip1) and p27(kip1), cyclin-dependent kinase inhibitors, was significantly higher in weaned rats when compared with control lactating rats. All the changes mentioned above also happen in the lactating mammary gland when propargylglycine, an inhibitor of the liver trans-sulphuration pathway, is administered. This effect is partially reversed by N -acetylcysteine administration. The administration of buthionine sulphoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, to lactating rats produces a decrease in GSH levels and changes in protein concentrations and gene transcripts similar to those in rats with impaired trans-sulphuration pathway. These data suggest that the inter-tissue flux of GSH is an important mechanism of L-cysteine delivery to the lactating mammary gland and emphasize the importance of this physiological event in maintaining the gene expression required to sustain lactation. PMID:12723969

  5. 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

  6. Fermented Acanthopanax koreanum Root Extract Reduces UVB- and H2O2-Induced Senescence in Human Skin Fibroblast Cells.

    PubMed

    Park, Min-Ja; Bae, Young-Seuk

    2016-07-28

    The present study assessed the effects of an aqueous extract of Acanthopanax koreanum root (AE) and of AE following fermentation by lactic acid bacteria (Lactobacillus plantarum and Bifidobacterium bifidum) (AEF) on human skin fibroblast HS68 cells exposed to ultraviolet B (UVB) irradiation and oxidative stress. AEF effectively antagonized the senescence-associated β-galactosidase staining and upregulation of p53 and p21(Cip1/WAF1) induced by UVB or H2O2 treatment in HS68 cells. It also exhibited excellent antioxidant activities in radical scavenging assays and reduced the intracellular level of reactive oxygen species induced by UVB or H2O2 treatment. The antioxidant and antisenescent activities of AEF were greater than those of nonfermented A. koreanum extract. AEF significantly repressed the UVB- or H2O2-induced activities of matrix metalloproteinase (MMP)-1 and -3, overexpression of MMP-1, and nuclear factor κB (NF-κB) activation. This repression of NF-κB activation and MMP-1 overexpression was attenuated by a mitogen-activated protein kinase activator, suggesting that this AEF activity was dependent on this signaling pathway. Taken together, these data indicated that AEF-mediated antioxidant and anti-photoaging activities may produce anti-wrinkle effects on human skin. PMID:27090187

  7. 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.

  8. Glutathione biosynthesis is a metabolic vulnerability in PI3K/Akt-driven breast cancer

    PubMed Central

    Lien, Evan C.; Lyssiotis, Costas A.; Juvekar, Ashish; Hu, Hai; Asara, John M.; Cantley, Lewis C.; Toker, Alex

    2016-01-01

    Cancer cells often select for mutations that enhance signaling through pathways that promote anabolic metabolism1. Although the PI3K/Akt signaling pathway, which is frequently dysregulated in breast cancer2, is a well-established regulator of central glucose metabolism and aerobic glycolysis3,4, its regulation of other metabolic processes required for tumor growth is not well defined. Here we report that in mammary epithelial cells, oncogenic PI3K/Akt stimulates glutathione (GSH) biosynthesis by stabilizing and activating Nrf2 to up-regulate the GSH biosynthetic genes. Increased Nrf2 stability is dependent on the Akt-mediated accumulation of p21Cip1/WAF1 and GSK-3 inhibition. Consistently, in human breast tumors, up-regulation of Nrf2 targets is associated with PI3K pathway mutation status and oncogenic Akt activation. Elevated GSH biosynthesis is required for PI3K/Akt-driven resistance to oxidative stress, initiation of tumor spheroids, and anchorage-independent growth. Furthermore, inhibition of GSH biosynthesis with buthionine sulfoximine (BSO) synergizes with cisplatin (CDDP) to selectively induce tumor regression in PI3K pathway mutant breast cancer cells, both in vitro and in vivo. Our findings provide insight into GSH biosynthesis as a metabolic vulnerability associated with PI3K pathway mutant breast cancers. PMID:27088857

  9. Role of RUNX2 in Breast Carcinogenesis

    PubMed Central

    Wysokinski, Daniel; Blasiak, Janusz; Pawlowska, Elzbieta

    2015-01-01

    RUNX2 is a transcription factor playing the major role in osteogenesis, but it can be involved in DNA damage response, which is crucial for cancer transformation. RUNX2 can interact with cell cycle regulators: cyclin-dependent kinases, pRB and p21Cip1 proteins, as well as the master regulator of the cell cycle, the p53 tumor suppressor. RUNX2 is involved in many signaling pathways, including those important for estrogen signaling, which, in turn, are significant for breast carcinogenesis. RUNX2 can promote breast cancer development through Wnt and Tgfβ signaling pathways, especially in estrogen receptor (ER)-negative cases. ERα interacts directly with RUNX2 and regulates its activity. Moreover, the ERα gene has a RUNX2 binding site within its promoter. RUNX2 stimulates the expression of aromatase, an estrogen producing enzyme, increasing the level of estrogens, which in turn stimulate cell proliferation and replication errors, which can be turned into carcinogenic mutations. Exploring the role of RUNX2 in the pathogenesis of breast cancer can lead to revealing new therapeutic targets. PMID:26404249

  10. 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.

  11. Characterization of the cytotoxic activity of [2]rotaxane (TRO-A0001), a novel supramolecular compound, in cancer cells.

    PubMed

    Fujita, Yoshihiko; Kimura, Masahiko; Sato, Hiroki; Takata, Toshikazu; Ono, Nobufumi; Nishio, Kazuto

    2016-06-01

    Rotaxanes comprise a class of interlocked molecules containing a wheel threaded onto an axle with blocking groups on the ends to keep the wheel from sliding off. Here, we show that [2][bis(2-(3,5-dimethylphenylcarbonyloxy)ethyl) ammoniumtrifluoromethanesulfonate]-[dibenzo-24-crown-8] rotaxane (TRO-A0001), a rotaxane compound, exerted a growth inhibitory effect on several human cancer cell lines. An MTT assay revealed an IC50 of 14-830 nM for TRO-A0001 in these cells. Neither the wheel nor the axle part alone inhibited tumor cell growth, suggesting that the complete rotaxane molecule with its unique "intramolecular mobility" is required to inhibit cell growth. Annexin-V/PI staining provided evidence of the induction of apoptosis, which was further confirmed by the observation of poly (ADP-ribose) polymerase cleavage. Furthermore, a cell cycle analysis using flow cytometry showed that TRO-A0001 treatment resulted in G1 arrest in glioblastoma T98G and melanoma G361 cells. An immunoblot analysis revealed that in both cell lines, TRO-A0001 treatment caused the induction of p21/Cip1, thereby down-regulating Cdks 2, 4 and 6 and reducing Cyclins D1 and E. The results presented in this study demonstrate cytotoxicity of the rotaxane compound and its potential as a lead compound for the development of a chemotherapeutic agent against cancer. PMID:27052614

  12. 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.

  13. Ameloblastin in Hertwig's epithelial root sheath regulates tooth root formation and development.

    PubMed

    Hirose, Naoto; Shimazu, Atsushi; Watanabe, Mineo; Tanimoto, Kotaro; Koyota, Souichi; Sugiyama, Toshihiro; Uchida, Takashi; Tanne, Kazuo

    2013-01-01

    Tooth root formation begins after the completion of crown morphogenesis. At the end edge of the tooth crown, inner and outer enamel epithelia form Hertwig's epithelial root sheath (HERS). HERS extends along with dental follicular tissue for root formation. Ameloblastin (AMBN) is an enamel matrix protein secreted by ameloblasts and HERS derived cells. A number of enamel proteins are eliminated in root formation, except for AMBN. AMBN may be related to tooth root formation; however, its role in this process remains unclear. In this study, we found AMBN in the basal portion of HERS of lower first molar in mice, but not at the tip. We designed and synthesized small interfering RNA (siRNA) targeting AMBN based on the mouse sequence. When AMBN siRNA was injected into a prospective mandibular first molar of postnatal day 10 mice, the root became shorter 10 days later. Furthermore, HERS in these mice revealed a multilayered appearance and 5-bromo-2'-deoxyuridine (BrdU) positive cells increased in the outer layers. In vitro experiments, when cells were compared with and without transiently expressing AMBN mRNA, expression of growth suppressor genes such as p21(Cip1) and p27(Kip1) was enhanced without AMBN and BrdU incorporation increased. Thus, AMBN may regulate differentiation state of HERS derived cells. Moreover, our results suggest that the expression of AMBN in HERS functions as a trigger for normal root formation.

  14. From Immunity and Vaccines to Mammalian Regeneration.

    PubMed

    Heber-Katz, Ellen

    2015-07-15

    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 p21(cip1/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

  15. 5-demethyltangeretin inhibits human non-small cell lung cancer cell growth by inducing G2/M cell cycle arrest and apoptosis

    PubMed Central

    Charoensinphon, Noppawat; Qiu, Peiju; Dong, Ping; Zheng, Jinkai; Ngauv, Pearline; Cao, Yong; Li, Shiming; Ho, Chi-Tang; Xiao, Hang

    2013-01-01

    Scope Tangeretin and 5-demethyltangeretin (5DT) are two closely related polymethoxyflavones found in citrus fruits. We investigated growth inhibitory effects on three human non-small cell lung cancer (NSCLC) cells. Methods and results Cell viability assay demonstrated that 5DT inhibited NSCLC cell growth in a time- and dose-dependent manner, and IC50s of 5DT were 79-fold, 57-fold and 56-fold lower than those of tangeretin in A549, H460, and H1299 cells, respectively. Flow cytometry analysis showed that 5DT induced extensive G2/M cell cycle arrest and apoptosis in NSCLC cells, while tangeretin at 10-fold higher concentrations did not. The apoptosis induced by 5DT was further confirmed by activation of caspase-3 and cleavage of PARP. Moreover, 5DT dose-dependently upregulated p53 and p21Cip1/Waf1, and downregulated Cdc-2 (Cdk-1) and cyclin B1. HPLC analysis revealed that the intracellular levels of 5DT in NSCLC cells were 2.7 - 4.9-fold higher than those of tangeretin after the cells were treated with 5DT or tangeretin at the same concentration. Conclusions our results demonstrated that 5DT inhibited NSCLC cell growth by inducing G2/M cell cycle arrest and apoptosis. These effects were much stronger than those produced by tangeretin, which is partially due to the higher intracellular uptake of 5DT than tangeretin. PMID:23926120

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

    PubMed

    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

    2015-03-01

    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. Penta-1,2,3,4,6-O-galloyl-beta-D-glucose induces senescence-like terminal S-phase arrest in human hepatoma and breast cancer cells.

    PubMed

    Yin, Shutao; Dong, Yinhui; Li, Jinhua; Lü, Junxuan; Hu, Hongbo

    2011-08-01

    Senescence is a permanent growth arrest and has been implicated as an efficient anti-carcinogenesis mechanism. The purpose of this study was designed to test the hypothesis that penta-1,2,3,4,6-O-galloyl-beta-D-glucose (PGG), a naturally occurring polyphonolic gallotannin compound, might induce this type of permanent growth arrest in cancer cells. Our results show, for the first time, that PGG-induced senescence-like S-phase arrest in HepG2, Huh-7 human hepatoma cells, and SKBr3 human breast cancer cells at sublethal doses, judged by cellular morphological changes, increased senescence-associated β-galactosidase (SA-β-gal) activity, together with loss of proliferative capacity after being released from the treatment. This senescence-like response was mediated by intracellular ROS generation, but was not attributed to p53 Ser15 phosphorylative activation and was uncoupled from the p21cip1 axis, which has been shown to mediate Pten loss-induced cellular senescence or oncogene-driven senescence. The findings of the present study implicate a novel mechanism of PGG action to induce an atypical cellular senescence, adding to its promise as a potential chemopreventive agent.

  18. Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity

    PubMed Central

    Ullah, Zakir; Kohn, Matthew J.; Yagi, Rieko; Vassilev, Lyubomir T.; DePamphilis, Melvin L.

    2008-01-01

    Genome endoreduplication during mammalian development is a rare event for which the mechanism is unknown. It first appears when fibroblast growth factor 4 (FGF4) deprivation induces differentiation of trophoblast stem (TS) cells into the nonproliferating trophoblast giant (TG) cells required for embryo implantation. Here we show that RO3306 inhibition of cyclin-dependent protein kinase 1 (CDK1), the enzyme required to enter mitosis, induced differentiation of TS cells into TG cells. In contrast, RO3306 induced abortive endoreduplication and apoptosis in embryonic stem cells, revealing that inactivation of CDK1 triggers endoreduplication only in cells programmed to differentiate into polyploid cells. Similarly, FGF4 deprivation resulted in CDK1 inhibition by overexpressing two CDK-specific inhibitors, p57/KIP2 and p21/CIP1. TS cell mutants revealed that p57 was required to trigger endoreduplication by inhibiting CDK1, while p21 suppressed expression of the checkpoint protein kinase CHK1, thereby preventing induction of apoptosis. Furthermore, Cdk2−/− TS cells revealed that CDK2 is required for endoreduplication when CDK1 is inhibited. Expression of p57 in TG cells was restricted to G-phase nuclei to allow CDK activation of S phase. Thus, endoreduplication in TS cells is triggered by p57 inhibition of CDK1 with concomitant suppression of the DNA damage response by p21. PMID:18981479

  19. Cip/Kip cyclin-dependent protein kinase inhibitors and the road to polyploidy

    PubMed Central

    Ullah, Zakir; Lee, Chrissie Y; DePamphilis, Melvin L

    2009-01-01

    Cyclin-dependent kinases (CDKs) play a central role in the orderly transition from one phase of the eukaryotic mitotic cell division cycle to the next. In this context, p27Kip1 (one of the CIP/KIP family of CDK specific inhibitors in mammals) or its functional analogue in other eukarya prevents a premature transition from G1 to S-phase. Recent studies have revealed that expression of a second member of this family, p57Kip2, is induced as trophoblast stem (TS) cells differentiate into trophoblast giant (TG) cells. p57 then inhibits CDK1 activity, an enzyme essential for initiating mitosis, thereby triggering genome endoreduplication (multiple S-phases without an intervening mitosis). Expression of p21Cip1, the third member of this family, is also induced in during differentiation of TS cells into TG cells where it appears to play a role in suppressing the DNA damage response pathway. Given the fact that p21 and p57 are unique to mammals, the question arises as to whether one or both of these proteins are responsible for the induction and maintenance of polyploidy during mammalian development. PMID:19490616

  20. 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

  1. Interactions of ErbB4 and Kap1 Connect the Growth Factor and DNA Damage Response Pathways

    PubMed Central

    Gilmore-Hebert, Maureen; Ramabhadran, Rajani; Stern, David F.

    2014-01-01

    ErbB4 is unusual among receptor tyrosine kinases because some isoforms can be efficiently cleaved at the plasma membrane to release a soluble intracellular domain. The cleavage product has high kinase activity and homes to the nucleus. A screen for proteins that associate with the ErbB4 intracellular domain identified candidate interactors including ITCH, WWP2, Nucleolin, and Krab-associated protein 1 (Kap1). Kap1 binds to multiple isoforms of ErbB4 but does not require ErbB4 kinase activity for binding, nor is it an ErbB4 substrate. Kap1 reduces ERBB4 transcription and either directly or indirectly modulates the expression of genes that are themselves regulated by ErbB4. Upregulation of ErbB4 and suppression of MDM2 jointly enhance and accelerate the accumulation of p21CIP1 in response to DNA damage. Overall, these findings further substantiate the role of ErbB4 in conjoint regulation of growth factor signaling and DNA damage responses. PMID:20858735

  2. Clioquinol induces DNA double-strand breaks, activation of ATM, and subsequent activation of p53 signaling.

    PubMed

    Katsuyama, Masato; Iwata, Kazumi; Ibi, Masakazu; Matsuno, Kuniharu; Matsumoto, Misaki; Yabe-Nishimura, Chihiro

    2012-09-01

    Clioquinol, a Cu²⁺/Zn²⁺/Fe²⁺ chelator/ionophor, was used extensively in the mid 1900s as an amebicide for treating indigestion and diarrhea. It was eventually withdrawn from the market because of a link to subacute myelo-optic neuropathy (SMON) in Japan. The pathogenesis of SMON, however, is not fully understood. To clarify the molecular mechanisms of clioquinol-induced neurotoxicity, a global analysis using DNA chips was carried out on human neuroblastoma cells. The global analysis and quantitative PCR demonstrated that mRNA levels of p21(Cip1), an inhibitor of cyclins D and E, and of GADD45α, a growth arrest and DNA damage-inducible protein, were significantly increased by clioquinol treatment in SH-SY5Y and IMR-32 neuroblastoma cells. Activation of p53 by clioquinol was suggested, since clioquinol induced phosphorylation of p53 at Ser15 to enhance its stabilization. The phosphorylation of p53 was inhibited by KU-55933, an inhibitor of ataxia-telangiectasia mutated kinase (ATM), but not by NU7026, an inhibitor of DNA-dependent protein kinase (DNA-PK). Clioquinol in fact induced phosphorylation of ATM and histone H2AX, a marker of DNA double-strand breaks (DSBs). These results suggest that clioquinol-induced neurotoxicity is mediated by DSBs and subsequent activation of ATM/p53 signaling. PMID:22627294

  3. The Cyclin-dependent Kinase Inhibitor Dacapo Promotes Genomic Stability during Premeiotic S Phase

    PubMed Central

    Narbonne-Reveau, Karine

    2009-01-01

    The proper execution of premeiotic S phase is essential to both the maintenance of genomic integrity and accurate chromosome segregation during the meiotic divisions. However, the regulation of premeiotic S phase remains poorly defined in metazoa. Here, we identify the p21Cip1/p27Kip1/p57Kip2-like cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) as a key regulator of premeiotic S phase and genomic stability during Drosophila oogenesis. In dap−/− females, ovarian cysts enter the meiotic cycle with high levels of Cyclin E/cyclin-dependent kinase (Cdk)2 activity and accumulate DNA damage during the premeiotic S phase. High Cyclin E/Cdk2 activity inhibits the accumulation of the replication-licensing factor Doubleparked/Cdt1 (Dup/Cdt1). Accordingly, we find that dap−/− ovarian cysts have low levels of Dup/Cdt1. Moreover, mutations in dup/cdt1 dominantly enhance the dap−/− DNA damage phenotype. Importantly, the DNA damage observed in dap−/− ovarian cysts is independent of the DNA double-strands breaks that initiate meiotic recombination. Together, our data suggest that the CKI Dap promotes the licensing of DNA replication origins for the premeiotic S phase by restricting Cdk activity in the early meiotic cycle. Finally, we report that dap−/− ovarian cysts frequently undergo an extramitotic division before meiotic entry, indicating that Dap influences the timing of the mitotic/meiotic transition. PMID:19211840

  4. Critical roles of DMP1 in human epidermal growth factor receptor 2/neu-Arf-p53 signaling and breast cancer development.

    PubMed

    Taneja, Pankaj; Maglic, Dejan; Kai, Fumitake; Sugiyama, Takayuki; Kendig, Robert D; Frazier, Donna P; Willingham, Mark C; Inoue, Kazushi

    2010-11-15

    Human epidermal growth factor receptor 2 (HER2) overexpression stimulates cell growth in p53-mutated cells while it inhibits cell proliferation in those with wild-type p53, but the molecular mechanism is unknown. The Dmp1 promoter was activated by HER2/neu through the phosphatidylinositol-3'-kinase-Akt-NF-κB pathway, which in turn stimulated Arf transcription. Binding of p65 and p52 subunits of NF-κB was shown to the Dmp1 promoter and that of Dmp1 to the Arf promoter on HER2/neu overexpression. Both Dmp1 and p53 were induced in premalignant lesions from mouse mammary tumor virus-neu mice, and mammary tumorigenesis was significantly accelerated in both Dmp1+/- and Dmp1-/- mice. Selective deletion of Dmp1 and/or overexpression of Tbx2/Pokemon was found in >50% of wild-type HER2/neu carcinomas, although the involvement of Arf, Mdm2, or p53 was rare. Tumors from Dmp1+/-, Dmp1-/-, and wild-type neu mice with hemizygous Dmp1 deletion showed significant downregulation of Arf and p21Cip1/WAF1, showing p53 inactivity and more aggressive phenotypes than tumors without Dmp1 deletion. Notably, endogenous hDMP1 mRNA decreased when HER2 was depleted in human breast cancer cells. Our stu