BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase ( TERT ) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT . However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16 -specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION P16 methylation drives TERT -mediated immortalization and transformation of normal human cells that may contribute to cancer development.
Humans ; Telomerase/genetics* ; DNA Methylation/physiology* ; Fibroblasts/cytology* ; Cyclin-Dependent Kinase Inhibitor p16/metabolism* ; Cell Line ; Cell Transformation, Neoplastic/genetics*

OBJECTIVETo determine whether testosterone modulates markers of cardiomyocytes aging via its classic androgen receptor (AR)-dependent pathway or conversion to estradiol.

METHODSMale littermates and testicular feminized (Tfm) mice were randomly separated into 4 experimental groups littermate controls (n=8), Tfm mice (n=7), testosterone-treated Tfm mice (n=8), and Tfm mice treated with testosterone in combination with the aromatase inhibitor anastrazole (n=7). Cardiomyocytes were isolated from mouse left ventricles, the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the amount of malondialdehyde (MDA) were measured using colorimetry method, and expression of p16(INK4α) and retinoblastoma (Rb) proteins were detected by Western blotting.

RESULTSThe SOD and GSH-Px enzyme activities of cardiomyocytes were decreased, and the MDA levels and the expression of p16(INK4α) and Rb proteins were increased in Tfm mice compared with control mice. An increase was observed in the activities of SOD and GSH-Px enzyme as well as a decrease in MDA levels and the expression of p16(INK4α) and Rb proteins in the testosterone-treated Tfm mice. After co-treatment with anastrazole in Tfm mice, these improvement were partly inhibited.

CONCLUSIONPhysiological testosterone replacement can delay cardiomyocyte aging in Tfm mice, an effect that is independent of the AR pathway and in part conversion to estradiol.

Androgen-Insensitivity Syndrome ; metabolism ; Animals ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Female ; Glutathione Peroxidase ; metabolism ; Male ; Mice ; Myocytes, Cardiac ; physiology ; Receptors, Androgen ; physiology ; Superoxide Dismutase ; metabolism ; Testosterone ; physiology

Cellular senescence is an irreversible cell cycle arrest triggered by the activation of oncogenes or mitogenic signaling as well as the enforced expression of tumor suppressors such as p53, p16(INK4A) and promyelocytic leukemia protein (PML) in normal cells. E2F-binding protein 1 (E2FBP1), a transcription regulator for E2F, induces PML reduction and suppresses the formation of PML-nuclear bodies, whereas the down-regulation of E2FBP1 provokes the PML-dependent premature senescence in human normal fibroblasts. Here we report that the depletion of E2FBP1 induces the accumulation of PML through the Ras-dependent activation of MAP kinase signaling. The cellular levels of p16(INK4A) and p53 are elevated during premature senescence induced by depletion of E2FBP1, and the depletion of p16(INK4A), but not p53 rescued senescent cells from growth arrest. Therefore, the premature senescence induced by E2FBP1 depletion is achieved through the p16(INK4A)-Rb pathway. Similar to human normal fibroblasts, the growth inhibition induced by E2FBP1 depletion is also observed in human tumor cells with intact p16(INK4A) and Rb. These results suggest that E2FBP1 functions as a critical antagonist to the p16(INK4A)-Rb tumor suppressor machinery by regulating PML stability.
Cell Line, Tumor ; Cells, Cultured ; Cellular Senescence ; genetics ; physiology ; Cyclin-Dependent Kinase Inhibitor p16 ; antagonists & inhibitors ; genetics ; physiology ; DNA-Binding Proteins ; deficiency ; genetics ; physiology ; Down-Regulation ; Fibroblasts ; Gene Expression Regulation ; Humans ; Intranuclear Inclusion Bodies ; metabolism ; MAP Kinase Signaling System ; Nuclear Proteins ; genetics ; metabolism ; physiology ; Promyelocytic Leukemia Protein ; Protein Isoforms ; Protein Stability ; RNA Interference ; Retinoblastoma Protein ; antagonists & inhibitors ; genetics ; physiology ; Transcription Factors ; deficiency ; genetics ; metabolism ; physiology ; Transfection ; Tumor Suppressor Protein p53 ; physiology ; Tumor Suppressor Proteins ; genetics ; metabolism ; physiology ; Ubiquitination ; ras Proteins ; metabolism

BACKGROUND AND OBJECTIVEPrevious studies have shown that Bmi-1 is overexpressed in a variety of tumors, suggesting that Bmi-1 plays an important role in tumorigenesis. In this study, we investigated the effect of Bim-1 siRNA on cell proliferation, cell cycle, cell apoptosis and migration of human esophageal carcinoma EC9706 cells, and explored its potential mechanisms.

METHODSBmi-1 small interfering RNA (siRNA) was transferred into EC9706 cells. Then, cell proliferation was measured using cell counting kit-8 (CCK-8), cell cycle and cell apoptosis were analyzed by flow cytometry, cell migration ability was detected using Boyden chamber assay, and the mRNA and protein expression levels of Bmi-1, p16, Bcl-2, Bax, and MMP-2 were determined using real-time polymerase chain reaction (PCR) and Western blot analysis, respectively.

RESULTSBmi-1 siRNA treatment significantly inhibited the expression of Bmi-1 at both mRNA and protein levels in EC9706 cells. Cell proliferation rate decreased dramatically in the Bmi-1 siRNA treated group than in the untreated group and in the scrambled siRNA treated group (both P < 0.001). In Bmi-1 treated group, the percentage of cells at G(0)/G(1) stage was 71.93%, which was higher than that in the untreated group (47.36%) or scramble siRNA treated group (48.47%) (both P < 0.001). Early cell apoptosis rate also increased significantly in the Bmi-1 siRNA treated group (both 17.32%) than in the untreated group (2.61%) and in the scramble siRNA treated group (2.73%) (both P < 0.001). Further experiment suggested that downregulation of Bmi-1 led to less cell migration. In EC9706 cells transfected by Bmi-1 siRNA, the expression levels of p16 and Bax increased, while the expression level of Bcl-2 decreased.

CONCLUSIONSBmi-1 downregulation in esophageal carcinoma cells inhibits cell proliferation, cell cycle, and cell migration, while increases cell apoptosis. These results suggest that Bmi-1 is a potential molecular target of treating esophageal cancer.

Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Down-Regulation ; Esophageal Neoplasms ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Nuclear Proteins ; genetics ; metabolism ; physiology ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; genetics ; metabolism ; physiology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Repressor Proteins ; genetics ; metabolism ; physiology ; Transfection ; bcl-2-Associated X Protein ; metabolism

The expression of tumor suppressor gene p16INK4a in mouse endometrium during early pregnancy and its possible role in blastocyst implantation were investigated in the present study. Real-time fluorescent quantitative PCR (FQ-PCR) and immunohistochemistry were applied to detect p16INK4a mRNA and protein expressions in endometrium of un-pregnant and pregnant mice on day 2, 3, 4, 5, 7, respectively. In addition, p16INK4a antibody was injected into the horns of uteri in pregnant mice on day 3 and its effect during blastocyst implantation was detected in vivo. The higher expressions of p16INK4a mRNA and protein were observed in pregnant mice compared with that in un-pregnant mice, with a steady increase from day 2 to day 5 and reaching the maximal level on day 5 of pregnancy and then decreasing. p16INK4a antibody decreased the number of implanted blastocysts compared with that of saline-injected group. The results suggest that p16INK4a may be associated with apoptosis of luminal epithelial cells and decidual cells, coordinating decidualization of endometrium and invasion of trophoblastic cells. Thus, we presume that p16INK4a participates in the process of blastocyst implantation in mice.
Animals ; Blastocyst ; physiology ; Cyclin-Dependent Kinase Inhibitor p16 ; physiology ; Embryo Implantation ; Endometrium ; physiology ; Female ; Immunohistochemistry ; Mice ; Pregnancy ; RNA, Messenger ; Real-Time Polymerase Chain Reaction

BACKGROUNDBoth p16(INK4) and p21(Waf1) are tumor suppressors with similar biological functions in the regulation of cellular senescence. Previous reports showed that p16(INK4) could be activated by p21(Waf1) through transcriptional factor Sp1 in HeLa cells. This study was undertaken to determine the effects of p16(INK4) on the expression and functions of p21(Waf1).

METHODSHuman diploid fibroblast 2BS cells were stably transfected with sense (2BS/p16(INK4)), antisense p16(INK4) (2BS/asp16(INK4)) or empty vector (2BS/neo). Then they were assayed by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence activated cell sorting (FACS) and Western blot.

RESULTS2BS/p16(INK4) cells exhibited cell cycle arrest in both G1 and G2/M phases. Endogenous p21(Waf1) protein levels increased twofold in the 2BS/p16(INK4) cells, but not decreased in the 2BS/asp16(INK4) cells. p21(Waf1) mRNA levels were not affected in neither 2BS/p16(INK4) nor 2BS/asp16(INK4) cells.

CONCLUSIONp16(INK4) may play an important role in the regulation of cellular senescence by modulating the p21(Waf1) protein level via the posttranscriptional mechanism.

Cell Cycle ; Cells, Cultured ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; physiology ; Cyclin-Dependent Kinase Inhibitor p21 ; physiology ; Fibroblasts ; metabolism ; Humans ; Transcription, Genetic

OBJECTIVETo confirm the relations between the expression of cyclin E, p16ink4, ki67 and HPV16/18 infection using cervical exfoliated cells, and evaluate the usefulness of cyclin E, p16ink4 and ki67 as biomarkers for screening of cervical carcinomas.

METHODSThe expression of cyclin E, p16ink4 oncoproteins and ki67 proliferative activity was evaluated immunohistochemically in 78 cervical exfoliated epithelial specimens. Human papillomavirus type16 and 18 (HPV16/18) infection was assessed by polymerase chain reaction (PCR) using type specific primers.

RESULTSCyclin E, p16ink4 and ki67 were all overexpressed in cervical preneoplasia and neoplasia cells, compared with little expressed in ASCUS (P less than 0.005). Overexpression of cyclin E was observed in CIN, (P less than 0.01), p16ink4 and ki67 overexpressed in invasive carcinoma(100 percent and 90.9 percent respectively). The degree of p16ink4 and ki67 expression correlated well with the degree of cervical neoplasia (P less than 0.005). HPV16 infection was assessed at all stages of cervical neoplasia samples, and a significant relationship with the degree of cervical epithelial lession was observed at the same time. The expression level of p16ink4 and ki67 seemed to be more closely associated with HPV16 infection than cyclin E did (rs=1.0 vs rs=0.4). HPV18 was found positive in only 1 case in CIN1 and in 4 cases in CIN2-3. Therefore no significance was found on statistical analysis (P less than 0.005).

CONCLUSIONCyclin E, p16ink4 and ki67 should be regarded as useful biomarkers of HPV-related cervical neoplasias, and be used for screening patients at high risk for developing cervical carcinomas. Moreover, cyclin E might be a significant cytologic marker for the primary screening of cervical carcinomas.

Adult ; Aged ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; virology ; Cervix Uteri ; cytology ; metabolism ; virology ; Cyclin E ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; DNA, Viral ; genetics ; Female ; Host-Pathogen Interactions ; Human papillomavirus 16 ; genetics ; physiology ; Human papillomavirus 18 ; genetics ; physiology ; Humans ; Immunohistochemistry ; Ki-67 Antigen ; biosynthesis ; Middle Aged ; Papillomavirus Infections ; metabolism ; pathology ; virology ; Polymerase Chain Reaction ; Uterine Cervical Neoplasms ; metabolism ; pathology

BACKGROUNDBmi-1 gene determines the proliferative capacity of normal and leukemia stem cells. Expression of Bmi-1 has been found in all types of myeloid leukemia cells in both humans and mice. This study aimed at assessing the effect of antisense Bmi-1 expression on K562 cells proliferation and p16 protein (p16) expression.

METHODSA transcriptional repressor, Bmi-1 cDNA was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) of its mRNA from K562 cells. A plasmid expressing antisense Bmi-1 mRNA was then constructed by reverse design of PCR primers and cloned to the plasmid pLNCX2; G418 was added to the medium after the plasmid was successfully introduced in K562 cells by lipofectin-mediated DNA transfection. The effects of the antisense expression on the proliferation of K562 cells were analyzed by using microculture tetrazolium and colony forming. Cell cycle was analyzed by using flow cytometry. The p16 expression of K562 cells was observed by immunofluorescence histochemical stain.

RESULTSK562 cells transfected with antisense Bmi-1 plasmid grew significantly slower than that of controls (the parental K562 and cells transfected with empty plasmid). The colony forming ability of antisense Bmi-1 plasmid transfected cells decreased significantly (P < 0.01) compared with controls. The p16 expression of cells transfected with antisense Bmi-1 was upgraded more apparently than that of controls.

CONCLUSIONThe antisense Bmi-1 gene can inhibit the growth of K562 cell and upgrade expression of p16 in K562 cells.

Cell Cycle ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Humans ; K562 Cells ; Nuclear Proteins ; antagonists & inhibitors ; genetics ; Plasmids ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; antagonists & inhibitors ; genetics ; RNA, Antisense ; physiology ; Repressor Proteins ; antagonists & inhibitors ; genetics

Carcinoma of the uterine cervix is one of the most common malignancies among women worldwide. Human papillomaviruses (HPV) have been identified as the major etiological factor in cervical carcinogenesis. However, the time lag between HPV infection and the diagnosis of cancer indicates that multiple steps, as well as multiple factors, may be necessary for the development of cervical cancer. The development and progression of cervical carcinoma have been shown to be dependent on various genetic and epigenetic events, especially alterations in the cell cycle checkpoint machinery. In mammalian cells, control of the cell cycle is regulated by the activity of cyclin-dependent kinases (CDKs) and their essential activating coenzymes, the cyclins. Generally, CDKs, cyclins, and CDK inhibitors function within several pathways, including the p16INK4A-cyclin D1-CDK4/6-pRb-E2F, p21WAF1-p27KIP1-cyclinE-CDK2, and p14ARF-MDM2-p53 pathways. The results from several studies showed aberrant regulation of several cell cycle proteins, such as cyclin D, cyclin E, p16 INK4A, p21WAF1, and p27KIP1, as characteristic features of HPV- infected and HPV E6/E7 oncogene-expressing cervical carcinomas and their precursors. These data suggested further that interactions of viral proteins with host cellular proteins, particularly cell cycle proteins, are involved in the activation or repression of cell cycle progression in cervical carcinogenesis.
Uterine Cervical Neoplasms/*pathology ; Tumor Suppressor Protein p53/physiology ; Tumor Suppressor Protein p14ARF/physiology ; Retinoblastoma Protein/physiology ; Proto-Oncogene Proteins c-mdm2/physiology ; Humans ; Female ; E2F Transcription Factors/physiology ; Cyclin-Dependent Kinase Inhibitor p27/physiology ; Cyclin-Dependent Kinase Inhibitor p21/physiology ; Cyclin-Dependent Kinase Inhibitor p16/physiology ; Cyclin-Dependent Kinase 4/physiology ; Cyclin-Dependent Kinase 2/physiology ; Cyclin E/physiology ; Cyclin D1/physiology ; Cell Cycle/*physiology

OBJECTIVETo construct E1-deletion and replication-defective human type 5 recombinant adenovirus vector and to study the effect of p16INK4a on proliferation and aging of A549 cells.

METHODSp16INK4a cDNA was cloned into pAdCMV to construct recombinant pAdCMV p16INK4a, which was co-transfected into 293 cell together with pJM17. The recombinant p16INK4a adenovirus (Ad-p16INK4a) was generated by homologous recombination and identified with duplex PCR. Lung cancer cell A549, which has a homozygous deletion of p16INK4a gene, was infected with the prepared Ad-p16INK4a virus. X-gal staining and TRAP-ELISA were used for detecting senescence-associated beta-galactosidase and telomerase activities in A549 cells.

RESULTSImmunohistochemical staining and Western blot indicated that p16INK4a gene was transferred into A549 cell with more than 95% efficiency by recombinant adenovirus and p16INK4a protein was expressed at a high level- p16INK4a could markedly inhibit growth of A549 cells, induced expression of senescence-associated beta-galactosidase and suppressed telomerase activity in A549 cells.

CONCLUSIONRecombinant adenovirus vector could efficiently mediate transfer and expression of foreign genes in human cell and could be used for gene immunization and gene therapy; p16INK4a could inhibit A549 cell growth and induce its replicative senescence.

Adenoviridae ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; physiology ; Gene Expression ; Genetic Vectors ; Humans ; Recombination, Genetic ; Transfection