Induction of apoptosis in p16INK4A mutant cell lines by adenovirus-mediated overexpression of p16INK4A protein

The tumor suppressor gene p16INK4A is a cyclin-dependent kinase inhibitor (CDKI) and an important cell cycle regulator. We have previously constructed a recombinant adenovirus which expresses p16 (Adp16) and shown that infection in a variety of human tumor cell lines with this recombinant virus results in high levels of p16INK4A protein expression resulting in cell cycle arrest and loss of cyclin-cdk activity. Furthermore, adenoviral-mediated overexpression of wild-type p16INK4A is more toxic in cancer cells which express mutant forms of p16INK4A compared to cancer cell lines containing endogenous wild-type p16. TUNEL assay and DAPI staining following infection of MDA-MB 231 breast cancer cells with Adp16 indicate that p16INK4A-mediated cytotoxicity was associated with apoptosis. This is supported by studies demonstrating a decrease in cpp32 and cyclinB1 protein levels and induction of poly (ADP-ribose) polymerase (PARP) cleavage following infection of MDA-MB-231 cells with Adp16. These results suggest that gene therapy using Adp16 may be a promising treatment option for human cancers containing alterations in p16 expression.     

Rb independent inhibition of cell growth by p15(INK4B).

The INK4 cyclin dependent kinase inhibitors (CDKI), such as p15(INK4B) and p16(INK4A), block cell cycle progression from G to S phase. This is mediated by inhibition of phosphorylation of proteins, including the retinoblastoma susceptibility protein (Rb), by cyclin dependent kinases. Ectopic over-expression of the p16(INK4A) CDKI can inhibit growth of cell lines depending on Rb status. Cell lines lacking Rb, with few exceptions, are resistant to growth inhibition by p16(INK4A). The effects of ectopic over-expression of p15(INK4B) in cell lines with and without wild type Rb were examined by measuring cell recovery. Proliferation was inhibited in cells lacking Rb as well as in cells with wild type Rb expression. Experiments analyzing the effectiveness of chimeric p15(INK4B)/p16(INK4A) proteins indicated that the Rb independent growth inhibition required N-terminal residues of p15(INK4B). Linker insertion mutation of p15(INK4B) showed that the inhibition was dependent on intact ankyrin structures. Double staining flow cytometry found that the growth inhibition correlated with a decrease in cells in G2/M phases of the cell cycle. These findings are consistent with Rb independent inhibition of the progression from G1 to S caused by overexpression of p15(INK4B).     

A two-stage,p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status

With increasing frequency during serial passage in culture, primary human keratinocytes express p16(INK4A) (p16) and undergo senescence arrest. Keratinocytes engineered to express hTERT maintain long telomeres but typically are not immortalized unless, by mutation or other heritable event, they avoid or greatly reduce p16 expression. We have confirmed that keratinocytes undergo p16-related senescence during growth in culture, whether in the fibroblast feeder cell system or in the specialized K-sfm medium formulation, and that this mechanism can act as a barrier to immortalization following hTERT expression. We have characterized the p16-related arrest mechanism more precisely by interfering specifically with several regulators of cell cycle control. Epidermal, oral mucosal, corneal limbal, and conjunctival keratinocytes were transduced to express a p16-insensitive mutant cdk4 (cdk4(R24C)), to abolish p16 control, and/or a dominant negative mutant p53 (p53DD), to abolish p53 function. Expression of either cdk4(R24C) or p53DD alone had little effect on life span, but expression of both permitted cells to divide 25 to 43 population doublings (PD) beyond their normal limit. Keratinocytes from a p16(+/-) individual transduced to express p53DD alone displayed a 31-PD life span extension associated with selective growth of variants that had lost the wild-type p16 allele. Cells in which both p53 and p16 were nonfunctional divided rapidly during their extended life span but experienced telomere erosion and ultimately ceased growth with very short telomeres. Expression of hTERT in these cells immortalized them. Keratinocytes engineered to express cdk4(R24C) and hTERT but not p53DD did not exhibit an extended life span. Rare immortal variants exhibiting p53 pathway defects arose from them, however, indicating that the p53-dependent component of keratinocyte senescence is telomere independent. Mutational loss of p16 and p53 has been found to be a frequent early event in the development of squamous cell carcinoma. Our results suggest that such mutations endow keratinocytes with extended replicative potential which may serve to increase the probability of neoplastic progression.     

Tumor suppressor INK4: refinement of p16INK4A structure and determination of p15INK4B structure by comparative modeling and NMR data

Within the tumor suppressor protein INK4 (inhibitor of cyclin-dependent kinase 4) family, p15INK4B is the smallest and the only one whose structure has not been determined previously, probably due to the protein's conformational flexibility and instability. In this work, multidimensional NMR studies were performed on this protein. The first tertiary structure was built by comparative modeling with p16INK4A as the template, followed by restrained energy minimization with NMR constraints (NOE and H-bonds). For this purpose, the solution structure of pl6INK4A, whose quality was also limited by similar problems, was refined with additional NMR experiments conducted on an 800 MHz spectrometer and by structure-based iterative NOE assignments. The nonhelical regions showed major improvement with root-mean-square deviation (RMSD) improved from 1.23 to 0.68 A for backbone heavy atoms. The completion of p15INK4B coupled with refinement of p16INK4A made it possible to compare the structures of the four INK4 members in depth, and to compare the structures of p16INK4A in the free form and in the p16INK4A-CDK6 complex. This is an important step toward a comprehensive understanding of the precise functional roles of each INK4 member.     

Retinoic acid inhibits the growth of bone marrow mesenchymal stem cells and induces p27Kip1 and p16INK4A up-regulation

The importance of bone marrow mesenchymal stem cells in hemopoiesis has been definitely demonstrated. Thus, their impairment might cause profound alteration on production and maturation of blood cells. In the present paper, we investigated, for the first time, the effect of retinoic acid, an important antileukemic molecule, on the proliferation of primary cultures of human bone marrow mesenchymal stem cells. We demonstrated that retinoic acid, at a pharmacological concentration, hampers strongly the growth of the cells, without inducing osteoblastic differentiation. The analysis of cell division cycle machinery showed that the antiproliferative effect is associated with (i) the up-regulation of two cyclin-dependent kinase inhibitors, namely p27^Kip1 and p16^INK4A, and (ii) the down-regulation of cyclin-dependent kinase 2 activity and pRB phosphorylation. The reported findings represent novel insights into the antileukemic effects of the drug and contribute in clarifying the molecular mechanism of its pharmacological activity.     

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

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

p16(INK4A) immunohistochemical overexpression in premalignant and malignant oral lesions infected with human papillomavirus.

Human papillomavirus (HPV) is believed to promote the oncogenic process, and the correlation between viral oncoproteins and dysfunction of p16(INK4A) tumor suppressor protein in oral lesions is controversial. To test the hypothesis that anogenital HPV types participate in disruption of the regulation of p16(INK4A) suppressor protein in oral lesions, we analyzed 46 oral biopsy specimens for the presence of HPV 6/11 and 16/18 by in situ hybridization (ISH) and for p16(INK4A) expression by immunohistochemistry (IHC). Eighteen (39%) of the 46 oral lesions were HPV-positive and 28 (61%) were HPV-negative. HPV 6/11 DNA was found in 5 (11%) and HPV 16/18 in 13 (28%) of 46 biopsies. Nine of the 18 HPV-positive oral lesions (50%), assessed by catalyzed signal amplification coupled to ISH (CSA-ISH), gave high-intensity p16(INK4A) immunostaining. Focal and diffuse patterns were observed in 11/13 (77%) lesions with HPV 16/18, focal immunopositivity in 3/5 (80%) with HPV 6/11, and negative or sporadic p16-labeling in 18/28 (64%) without the presence of HPV DNA. These results showed a strong association between overexpression of p16 protein and malignant oral lesions, mainly those infected by HPV 16/18. We can conclude that high-risk HPV types are associated with p16 overexpression, and p16 may serve as a biomarker in oral cancer related to high-risk HPV infection.     

Pak4 induces premature senescence via a pathway requiring p16INK4/p19ARF and mitogen-activated protein kinase signaling

Exposure of primary cells to mitogenic stimuli or oncogenes often causes them to undergo premature senescence. This is most likely a protective function that prevents uncontrolled proliferation. Pak4 is a target for the Rho GTPase Cdc42. Pak4 is overexpressed in human tumor cell lines, and it is the only member of the Pak family that is highly transforming in immortalized fibroblasts. Here we show that in primary fibroblasts, activated Pak4 inhibits cell proliferation and promotes premature senescence. Furthermore, Pak4 expression levels are upregulated in response to stimuli that promote senescence. Pak4-induced arrest appears to be mediated by a pathway that requires the ERK mitogen-activated protein kinase, as well as the cell cycle inhibitors p16^INK4 and p19^ARF. These new results describing a role for Pak4 in senescence are important for understanding why this protein is associated with cancer and how it promotes transformation in immortalized cells.     

Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation

Normal human somatic cells have a finite life span and undergo replicative senescence after a limited number of cell divisions. Erosion of telomeric DNA has emerged as a key factor in senescence, which is antagonized during cell immortalization and transformation. To clarify the involvement of telomerase in the immortalization of keratinocytes, catalytic subunit of telomerase (hTERT) expression was restored in normal human esophageal epithelial cells (EPC2). EPC2-hTERT cells overcame senescence and were immortalized without p16INK4a genetic or epigenetic alterations. p16INK4a was expressed at moderate levels and remained functional as evidenced by induction with UV treatment and binding to cyclin-dependent kinase 4 and 6. There were no mutations in the p53 gene, and p53 was functionally intact. Importantly, senescence could be activated in the immortalized EPC2-hTERT cells by overexpression of oncogenic H-ras or p16INK4a. Furthermore, the EPC2-hTERT cells yielded basal cell hyperplasia in an innovative organotypic culture system in contrast to a normal epithelium from parental cells. These comprehensive results indicate that the expression of telomerase induces immortalization of normal human esophageal keratinocytes without inactivation of p16INK4a/pRb pathway or abrogation of the p53 pathway.     

A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy

p53 and INK4a/ARF mutations promote tumorigenesis and drug resistance, in part, by disabling apoptosis. We show that primary murine lymphomas also respond to chemotherapy by engaging a senescence program controlled by p53 and p16(INK4a). Hence, tumors with p53 or INK4a/ARF mutations-but not those lacking ARF alone-respond poorly to cyclophosphamide therapy in vivo. Moreover, tumors harboring a Bcl2-mediated apoptotic block undergo a drug-induced cytostasis involving the accumulation of p53, p16(INK4a), and senescence markers, and typically acquire p53 or INK4a mutations upon progression to a terminal stage. Finally, mice bearing tumors capable of drug-induced senescence have a much better prognosis following chemotherapy than those harboring tumors with senescence defects. Therefore, cellular senescence contributes to treatment outcome in vivo.     

Retroviral transfer of the p16INK4a cDNA inhibits C6 glioma formation in Wistar rats

BACKGROUND: The p16INK4A gene product halts cell proliferation by preventing phosphorylation of the Rb protein. The p16INK4a gene is often deleted in human glioblastoma multiforme, contributing to unchecked Rb phosphorylation and rapid cell division. We show here that transduction of the human p16INK4a cDNA using the pCL retroviral system is an efficient means of stopping the proliferation of the rat-derrived glioma cell line, C6, both in tissue culture and in an animal model. C6 cells were transduced with pCL retrovirus encoding the p16INK4a, p53, or Rb genes. These cells were analyzed by a colony formation assay. Expression of p16INK4a was confirmed by immunohistochemistry and Western blot analysis. The altered morphology of the p16-expressing cells was further characterized by the senescence-associated beta-galactosidase assay. C6 cells infected ex vivo were implanted by stereotaxic injection in order to assess tumor formation. RESULTS: The p16INK4a gene arrested C6 cells more efficiently than either p53 or Rb. Continued studies with the p16INK4a gene revealed that a large portion of infected cells expressed the p16INK4a protein and the morphology of these cells was altered. The enlarged, flat, and bi-polar shape indicated a senescence-like state, confirmed by the senescence-associated beta-galactosidase assay. The animal model revealed that cells infected with the pCLp16 virus did not form tumors. CONCLUSION: Our results show that retrovirus mediated transfer of p16INK4a halts glioma formation in a rat model. These results corroborate the idea that retrovirus-mediated transfer of the p16INK4a gene may be an effective means to arrest human glioma and glioblastoma.