Significant role for p16INK4a in p53-independent telomere-directed senescence

Telomere attrition in primary human fibroblasts induces replicative senescence accompanied by activation of the p53 and p16(INK4a)/RB tumor suppressor pathways. Although the contribution of p53 and its target, p21, to telomere-driven senescence have been well established, the role of p16(INK4a) is controversial. Attempts to dissect the significance of p16(INK4a) in response to telomere shortening have been hampered by the concomitant induction of p16(INK4a) by cell culture conditions. To circumvent this problem, we studied the role of p16(INK4a) in the cellular response to acute telomere damage induced by a dominant negative allele of TRF2, TRF2(Delta B Delta M). This approach avoids the confounding aspects of culture stress because parallel cultures with and without telomere damage can be compared. Telomere damage generated with TRF2(Delta B Delta M) resulted in induction of p16(INK4a) in the majority of cells as detected by immunohistochemistry. Inhibition of p16(INK4a) with shRNA or overexpression of BMI1 had a significant effect on the telomere damage response in p53-deficient cells. While p53 deficiency alone only partially abrogated the telomere damage-induced cell cycle arrest, combined inhibition of p16(INK4a) and p53 led to nearly complete bypass of telomere-directed senescence. We conclude that p16(INK4a) contributes to the p53-independent response to telomere damage.     

MYCL1, FHIT,SPARC, p16(INK4) and TP53 genes associated to lung cancer in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic interstitial pneumonia limited to the lung and characterized by a fibroproliferative response with only minor signs of inflammation, which almost always causes rapid fibrotic destruction of the lung. In this study, we investigated genomic instability in IPF, using microsatellite DNA analysis, aiming to detect any specific genetic alterations for this disease. We used 40 highly polymorphic microsatellite DNA markers, in multiplex PCR assays, to examine 52 sputum specimens from IPF patients versus correspondent venous blood. Loss of heterozygosity (LOH) was found in 20 (38.5%) patients in at least one locus. These alterations were found on markers previously associated with lung cancer located on 1p34.3, 3p21.32-p21.1, 5q32-q33.1, 9p21 and 17p13.1 where MYCL1, FHIT, SPARC, p^16Ink4 and TP53 genes have been mapped respectively. These data provide new insights into IPF pathogenesis and a new perspective for its correlation with lung cancer.     

Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a)

Cellular senescence can be triggered by telomere shortening as well as a variety of stresses and signaling imbalances. We used multiparameter single-cell detection methods to investigate upstream signaling pathways and ensuing cell cycle checkpoint responses in human fibroblasts. Telomeric foci containing multiple DNA damage response factors were assembled in a subset of senescent cells and signaled through ATM to p53, upregulating p21 and causing G1 phase arrest. Inhibition of ATM expression or activity resulted in cell cycle reentry, indicating that stable arrest requires continuous signaling. ATR kinase appears to play a minor role in normal cells but in the absence of ATM elicited a delayed G2 phase arrest. These pathways do not affect expression of p16, which was upregulated in a telomere- and DNA damage-independent manner in a subset of cells. Distinct senescence programs can thus progress in parallel, resulting in mosaic cultures as well as individual cells responding to multiple signals.     

p16INK4A在黑素瘤中的研究进展

黑素瘤是人类恶性皮肤肿瘤之一,对药物治疗不敏感且死亡率高.近年来随着分子生物学的发展,人们对黑素瘤的研究在基因水平取得了很大进步.p16INK4A作为一种多肿瘤抑制基因,与黑素瘤的发生、发展有关.P16 INK4A在黑素瘤中的失活形式主要是缺失、突变和甲基化,进一步明确p16INK4A基因的失活机制及其与黑素瘤的相关性,可能为黑素瘤的临床诊断、治疗和判断预后等方面提供一种新路径.     

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.     

Possible involvement of p21 but not of p16 or p53 in keratinocyte senescence

It has been reported that p21, p53, and p16 affect the cell cycle and cell senescence. However, their roles in keratinocyte senescence are not clear. We established primary keratinocyte strains from 15 donors and maintained them until replicative senescence; their population doublings ranged from 5.7-45.2. These strains were classified based on their population doublings as short (5.7-10.4), intermediate (13.9-17.4), and long (21.5-45.2). To investigate the roles of p21, p53, and p16 in the cellular senescence of the cultured keratinocytes, we quantitatively analyzed p21, p53, and p16 levels of keratinocyte strains with different life spans by Western blot with Fluorol mager. p21 levels increased in the senescent phase but not in the nonsenescent phase in all of the short, intermediate, and long life-span strains. Northern blot analysis also revealed induction of p21 mRNA was similar to that of p21 protein levels. There were no apparent differences in p53 levels between senescent and nonsenescent cells. The short life-span strains exhibited a significant increase in p16 levels in the senescent phase (eighth or tenth passage). However, in two long life-span strains, p16 levels were increased in the nonsenescent phase (eighth passage) but then declined as the cells reached senescence (twenty-seventh passage). Therefore, induction of p16 appeared not to be associated with senescence in long life-span strains. In conclusion, p21 but not p16 or p53 may play roles in keratinocyte senescence.     

p53 checkpoint-defective cells are sensitive to X rays, but not hypoxia

X-ray-induced damage leads to cell-cycle "checkpoint" arrest by p53-dependent induction of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1). Human tumor cells that lack this response fail to arrest after exposure to DNA-damaging agents, undergo multiple rounds of endoreduplicative DNA synthesis, and eventually commit to an apoptotic cell death. Since low oxygen tension can also induce p53 protein accumulation, and can lead to cell-cycle arrest or apoptosis, we examined the expression of p21 in tumor cells under normoxic and hypoxic conditions. In a survey of cells, mRNA for the p21 gene was induced two- to threefold in response to hypoxia in a seemingly p53-independent manner. We therefore examined genetically matched cells that differ in their p21 and p53 status for response to ionizing radiation and hypoxia. We found that both p21-deficient and p53-deficient cells exhibit an increase in chromosome instability, an increased level of apoptosis, and a failure to arrest after exposure to ionizing radiation. However, cells that lack either p21 or p53 exhibit no increase in chromosome instability or elevated apoptosis and still arrest in response to hypoxia. Thus, the mechanism responsible for the differential response to either hypoxia or X rays presumably lies in the control of cell-cycle progression in response to stress and its dependence on p21. Since the loss of a DNA-damage-dependent checkpoint does not sensitize cells to killing by stresses that elicit a DNA-damage-independent checkpoint, targeting the function of p21 pharmacologically will not kill tumor cells in situ in the absence of a DNA damage signal.     

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.     

Tumor suppressor INK4: determination of the solution structure of p18INK4C and demonstration of the functional significance of loops in p18INK4C and p16INK4A

Since the structures of several ankyrin-repeat proteins including the INK4 (inhibitor of cyclin-dependent kinase 4) family have been reported recently, the detailed structures and the functional roles of the loops have drawn considerable interest. This paper addresses the potential importance of the loops of ankyrin-repeat proteins in three aspects. First, the solution structure of p18INK4C was determined by NMR, and the loop structures were analyzed in detail. The loops adapt nascent antiparallel beta-sheet structures, but the positions are slightly different from those in the crystal structure. A detailed comparison between the solution structures of p16 and p18 has also been presented. The determination of the p18 solution structure made such detailed comparisons possible for the first time. Second, the [1H,15N]HSQC NMR experiment was used to probe the interactions between p18INK4C and other proteins. The results suggest that p18INK4C interacts very weakly with dna K and glutathione S-transferase via the loops. The third aspect employed site-specific mutagenesis and functional assays. Three mutants of p18 and 11 mutants of p16 were constructed to test functional importance of loops and helices. The results suggest that loop 2 is likely to be part of the recognition surface of p18INK4C or p16INK4A for CDK4, and they provide quantitative functional contributions of specific residues. Overall, our results enhance understanding of the structural and functional roles of the loops in INK4 tumor suppressors in particular and in ankyrin-repeat proteins in general.     

Phosphorylation of p16INK4A correlates with Cdk4 association

Progression through the eukaryotic cell cycle is driven by the activity of cyclin-dependent kinases. The cyclin D-dependent kinase Cdk4 promotes progression through the G(1) phase of the cell cycle and is deregulated in many human tumors. The tumor suppressor protein p16(INK4A) (p16) forms a complex with Cdk4 and inhibits kinase activity. Here we report that p16 is phosphorylated, and the phosphorylated form of p16 is preferentially associated with Cdk4 in normal human fibroblasts. We mapped phosphorylation sites on exogenously overexpressed p16 to serines 7, 8, 140, and 152 and found that endogenous p16 associated with Cdk4 is phosphorylated at serine 152. All mapped phosphorylation sites lie outside of the conserved kinase-binding domain of p16 but in regions of the protein affected by mutations in familial and sporadic cancer. Our results suggest a novel regulation of p16 activity.     

p16INK4a基因的功能及其调控

p16INK4a蛋白能抑制CDK4和CDK6的活性,使pRb处于非磷酸化或低磷酸化状态而能与转录因子E2Fs结合,从而抑制DNA 的合成,阻止细胞由G1期进入S期.p16INK4a的表达受Ets1和Ets2的正调控,受Bmi-1的负调控.p16INK4a基因缺失、突变、甲基化、RNA剪接加工错误可导致细胞周期失控和癌变.应用p16INK4a对某些肿瘤进行基因治疗的研究正在进行中.     

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(MTS-1/CDKN2/INK4a) in cancer progression

Since its discovery as an inhibitor of cyclin-dependent kinases 4 and 6, the tumor suppressor p16 has continued to gain widespread importance in cancer. The high frequency of deletions of p16 in tumor cell lines first suggested an important role for p16 in carcinogenesis. This initial genetic evidence was subsequently strengthened by numerous studies documenting p16 inactivation in kindreds with familial melanoma. Moreover, a high frequency of p16 gene alterations was found in primary tumors, while recent studies have identified p16 promoter methylation as a major mechanism of tumor-suppressor-gene silencing. Additional insight into p16's role in cancer has come from the genetic analysis of precancerous lesions and various tissue culture models. It is now believed that loss of p16 is an early and often critical event in tumor progression. Consequently, p16 is a major tumor-suppressor gene whose frequent loss occurs early in many human cancers.     

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.