Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts

Abstract

Human diploid fibroblasts (HDFs) can be grown in culture for a finite number of population doublings before they cease proliferation and enter a growth-arrest state termed replicative senescence. The retinoblastoma gene product, Rb, expressed in these cells is hypophosphorylated. To determine a possible mechanism by which senescent human fibroblasts maintain a hypophosphorylated Rb, we examined the expression levels and interaction of the Rb kinases, CDK4 and CDK6, and the cyclin-dependent kinase inhibitors p21 and p16 in senescent HDFs. Cellular p21 protein expression increased dramatically during the final two to three passages when the majority of cells lost their growth potential and neared senescence but p21 levels declined in senescent HDFs. During this period, p16 mRNA and cellular protein levels gradually rose with the protein levels in senescent HDFs reaching nearly 40-fold higher than early passage cells. In senescent HDFs, p16 was shown to be complexed to both CDK4 and CDK6. Immunodepletion analysis of p21 and p16 from the senescent cell extracts revealed that p16 is the major CDK inhibitor for both CDK4 and CDK6 kinases. Immunoprecipitation of CDK4 and CDK6 and their associated proteins from radiolabeled extracts from senescent HDFs showed no other CDK inhibitors. Based upon these results, we propose that senescence is a multistep process requiring the expression of both p21 and p16. p16 up-regulation is a key event in the terminal stages of growth arrest in senescence, which may explain why p16 but not p21 is commonly mutated in immortal cells and human tumors.Growth and cell division of human diploid fibroblasts (HDFs) in culture eventually generates a metabolically active but nondividing population of senescent cells. During replicative senescence, as described by Hayflick over three decades ago (1), human embryonic fibroblasts will undergo a total of 60-80 cumulative population doublings. Two tumor suppressor genes, the retinoblastoma gene product (Rb) and p53, have been implicated in the induction of the senescent state. Inactivation of p53 and Rb function by infection with simian virus 40 (SV40), expression of human papilloma viral proteins, E6 and E7, (2) or down-regulation of protein expression with anti-sense oligomers extends the life span of HDFs (3). Rb is regulated by phosphorylation, and in senescent cells it is found in its growth-suppressing hypophosphorylated state even in the presence of growth factors (4). Rb inactivation leads to expression of E2F-dependent genes such as thymidine kinase, DNA polymerase-␣, cdc2, and cyclin A (5), which are not expressed in senescent cells (6), indicating that the failure to phosphorylate Rb is important in the growth arrest of senescent cells.Three cyclin-dependent kinases, CDK2, CDK4, and CDK6, are involved in the phosphorylation of the Rb protein (reviewed in ref. 5). In senescent fibroblasts, CDK2 is catalytically inactive and the protein down-regulated (7). CDK4 is also reported to be down-regulated in senescent cells (8), while the st...

Characterization of the cyclin-dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule

“…The very low turnover of p16 mRNA and the observations of high levels of p16 in late passage number ®broblasts and keratinocytes are both in line with the idea that once p16 expression is induced it will lead to an irreversible exit from the cell cycle as part of the senescence mechanism Loughran et al, 1996;Alcorta et al, 1996). This hypothesis is supported by data suggesting the existence of a gene involved in senescence mapping to the 9p21 locus (Loughran et al, 1994) and the loss of senescence in p16 7/7 mouse embryo ®broblasts .…”

“…Di erent signalling pathways have been suggested to control the levels of INK4a proteins in the cells. p15 expression is induced after the cells have been treated with TGF-b (Hannon and Beach 1995) and increased levels of p16 and p18 are observed in cells of late passage numbers (Hara et al, 1996;Alcorta et al, 1996;Loughran et al, 1996) and in skeletal muscle cells undergoing di erentiation (Franklin and Xiong, 1996), respectively. This might suggest that in spite of similar biochemical activity, it is possible that the e ect of CDK inhibition in the cell by the INK proteins can vary depending on the cell phenotype.…”

Re-expression of endogenous p16ink4a in oral squamous cell carcinoma lines by 5-aza-2′-deoxycytidine treatment induces a senescence-like state

“…The p16 ink4a protein has been linked to senescence (Alcorta et al, 1996;Loughran et al, 1996;Serrano et al, 1997;Foster et al, 1998), and as high levels of p16 ink4a were re-expressed in SCC15 after 5-Aza-CdR treatment, we investigated whether re-expression of p16 ink4a in this cancer cell line would prompt the cells to undergo senescence.…”

“…In addition, the ink4 family of kinase inhibitors also critically contributes to the regulation of cdk4 and cdk6-associated kinase activity (Serrano et al, 1993;Hannon and Beach, 1994). Moreover, upregulation of p16 ink4a a member of the ink4 family, has been observed during the late stages of senescence, where proliferation ceases terminally (Alcorta et al, 1996). Consistent with a role in regulating senescence, p16 ink4a expression was lost in mammary epithelial cells that escaped from the M0 proliferation block (Foster et al, 1998) and ectopic expression of p16 ink4a induced termination of proliferation and a senescence-like state in human glioma cells (Uhrbom et al, 1997).…”

Tissue-specific alternative splicing in the human INK4a/ARF cell cycle regulatory locus

“…An identical hybridization pattern was observed with an exon 1a-speci®c probe (data not shown). It was particularly notable that INK4a mRNA was detectable in normal pancreas as it has been shown in numerous situations that the level of INK4a mRNA is below the detection limit of the standard Northern blot and is only detectable by RT ± PCR methods in normal cells (Alcorta et al, 1996;Khleif et al, 1996;Valle et al, 1997;Yeager et al, 1995;Zindy et al, 1997), indicating a very high level of INK4a mRNA in the pancreas as compared to other normal tissues. The p12 transcript, while approximately 30% of the intensity of the INK4a transcript, was also detectable.…”