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.

Figure 1

p16 and p21 protein expression in proliferating and senescent HDFs. Total extracts from the same number of cells from NHF1 (lanes 1 and 2), MRC-5 (lanes 3 and 4), IMR-90 (lanes 5 and 6), and WI-38 (lanes 7 and 8) strains of HDFs were analyzed for expression of p16 (A) and p21 (B) under conditions of log phase growth (lanes 1, 3, 5, and 7) and senescence (lanes 2, 4, 6, and 8).

Figure 4

Relative levels of CDK inhibitors, p21 and p16 in total extracts and in CDK complexes in human fibroblasts. Extracts from early passage MRC-5 fibroblasts logarithmically growing (lane 1), arrested by growth in LS (lane 2), or 20 hr after gamma irradiation (Irrad) (lane 3), and senescent MRC-5 cells (Sen) (lanes 4) or a SV40-transformed immortal human fibroblast line, GM639 (lane 5) were either analyzed directly (Total lysates) (A) or immunoprecipitated under nondenaturing conditions with anti-CDK4 (B) or anti-CDK6 (C) antibodies. Total and immunoprecipitated lysates were analyzed for the levels of CDK4, CDK6, p21, and p16 present. The approximate relative levels in total extracts were determined directly by densitometry. For immunoprecipitated extracts, relative levels were normalized for the amount of the appropriate CDK immunoprecipitated.

Figure 2

Growth and age related expression of cell cycle related proteins in MRC-5 fibroblasts. Extracts representing the same number of cells from early passage MRC-5 fibroblasts in log phase (lane 1), arrested by growth in reduced serum (LS) (lane 2) or to HD (lane 3) and from MRC-5 fibroblasts at increasing age in culture (lanes 4–8) were analyzed for expression of CDK4 and CDK6, MAP kinase, cyclin D1, the cyclin-dependent inhibitors, p21 and p16. The percentage of the population actively undergoing DNA synthesis during the 48-hr period before the extracts were made is shown as the LI. The degree of senescence is shown as the number of cumulative population doublings remaining before loss of proliferative potential (Remaining CPD) and is denoted as late, near senescent (Near Sen) and senescent (Sen) populations. Lanes 1–8 represent 25, 42, 43, 53, 95, 105, 115, and 100 μg of protein, respectively.

Figure 5

Relative amounts of CDK4 and CDK6 complexed with p16 and p21 in senescent MRC-5. Extracts of senescent MRC-5 fibroblasts were immunodepleted for either p16 or p21 by three serial precipitations with p16 and p21 antisera. The protein levels of CDK4, CDK6 and MAP kinase (A) or p16 and p21 (B) were examined. (A) Lanes 1 and 2 represent the same number of early passage and senescent cells while lanes 3–6 represent 150 μg of the untreated (lane 3), p16-depleted (lane 4), and p21-depleted extracts (lane 5). (B) Protein (150 μg) was analyzed for total extracts of early passage (lane 1), senescent cells (lane 2), and untreated (lane 4), p16-depleted (lane 5) and p21-depleted (lane 6) extracts. The extracted cell pellet was resuspended in a similar volume of Laemmli sample buffer and a fraction representing the same fraction as loaded for the untreated extract representing an undefined amount of protein (lane 3) was analyzed.

Figure 3

Radiolabeled proteins in immunoprecipitated native CDK4 and CDK6 complexes from MRC-5 extracts under different growth states. Extracts from radiolabeled early passage MRC-5 fibroblasts logarithmically growing (lanes 1 and 2), arrested by growth in LS (lanes 3 and 4), 20 hr after gamma irradiation (Irrad) (lanes 5 and 6), and senescent MRC-5 cells (Sen) (lanes 7 and 8) or a SV40-transformed immortal human fibroblast line, GM639 (lanes 9 and 10) were immunoprecipitated under nondenaturing conditions with anti-CDK4 (A) or anti-CDK6 (B). Specificity of the precipitations was shown by competing the antibody with either a buffered saline solution (−) or peptides used to generate the antibodies in PBS (+). Extracts from the same number of cells were immunoprecipitated. Lanes 7–10 for both A and B were exposed three times longer to compensate for differences in radiolabel incorporation between extracts.

Figure 6

Expression of p16, p21 and glyceraldehyde phosphate dehydrogenase mRNAs in MRC-5 cells. RNA from cells in log phase (lane 1, log), growth arrested by incubation in reduced serum (lane 2, LS), growth arrested by growth to HD (lane 3) or from cells approaching and in senescence (lanes 4 to 7, CPD Remaining) were probed for levels of p16α (A), p21 (B), and glyceraldehyde phosphate dehydrogenase (C) mRNAs. The LIs for the log and increasing age (lanes 1, 4–7) were 72, 49, 39, 33, and 5%, respectively. Increases are expressed relative log phase cell expression based upon normalization to glyceraldehyde phosphate dehydrogenase expression.