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...

Terminal differentiation of many cell types involves permanent withdrawal from the cell division cycle. The p18INK4c protein, a member of the p16/INK4 cyclin-dependent kinase (CDK) inhibitor family, is induced more than 50-fold during myogenic differentiation of mouse C2C12 myoblasts to become the predominant CDK inhibitor complexed with CDK4 and CDK6 in terminally differentiated myotubes. We have found that the p18INK4c gene expresses two mRNA transcripts-a 2.4-kb transcript, p18(L), and a 1.2-kb transcript, p18(S). In proliferating C2C12 myoblasts, only the larger p18(L) transcript is expressed from an upstream promoter. Work over the past decade with several model systems has identified a number of transcription factors that play a critical role in initiating a cascade of events leading to activation of lineage-specific genes and, ultimately, to conversion of precursor cells into functionally specialized cells. Coupled with this process is withdrawal of proliferating undifferentiated cells from the mitotic cell cycle at a specific point in G 1 phase to become permanently arrested, terminally differentiated cells. Myogenesis is a complex, multistep process in which determined muscle precursor cells first enter the differentiation pathway and then undergo phenotypic differentiation which is characterized by the expression of muscle structural genes before fusing to form multinucleated myotubes (reviewed in references 23, 32, and 35). Irreversible withdrawal from the cell cycle occurs after myogenin induction, and establishment of the postmitotic state is required for the expression of musclespecific contractile proteins. The MyoD family of basic helixloop-helix transcription factors regulates the determination and differentiation of muscle precursor cells and, in conjunction with the MEF2 family of MADS box transcription factors, also activates muscle structural genes. In contrast to the progress in understanding the mechanisms that regulate the initiation of differentiation and the subsequent expression of lineage-specific genes, little is known about how cell cycle arrest is initiated and maintained during terminal differentiation.Primary control of the eukaryotic cell cycle is provided by the activity of a family of serine/threonine protein kinases, CDKs (cyclin-dependent kinases; see two recent reviews in references 15 and 30). The enzymatic activity of a CDK is regulated by several mechanisms, including positively by the binding of a cyclin and negatively by the binding of a CDK inhibitor. In mammalian cells, there exist at least two distinct families of CDK inhibitors, represented by the two prototype CDK inhibitors p21 and p16 (31, 34). p21 (also variously known as CIP1, WAF1, SDI1, and CDKN1), first identified in normal human fibroblasts as a component of quaternary cyclin D-CDK complexes that also contain proliferating cell nuclear antigen, is a potent inhibitor of multiple cyclin-CDK enzymes. The p21 family contains two other related CDK inhibitor genes, p27 Kip1 and p57Kip2 . Expression of the p21 ge...

The Pax genes encode a family of developmental transcription factors that bind to specific DNA sequences via the paired domain and are necessary for the morphogenesis of a variety of tissues. The murine Pax-2 gene, through alternative splicing, encodes two nuclear proteins, Pax-2A and Pax-2B, which are transiently expressed during the differentiation of specific neural cell types and early kidney formation. In order to identify potential in vivo Pax-2 target sequences, chromatin from embryonic neural tube was immunoprecipitated with Pax-2 specific antibodies and cloned. Two unique immunoprecipitated clones containing three specific Pax-2 binding sites were identified by functional binding assays using Pax-2 proteins produced in both Escherichia coli and eukaryotic cells. In vitro DNA binding assays, using Pax-5 and Pax-8 DNA recognition sequences as well as the three immunopurified Pax-2 binding sites, demonstrated that both forms of the Pax-2 protein bind DNA with a similar specificity and that this binding is mediated by the paired domain. The binding sites identified in this report share significant homology among themselves and with previously defined consensus sequences for Pax-5 and Pax-2. The genomic clones can now be used as sequence tags to identify potential target loci.