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The occurrence of DNA double strand breaks induces cell cycle arrest in mortal and immortal human cells. In normal, mortal ®broblasts this block to proliferation is permanent. It depends on the growth regulator p53 and a protein p53 induces, the cyclin dependent kinase inhibitor, p21. We show here that following DNA damage in mortal ®broblasts, the induction of p21 and p53 is to a large degree shortlived. By 8 days after a brief exposure to DNA strand breaking agents, bleomycin or actinomycin D, p53 protein is at baseline levels, while the p53 transactivation level is only slightly above its baseline. By this time the concentration of p21 protein, which goes up as high as 100-fold shortly after treatment, is down to just 2 ± 4-fold over baseline levels. Following the drop in p21 concentration a large increase in the expression level of the tumor suppressor gene p16INK4a is observed. This scenario, where a transient increase in p21 is followed by a delayed induction of p16 INK4a , also happens with the permanent arrest that occurs with cellular senescence. In fact, these cells treated with agents that cause DNA double strand breaks share a number of additional markers with senescent cells. Our ®ndings indicate that these cells are very similar to senescent cells and that they have additional factor(s) beside p21 and p53 that maintain cell cycle arrest.

Recent liver regeneration studies indicate that maintaining hepatic Forkhead Box M1B (FoxM1B) expression in 12-month-old (old-aged) Transthyretin-FoxM1B transgenic mice increases hepatocyte proliferation and expression of cell cycle regulatory genes. Because these transgenic CD-1 mice maintain FoxM1B levels during the aging process, we conducted the current study to determine whether adenovirus delivery of the FoxM1B gene (AdFoxM1B) is sufficient to stimulate liver regeneration in old-aged Balb/c mice. Here we show that AdFoxM1B infection of old-aged mice caused a significant increase in FoxM1B expression, hepatocyte DNA replication, and mitosis following partial hepatectomy. This stimulation in hepatocyte S-phase progression was associated with diminished protein expression and perinuclear localization of cyclin-dependent kinase (Cdk) inhibitor p27Kip1 (p27) protein following partial hepatectomy. In contrast, old-aged mice infected with control virus displayed high hepatocyte levels of p27 protein, which had been localized to the nucleus prior to S-phase. Furthermore, we found that restoring FoxM1B expression did not influence p27 mRNA levels, and this new finding implicates FoxM1B in regulation of p27 protein levels. Likewise, AdFoxM1B-infected regenerating livers displayed elevated S-phase levels of Cdk2 kinase activity compared with old-aged mice infected with control virus. Furthermore, restoring FoxM1B expression in old-aged mice caused elevated levels of Cyclin B1, Cyclin B2, Cdc25B, Cdk1, and p55CDC mRNA as well as stimulating Cdc25B nuclear localization during liver regeneration, all of which are required for mitosis. These studies indicated that an acute delivery of the FoxM1B gene in old-aged mice is sufficient to re-establish proliferation of regenerating hepatocytes, suggesting that FoxM1B can be used for therapeutic intervention to alleviate the reduction in cellular proliferation observed in the elderly.

The cloning of the negative growth regulatory gene, p21Sdi1, has led to the convergence of the fields of cellular senescence, cell cycle regulation and tumor suppression. This gene was first cloned as an inhibitor of DNA synthesis that was overexpressed in terminally non‐dividing senescent human fibroblasts (SD11) and later as a p53 transactivated gene (WAF1) and a Cdk‐interacting protein (CIP1, p21) that inhibited cyclin‐dependent kinase activity. To identify the active region(s) of p21Sdi1, cDNA constructs encoding various deleted forms of the protein were analyzed. Amino acids 22‐71 were found to be the minimal region required for DNA synthesis inhibition. Amino acids 49‐71 were involved in binding to Cdk2, and constructs deleted in this region expressed proteins that were unable to inhibit Cdk2 kinase activity in vitro. The latter stretch of amino acids shared sequence similarity with amino acids 60‐76 of the p27Kip1 protein, another Cdk inhibitor. Point mutations made in p21Sdi1 in this region confirmed that amino acids common to both proteins were involved in DNA synthesis inhibition. Additionally, a chimeric protein, in which amino acids 49‐65 of p21Sdi1 were substituted with amino acids 60‐76 of p27Kip1, had almost the same DNA synthesis inhibitory activity as the wild‐type protein. The results indicate that the region of sequence similarity between p21Sdi1 and p27Kip1 encodes an inhibitory motif characteristic of this family of Cdk inhibitors.

Mice lacking FoxM1 specifically in progenitor-like type II alveolar epithelial cells exhibit defective alveolar barrier repair after microbe-induced lung injury.

In nonproliferating or growth-arrested cells, the transcription factor E2F remains bound to the retinoblastoma-related protein p130. Accumulation of this E2F-p130 complex correlates with an arrest of the cell cycle progression. Progression through G 1 phase is associated with a cyclin-dependent binding of the cyclindependent kinase cdk2 to the E2F-p130 complex. By fractionating mouse L-cell extracts, we have obtained a partially purified preparation of the E2F-p130 complex that also contains cdk2. Incubation of this complex with recombinant p21 results in a disruption of the interaction between cdk2 and the E2F-p130 complex. We have also analyzed the interaction between cdk2 and the E2F-p130 complex in extracts of a cell line that expresses a temperature-sensitive mutant of p53. Incubation at the permissive temperature (32؇C) results in an induction of p21 synthesis. An increase in the level of p21 in these cells correlates with a loss of cdk2 from the cdk2-containing E2F-p130 complex. We also show that the expression of a reporter gene containing E2F sites in the promoter region is reduced by the coexpression of p21. Since p21 is believed to be a mediator of p53, we speculate that the p21-mediated disruption of the cdk2-containing E2F-p130 complex plays a role in the growth suppression function of p53.The transcription factors of the E2F family play a central role in cell proliferation. Several growth-associated genes, including c-myc, n-myc, B-myb, cdc2, dhfr, and the thymidine synthetase, thymidine kinase, and DNA polymerase ␣ genes, contain E2F-binding sites in their regulatory regions (6,12, 29,33,50,55,56,66,70). Some of these genes have been shown to depend on E2F for their cell cycle-regulated expression (6,12,34,38,43,49,53,64). The activity of E2F is inhibited by proteins of the retinoblastoma family, such as the retinoblastoma tumor suppressor protein (pRB), p107, and p130. Inhibition of E2F activity coincides with a suppression of cell growth by these retinoblastoma family proteins (2,3,4,5,7,8,10,11,14,23,31,32,44,45,57,59,63,67,68,71,73,75).The tumor suppression function of pRB also appears to correlate with its ability to repress E2F-dependent transcription. The naturally occurring loss-of-function mutants of pRB are unable to regulate E2F (2,31,32,57). Recent studies indicated that pRB, p107, and p130 suppress cell growth by biochemically distinct pathways involving different members of the E2F family. pRB regulates E2F1, E2F2, and E2F3, whereas p107 and p130 regulate E2F4 and E2F5 (17, 20, 27, 28, 39, 42, 46, 59, 61, 67, 73). The E2F-regulatory function and growth suppression function of pRB are inactivated by cyclincyclin-dependent kinase (cdk)-dependent phosphorylation of pRB (8,9,13,16,36). p107 and p130 also associate with cyclin-cdk2 complexes (7,11,14,19,24,45,47,48,63,73). Recent experiments indicated that phosphorylation of p107 by a cyclin-cdk complex could regulate its interaction with E2F (74). p107 has been shown to interact with cyclin A-cdk2 through sequences that are distinct fro...

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