Cell cycle arrest at the G1 checkpoint allows completion of critical macromolecular events prior to S phase. G1 and G2 checkpoints (4, 5). Phosphorylation of the RB protein by Cdk and the release of RB-associated proteins-e.g., the transcription factor E2F-is correlated with the transition across the G, checkpoint (6-10). The free E2F is then available to transcriptionally activate genes encoding proteins critical for S-phase function, including deoxynucleotide biosynthesis (11).

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

Nutlin-3, an MDM2 inhibitor, activates p53, resulting in several types of cancer cells undergoing apoptosis. Although p53 is mutated or deleted in f50% of all cancers, p53 is still functionally active in the other 50%. Consequently, nutlin-3 and similar drugs could be candidates for neoadjuvant therapy in cancers with a functional p53. Cellular senescence is also a phenotype induced by p53 activation and plays a critical role in protecting against tumor development. In this report, we found that nutlin-3a can induce senescence in normal human fibroblasts. Nutlin-3a activated and repressed a large number of p53-dependent genes, including those encoding microRNAs. mir-34a, mir-34b, and mir-34c, which have recently been shown to be downstream effectors of p53-mediated senescence, were up-regulated, and inhibitor of growth 2 (ING2) expression was suppressed by nutlin-3a treatment. Two candidates for a p53-DNA binding consensus sequence were found in the ING2 promoter regulatory region; thus, we performed chromatin immunoprecipitation and electrophoretic mobility shift assays and confirmed p53 binding directly to those sites. In addition, the luciferase activity of a construct containing the ING2 regulatory region was repressed after p53 activation. Antisense knockdown of ING2 induces p53-independent senescence, whereas overexpression of ING2 induces p53-dependent senescence. Taken together, we conclude that nutlin-3a induces senescence through p53 activation in normal human fibroblasts, and p53-mediated mir34a, mir34b, and mir34c upregulation and ING2 down-regulation may be involved in the senescence pathway. [Cancer Res 2008;68(9):3193-203]

Werner syndrome (WS) is a recessive disorder characterized by genomic instability and by the premature onset of a number of age-related diseases. To understand the molecular basis of this disease, we deleted a segment of the murine Wrn gene and created Wrn-deficient embryonic stem (ES) cells. At the molecular level, wild type-but not mutant-WS protein co-purifies through a series of centrifugation, chromatography, and sucrose gradient steps with the well characterized 17 S multiprotein DNA replication complex. Furthermore, wild type WS protein co-immunoprecipitates with a prominent component of the multiprotein replication complex, proliferating cell nuclear antigen (PCNA). In vitro studies also indicate that PCNA binds to a region in the N terminus portion of the WS protein containing a potential 3-5 exonuclease domain. Finally, human WS protein also co-immunoprecipitates with both PCNA and topoisomerase I. These results suggest that the WS protein interacts with several components of the DNA replication fork.Werner syndrome (WS) 1 is a rare disorder characterized by the premature onset of a number of processes associated with aging (1, 2). In addition, the proliferative life span of WS fibroblasts is reduced compared with age-matched controls (3)(4)(5). WS cells from patients also exhibit genomic abnormalities such as variegated chromosomal translocations and deletions (6, 7). Most recently, WS cells have been shown to have an attenuation of a p53-dependent apoptotic pathway (8).The WS gene (WRN) product contains seven helicase consensus domains that are 34 -38% identical to the Escherichia coli RecQ gene (9,10) and to the putative yeast helicase Sgs1p (11,12). The Sgs1p is known to interact with types I and II topoisomerases and is required for genome stability in Saccharomyces cerevisiae (11)(12)(13). In addition, an interesting model involving the promotion of extrachromosomal rDNA circles has been proposed for the aging phenotype in yeast Sgs1 mutants (14). Several studies suggest that the WS protein may be implicated in some aspect of DNA replication. WS cells show a prolongation of the S phase of the cell cycle and a decreased rate of DNA synthesis (15). Interestingly, the homologue of the human Werner syndrome gene product in Xenopus laevis is required for the formation of replication foci in egg extracts (16).To study the possible interaction of the mouse WS protein with the DNA replication apparatus, we recently created a deletion of part of the helicase domain of the murine homologue of the WS gene in embryonic stem (ES) cells (17). Biochemical studies of our ES cell lines allowed us to conclude that the WS protein co-purifies with the DNA replication complex and binds to certain of its specific components. EXPERIMENTAL PROCEDURESProtein Analysis-Generation and maintenance of the wild type and homozygous mutant embryonic stem cells have been described previously (17). Protein extraction, immunoprecipitations, and Western blotting analyses were performed as described (8,18). A rabbit polyclonal anti...