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p21Waf1/Cip1 is a major transcriptional target of p53 and has been shown to be one of the principal mediators of the p53 induced G1 cell cycle arrest. We show that in addition to the G1 block, p21Waf1/Cip1 can also contribute to a delay in G2 and expression of p21Waf1/Cip1 gives rise to cell cycle profiles essentially indistinguishable from those obtained following p53 expression. Arrest of cells in G2 likely reflects an inability to induce cyclin B1/cdc2 kinase activity in the presence of p21Waf1/Cip1, although the inefficient association of p21Waf1/Cip1 and cyclin B1 suggests that the mechanism of inhibition is indirect. Cells released from an S-phase block were not retarded in their ability to progress through S-phase by the presence of p21Waf1/Cip1, despite efficient inhibition of cyclin E, A and B1 dependent kinase activity, suggesting that p21Waf1/Cip1 is inefficient at inhibiting replicative DNA synthesis in vivo. Interestingly, significant numbers of cells released from the p21Waf1/Cip1 activated G2 block undergo endoreduplication, passing through another S-phase before undergoing mitosis. This supports a function of the mitotic kinases in both entry into mitosis, and also in preventing re-replication of DNA following S-phase and suggests a role for p21Waf1/Cip1 in coupling DNA synthesis and mitosis. Unlike p53, which induces apoptosis in these cells, extended expression of p21Waf1/Cip1 resulted in the expression of a senescent-like phenotype in these p53 null, pRB null tumor cells.

The c-myc proto-oncogene has been shown to cause blockages to differentiation in many cell lineages. Although the mechanism by which c-Myc affects this process remains unknown, it is considered that it might result indirectly as an outcome of the continued cell-cycle progression invoked by c-Myc in cells which must growth arrest in order to differentiate. However, as there is little evidence to support this hypothesis, it is equally possible that a differentiation blockage occurs through a mechanism independent of c-Myc's involvement in cell-cycle progression. To explore this possibility we utilised a differentiation-defective variant of the U937 cell line, which still responds to the differentiation inducer by undergoing rapid growth arrest. Analysis of this line during growth arrest revealed that, although the expression of the Myc target gene, ornithine decarboxylase (ODC) was down-regulated, the cells differed from those of the parental line in that they continued to express high levels of c-Myc protein, but did not maintain high levels of expression of the Myc antagonists, mad1 and mxi1. Moreover, antisense down-regulation of the c-Myc protein levels in these growth-arrested cells revealed that this continued c-Myc expression was essential for their differentiation blockage. These data therefore indicate that c-Myc can block differentiation by a mechanism dissociated from its ability to direct cell-cycle progression or the expression of ODC.