p53 regulates a G2 checkpoint through cyclin B1

The p53 tumor suppressor controls multiple cell cycle checkpoints regulating the mammalian response to DNA damage. To identify the mechanism by which p53 regulates G2, we have derived a human ovarian cell that undergoes p53-dependent G2 arrest at 32 degrees C. We have found that p53 prevents G2/M transition by decreasing intracellular levels of cyclin B1 protein and attenuating the activity of the cyclin B1 promoter. Cyclin B1 is the regulatory subunit of the cdc2 kinase and is a protein required for mitotic initiation. The ability of p53 to control mitotic initiation by regulating intracellular cyclin B1 levels suggests that the cyclin B-dependent G2 checkpoint has a role in preventing neoplastic transformation.     

NQO1 stabilizes p53 through a distinct pathway

Wild-type p53 is a tumor-suppressor gene that encodes a short-lived protein that, upon accumulation, induces growth arrest or apoptosis. Accumulation of p53 occurs mainly by posttranslational events that inhibit its proteosomal degradation. We have reported previously that inhibition of NAD(P)H: quinone oxidoreductase 1 (NQO1) activity by dicoumarol induces degradation of p53, indicating that NQO1 plays a role in p53 stabilization. We now have found that wild-type NQO1, but not the inactive polymorphic NQO1, can stabilize endogenous as well as transfected wild-type p53. NQO1-mediated p53 stabilization was especially prominent under induction of oxidative stress. NQO1 also partially inhibited p53 degradation mediated by the human papilloma virus E6 protein, but not when mediated by Mdm-2. Inhibitors of heat shock protein 90 (hsp90), radicicol and geldanamycin, induced degradation of p53 and suppressed p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Differences in the effectiveness of dicoumarol and hsp90 inhibitors to induce p53 degradation and suppress apoptosis in these cell types indicate that NQO1 and hsp90 stabilize p53 through different mechanisms. Our results indicate that NQO1 has a distinct role in the regulation of p53 stability, especially in response to oxidative stress. The present data on the genetic and pharmacologic regulation of the level of p53 have clinical implications for tumor development and therapy.     

Notch1 differentially regulates oncogenesis by wildtype p53 overexpression and p53 mutation in grade III hepatocellular carcinoma

The tumor suppressor p53 is a key prognostic factor in hepatocellular carcinoma (HCC), yet only 35% of grade III tumors exhibit mutation of p53. Several other pathways have been implicated in HCC and, among these, the role of the Notch1/Snail pathway remains unclear. Therefore, we investigated the expression of p53, Notch1, and Snail proteins in HCC with regard to both clinical grade and p53 mutational status. Immunoblotting for p53 revealed that, whereas in many tumors increased p53 was a result of p53 mutation, wildtype p53 (p53WT) expression was also frequently elevated in HCCs. Coordinated evaluation of p53, Notch1, and Snail expression suggests that grade III HCC can be subdivided based on the expression of these three proteins. We found that Notch1 expression in HCC tissues and cell lines is differentially affected by p53WT and mutant p53 (p53Mut). Notch1 expression was correlated with p53 expression in cells expressing p53WT, but was not elevated in p53Mut-expressing cells. Virally mediated expression or silencing of p53WT or p53Mut confirmed that p53WT overexpression causes Notch1 up-regulation in HCC. Surprisingly, the consequence of Notch1 overexpression for the proliferative and invasive capacity of HCC cells depends on both the p53 mutational status and activation of the Snail pathway. CONCLUSION: In the presence of p53WT, Snail/Notch1 activation increased the invasiveness of HCC cells. In contrast, in the absence of p53WT, Notch1 decreased the invasiveness of HCC. Taken together, these findings shed new light on the complex role of the Notch1/Snail axis in HCC and provide a framework for further classifying HCC based on the expression and mutational status of p53 and the expression of Notch1 and Snail.     

FEZ1/LZTS1 gene at 8p22 suppresses cancer cell growth and regulates mitosis

The FEZ1/LZTS1 gene maps to chromosome 8p22, a region that is frequently deleted in human tumors. Alterations in FEZ1/LZTS1 expression have been observed in esophageal, breast, and prostate cancers. Here, we show that introduction of FEZ1/LZTS1 into Fez1/Lzts1-negative cancer cells results in suppression of tumorigenicity and reduced cell growth with accumulation of cells at late S-G(2)/M stage of the cell cycle. Fez1/Lzts1 protein is hyperphosphorylated by cAMP-dependent kinase during cell-cycle progression. We found that Fez1/Lzts1 is associated with microtubule components and interacts with p34(cdc2) at late S-G(2)/M stage in vivo. Present data show that FEZ1/LZTS1 inhibits cancer cell growth through regulation of mitosis, and that its alterations result in abnormal cell growth.     

Survivin regulates hematopoietic progenitor cell proliferation through p21WAF1/Cip1-dependent and -independent pathways

The cyclin-dependent kinase inhibitor p21WAF1/Cip1 and Survivin enhance granulocyte macrophage colony-forming unit (CFU-GM) cell cycle and proliferation and have been implicated as antiapoptotic proteins. We investigated the relationships between p21 and Survivin in primary CFU-GM and c-kit+, lineage-negative (Lin-) cells and demonstrate p21-dependent and -independent pathways whereby Survivin regulates progenitor cell proliferation. Ectopic Survivin enhanced p21+/+ CFU-GM formation and expansion of c-kit+, Lin- cells, whereas p21 gene loss abrogated these effects, indicating a p21 requirement. A dominant-negative form of Survivin and p21 gene deletion accelerated the loss of CFU-GM upon growth factor deprivation, and wild-type Survivin overexpression inhibited apoptosis of p21+/+ CFU-GM and c-kit+, Lin- cells but not p21-/- cells, suggesting that both Survivin and p21 block apoptosis of progenitors and that Survivin-mediated antiapoptosis requires p21. In contrast to the p21-dependent antiapoptotic effects, Survivin increased the proportion of CFU-GM in S-phase in both p21+/+ and p21-/- cells. Furthermore, modulating Survivin expression increased polyploidy in c-kit+, Lin- cells, which was accentuated by p21 deficiency. These results suggest that the Survivin-p21 axis plays an important role in the proliferation of normal hematopoietic cells and that Survivin regulates apoptosis through a p21 WAF1/Cip1-dependent pathway but may control S-phase entry independent of p21.