Synergism between p53 and Mcl-1 in protecting from hepatic injury, fibrosis and cancer.

BACKGROUND & AIMS: Mcl-1-deficient hepatocytes are prone to undergo apoptosis. The tumor suppressor protein p53 plays an important role in apoptosis control as well as other cellular responses. This study was initially aimed to examine whether p53 was involved in Mcl-1 deficiency-induced apoptosis of hepatocytes. METHODS: Hepatocyte-specific Mcl-1 knockout (Alb-Mcl-1(-/-)) mice and Alb-Mcl-1(-/-) mice in wild-type or p53-deficient background were generated and characterized. RESULTS: Alb-Mcl-1(-/-) mice were viable, but their liver cells were prone to undergo apoptosis and manifested a slightly elevated level of p53. To examine the role of p53 in Alb-Mcl-1(-/-) livers, Alb-Mcl-1(-/-) mice without p53 (DKO mice) were characterized. Unexpectedly, although p53-deficient mice appeared to be developmentally normal, DKO mice were highly susceptible to neonatal death (∼60%). Further analysis revealed that such an early lethality was likely due to hepatic failure caused by a marked reduction of fully-differentiated hepatocytes at the perinatal/neonatal stage. Moreover, those DKO mice that did survive to adulthood manifested more severe liver damage than Alb-Mcl-1(-/-) mice, suggesting that p53 was activated in Alb-Mcl-1(-/-) livers to promote cell survival. Microarray followed by quantitative PCR analysis suggested that p21(Waf1/Cip1), one p53 target gene with apoptosis-inhibitory function, is likely involved in the protective role of p53 in Alb-Mcl-1(-/-) livers. Moreover, we demonstrated that loss of p53 promoted liver fibrosis and tumor development in Alb-Mcl-1(-/-) mice. CONCLUSIONS: This study revealed an unexpected synergism between Mcl-1 and p53 in protecting from hepatic injury, fibrosis, and cancer.

The candidate tumor suppressor BTG3 is a transcriptional target of p53 that inhibits E2F1.

Proper regulation of cell cycle progression is pivotal for maintaining genome stability. In a search for DNA damage-inducible, CHK1-modulated genes, we have identified BTG3 (B-cell translocation gene 3) as a direct p53 target. The p53 transcription factor binds to a consensus sequence located in intron 2 of the gene both in vitro and in vivo, and depletion of p53 by small interfering RNA (siRNA) abolishes DNA damage-induced expression of the gene. Furthermore, ablation of BTG3 by siRNA in cancer cells results in accelerated exit from the DNA damage-induced G2/M block. In vitro, BTG3 binds to and inhibits E2F1 through an N-terminal domain including the conserved box A. Deletion of the interaction domain in BTG3 abrogates not only its growth suppression activity, but also its repression on E2F1-mediated transactivation. We also present evidence that by disrupting the DNA binding activity of E2F1, BTG3 participates in the regulation of E2F1 target gene expression. Therefore, our studies have revealed a previously unidentified pathway through which the activity of E2F1 may be guarded by activated p53.

p53 C-terminal phosphorylation by CHK1 and CHK2 participates in the regulation of DNA-damage-induced C-terminal acetylation.

The tumor suppressor protein p53 mediates stress-induced growth arrest or apoptosis and plays a major role in safeguarding genome integrity. In response to DNA damage, p53 can be modified at multiple sites by phosphorylation and acetylation. We report on the characterization of p53 C-terminal phosphorylation by CHK1 and CHK2, two serine/threonine (Ser/Thr) protein kinases, previously implicated in the phosphorylation of the p53 N terminus. Using tryptic phosphopeptide mapping, we have identified six additional CHK1 and CHK2 sites residing in the final 100 amino acids of p53. Phosphorylation of at least three of these sites, Ser366, Ser378, and Thr387, was induced by DNA damage, and the induction at Ser366 and Thr387 was abrogated by small interfering RNA targeting chk1 and chk2. Furthermore, mutation of these phosphorylation sites has a different impact on p53 C-terminal acetylation and on the activation of p53-targeted promoters. Our results demonstrate a possible interplay between p53 C-terminal phosphorylation and acetylation, and they provide an additional mechanism for the control of the activity of p53 by CHK1 and CHK2.

TTK/hMps1 participates in the regulation of DNA damage checkpoint response by phosphorylating CHK2 on threonine 68.

CHK2/hCds1 plays important roles in the DNA damage-induced cell cycle checkpoint by phosphorylating several important targets, such as Cdc25 and p53. To obtain a better understanding of the CHK2 signaling pathway, we have carried out a yeast two-hybrid screen to search for potential CHK2-interacting proteins. Here, we report the identification of the mitotic checkpoint kinase, TTK/hMps1, as a novel CHK2-interacting protein. TTK/hMps1 directly phosphorylates CHK2 on Thr-68 in vitro. Expression of a TTK kinase-dead mutant, TTK(D647A), interferes with the G(2)/M arrest induced by either ionizing radiation or UV light. Interestingly, induction of CHK2 Thr-68 phosphorylation and of several downstream events, such as cyclin B1 accumulation and Cdc2 Tyr-15 phosphorylation, is also affected. Furthermore, ablation of TTK expression using small interfering RNA results not only in reduced CHK2 Thr-68 phosphorylation, but also in impaired growth arrest. Our results are consistent with a model in which TTK functions upstream from CHK2 in response to DNA damage and suggest possible cross-talk between the spindle assembly checkpoint and the DNA damage checkpoint.

Differential effects of two growth inhibitory K vitamin analogs on cell cycle regulating proteins in human hepatoma cells.

A comparison was made between two K vitamin analogs. Growth in vitro of Hep G2 hepatoma cells was inhibited both by Compound 5 (Cpd 5), a recently synthesized thioalkyl analog of vitamin K or 2-(2-mercaptoethanol)-3-methyl-1, 4-naphthoquinone, as well as by synthetic vitamin K3 (menadione). Using synchronized Hep G2 hepatoma cells, the actions of both Cpd 5 and vitamin K3 on cell cycle regulating proteins were examined. Cpd 5 decreased the levels of cyclin D1, Cdk4, p16, p21 and cyclin B1. By contrast, VK3 only decreased the level of cyclin D1, but had no effect on the levels of Cdk4, p16 or p21. Interestingly, both VK3 and VK2 increased the levels of p21. The naturally occurring K vitamins had little effect on cell growth and none on the cyclins or Cdks. Amounts and activity of the G1/S phase controlling Cdc25A were measured. We found that Cpd 5 directly inhibited both Cdc25A activity and its protein expression, whereas VK3 did not. Thus, the main effects of Cpd 5 were on G1 and S phase proteins, especially Cdk4 and Cdc25A amounts in contrast to VK3. Computer docking studies of Cpd 5 and VK3 to Cdc25A phosphatase showed three binding sites. In the best conformation, Cpd 5 was found to be closer to the enzyme active site than VK3. These findings show that Cpd 5 represents a new class of anticancer agent, being a protein tyrosine phosphatase (PTP) antagonist, that binds to Cdc25A with suppression of its activity. Tumors expressing high levels of oncogenic Cdc25A phosphatase may thus be susceptible to the growth inhibitory activities of this class of compound.