Mammalian cells have a remarkable diverse repertoire of response to genotoxic stress that damage DNA. Cellular responses to DNA damaging agents will initially exhibit gene induction, which is regulated by complex mechanism(s) and probably involves multiple signaling pathways. In this paper, we demonstrate that induction of ATF3 protein, a member of the ATF/CREB family of transcription factors, by ionizing radiation (IR) requires normal cellular p53 function. In contrast, induction of ATF3 after UV radiation (UV) or Methyl methanesulphonate (MMS) is independent of p53 status. Induction of ATF3 by DNA damage is rapid, transient, and through a transcriptional mechanism. The ATF3 promoter is induced by UV and MMS, but not by IR. In addition, ATF3 promoter can be activated by MEKK1, an upstream activator of the ERK and JNK kinase pathway, but not induced following p53 expression. Those results indicate that regulation of ATF3 induction after DNA damage utilizes both the p53-dependent andindependent pathways, and may also involve MAP kinase signaling pathways. Using the tetracyclineinducible system (tet-off), we have found that overexpression of ATF3 protein moderately suppresses cell growth. Interestingly, over-expression of ATF3 protein is able to slow down progression of cells from G1 to S phase, indicating that ATF3 protein might play a negative role in the control of cell cycle progression.

In response to DNA damage, the cell cycle checkpoint is an important biological event in maintaining genomic fidelity. Gadd45, a p53-regulated and DNA damage inducible protein, has recently been demonstrated to play a role in the G2-M checkpoint in response to DNA damage. In the current study, we further investigated the biochemical mechanism(s) involved in the GADD45-activated cell cycle G2-M arrest. Using the tetracycline-controlled system (tet-off), we established GADD45-inducible lines in HCT116 (wild-type p53) and Hela (inactivated p53 status) cells. Following inducible expression of the Gadd45 protein, cell growth was strongly suppressed in both HCT116 and Hela cells. Interestingly, HCT116 cells revealed a significant G2-M arrest but Hela cells failed to arrest at the G2-M phases, indicating that the GADD45-activated G2-M arrest requires normal p53 function. The GADD45-induced G2-M arrest was observed independent of p38 kinase activity. Importantly, induction of Gadd45 protein resulted in a reduction of nuclear cyclin B1 protein, whose nuclear localization is critical for the completion of G2-M transition. The reduced nuclear cyclin B1 levels correlated with inhibition of Cdc2/cyclin B1 kinase activity. Additionally, overexpression of cyclin B1 substantially abrogated the GADD45-induced cell growth suppression. Therefore, GADD45 inhibition of Cdc2 kinase activity through alteration of cyclin B1 subcellular localization may be an essential step in the GADD45-induced cell cycle G2-M arrest and growth suppression.

Gadd45a, a p53-and BRCA1-regulated stress protein, has been implicated in the maintenance of genomic fidelity, probably through its roles in the control of cell cycle checkpoint and apoptosis. However, the mechanism(s) by which Gadd45a is involved in the induction of apoptosis remains unclear. We show here that inducible expression of Gadd45a protein causes dissociation of Bim, a Bcl2 family member, from microtubuleassociated components and translocation to mitochondria. The Bim accumulation in mitochondria enhances interaction of Bim with Bcl-2, relieves Bax from Bcl-2-bound complexes, and subsequently results in release of cytochrome c into the cytoplasm. Suppression of endogenous Bim greatly inhibits Gadd45a induction of apoptosis. Interestingly, Gadd45a interacts with elongation factor 1␣ (EF-1␣), a microtubule-severing protein that plays an important role in maintaining cytoskeletal stability, and inhibits EF-1␣-mediated microtubule bundling, indicating that the interaction of Gadd45a with EF-1␣ disrupts cytoskeletal stability. A mutant form of Gadd45a harboring a deletion of EF-1␣-binding domain fails to inhibit microtubule stability and to induce Bim translocation to mitochondria. Furthermore, coexpression of EF-1␣ antagonizes Gadd45a's property of suppressing cell growth and inducing apoptosis. These findings identify a novel link that connects stress protein Gadd45a to the apoptotic machinery and address the importance of cytoskeletal stability in apoptotic response to DNA damage.

Intrahepatic expression of hepatitis B x antigen (HBxAg) is associated with the development of hepatocellular carcinoma (HCC), perhaps through trans-activation of selected cellular genes. When this was examined by PowerBlot analysis, upregulated levels of ␤-catenin and several known ␤-catenin effectors were observed in HBxAg-positive compared with HBxAg-negative HepG2 cells. When HBxAg was introduced into Hep3B cells, upregulated expression of wildtype ␤-catenin was observed. This was also observed in Hep3B cells overexpressing the HBxAg upregulated gene, URG11. Upregulated expression of URG11 and ␤-catenin correlated with HBxAg trans-activation function. Transient transfection assays with fragments of the ␤-catenin promoter showed that it was activated by both HBxAg and URG11 and inhibited by URG11-specific small inhibitory RNA. The latter also inhibited the growth of Hep3BX cells in a serumfree medium, which correlated with depressed levels of ␤-catenin. Activation of ␤-catenin effector genes was observed in cells stably expressing HBxAg or overexpressing URG11 compared with control cells transfected with the pTOPFLASH reporter plasmid. Extensive costaining between HBxAg, URG11, and ␤-catenin was observed in infected liver and HCC nodules, suggesting a close relationship in vivo. In conclusion, wild-type ␤-catenin is activated by HBxAg, in part, through the upregulated expression of the HBxAg effector URG11. URG11 stimulates the ␤-catenin promoter and hepatocellular growth and survival. These observations also suggest that URG11 may be a regulatory element in the ␤-catenin signaling pathway and may be a target for chemoprevention of HCC. (HEPATOLOGY 2006;43:415-424.)

Hepatitis B x antigen (HB x Ag) is a trans-activating protein that may be involved in hepatocarcinogenesis, although few natural effectors of HB x Ag that participate in this process have been identified. To identify additional effectors, whole cell RNA isolated from HB x Ag-positive and HB x Ag-negative HepG2 cells were compared by polymerase chain reaction select cDNA subtraction, and one clone, upregulated gene, clone 11 (URG11), was chosen for further characterization. Elevated levels of URG11 mRNA and protein were observed in HB x Ag-positive compared to HB x Ag-negative HepG2 cells. Costaining was observed in infected liver (P < 0.01). URG11 stimulated cell growth in culture (P < 0.01), anchorage-independent growth in soft agar (P < 0.001), and accelerated tumor formation (P < 0.01), and yielded larger tumors (P < 0.02) in SCID mice injected subcutaneously with HepG2 cells. These data suggest that URG11 is a natural effector of HB x Ag that may promote the development of hepatocellular carcinoma.