Shunqian Jin

ATF3 induction following DNA damage is regulated by distinct signaling pathways and over-expression of ATF3 protein suppresses cells growth

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 and -independent pathways, and may also involve MAP kinase signaling pathways. Using the tetracycline-inducible system (tet-off), we have found that over-expression 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.

GADD45 -induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity

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.

Transcription factors Oct-1 and NF-YA regulate the p53-independent induction of the GADD45 following DNA damage.

The p53-regulated GADD45 gene is one of the important players in cellular response to DNA damage, and probably involved in the control of cell cycle checkpoint, apoptosis and DNA repair. There are both the p53-dependent and -independent pathways that regulate GADD45 induction. Following ionizing radiation, induction of the GADD45 gene is regulated by p53 through the p53-binding motif located in the third intron of the GADD45 gene. In contrast, GADD45 induction by methyl methanesulfonate (MMS), UV radiation (UV), and medium starvation is independent of p53 status although p53 may contribute to these responses. However, the regulatory elements that control the p53-independent induction of GADD45 remain uncertain. In this report, we have performed detailed analyses to characterize the responsive components that are required for the induction of the GADD45 promoter. We have found that the region between −107 and −62 of the GADD45 promoter is crucial for the induction. Sequence analysis indicates that there are two OCT-1 sites and one CAAT box located in this region. Site-directed mutations of both OCT-1 and CAAT motifs substantially abrogate the induction of the GADD45 promoter by DNA damage. In addition, both Oct-1 protein (binding to OCT-1 site) and NF-YA protein (binding to CAAT box) are induced after cell exposure to DNA damaging agents. Moreover, the Electrophoretic Mobility Shift Assay (EMSA) has demonstrated the direct bindings of Oct-1 and NF-YA proteins to their consensus sequences in the GADD45 promoter. Therefore, these results have presented the novel observation that transcription factors Oct-1 and NF-YA participate in the cellular response to DNA damage and are involved in the regulation of stress-inducible genes.

BRCA1 activation of the GADD45 promoter.

Breast cancer susceptibility gene BRCA1 has been implicated in the control of gene regulation and such regulated genes are thought to mediate the biological role of BRCA1. Overexpression of BRCA1 induces GADD45, a p53-regulated and stress-inducible gene. However, the molecular mechanism by which BRCA1 induces the expression GADD45 remains unclear. In this report, we have shown that the GADD45 promoter is strongly activated following expression of wild-type BRCA1. In contrast, both the tumor-derived BRCA1 mutants (p1749R and Y1853insA) and truncated BRCA1 mutant protein (Δ500–1863 BRCA1), which lack transactivation activity, were unable to activate the GADD45 promoter, indicating that the BRCA1-mediated activation of the GADD45 promoter requires normal transcriptional properties of BRCA1. BRCA1 did not induce the c-Jun and c-fos promoters, which rules out a general effect of BRCA1 on other immediate-responsive genes. Expression of the human papillomavirus E6 and the dominant-negative mutant p53 proteins had no effect on the induction of the GADD45 promoter by BRCA1, suggesting that activation of the GADD45 promoter by BRCA1 is independent of cellular p53 function. With the 5′-deletion analysis, the BRCA1-responsive element of the GADD45 promoter was mapped at the region from −121 to −75. Disruption of this region resulted in the abrogation of BRCA1 activation of the GADD45 promoter. Taken together, these results demonstrate that the mechanism by which BRCA1 induces GADD45 is mainly through the transactivation of the GADD45 promoter, further demonstrating the evidence that GADD45 acts as one of the BRCA1-regulated genes.

Gadd45a Expression Induces Bim Dissociation from the Cytoskeleton and Translocation to Mitochondria

ABSTRACT 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 microtubule-associated 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 1α (EF-1α), a microtubule-severing protein that plays an important role in maintaining cytoskeletal stability, and inhibits EF-1α-mediated microtubule bundling, indicating that the interaction of Gadd45a with EF-1α disrupts cytoskeletal stability. A mutant form of Gadd45a harboring a deletion of EF-1α-binding domain fails to inhibit microtubule stability and to induce Bim translocation to mitochondria. Furthermore, coexpression of EF-1α antagonizes Gadd45as 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.

Gadd45a contributes to p53 stabilization in response to DNA damage.

p53 is an important molecule in cellular response to DNA damage. After genotoxic stress, p53 protein stabilizes transiently and accumulates in the nucleus, where it functions as a transcription factor and upregulates multiple downstream-targeted genes, including p21Waf1/Cip1, Gadd45a and Bax. However, regulation of p53 stabilization is complex and may mainly involve post-translational modification of p53, such as phosphorylation and acetylation. Using mouse embryonic fibroblasts (MEFs) derived from Gadd45a knockouts, we found that disruption of Gadd45a greatly abolished p53 protein stabilization following UVB treatment. Phosphorylation of p53 at Ser-15 was substantially reduced in Gadd45a−/− MEFs. In addition, p53 induction by UVB was shown to be greatly abrogated in the presence of p38 kinase inhibitor, but not c-Jun N-terminal kinase (JNK) and extracellular-signal regulated kinase (ERK), suggesting that p38 protein kinase is involved in the regulation of p53 induction. Along with the findings presented above, inducible expression of Gadd45a enhanced p53 accumulation after cell exposure to UVB. Taken together, the current study demonstrates that Gadd45a, a conventional downstream gene of p53, may play a role as an upstream effector in p53 stabilization following DNA damage, and thus has defined a positive feedback signal in the activation of the p53 pathway.

Gadd45a Interacts with Aurora-A and Inhibits Its Kinase Activity

Centrosome stability is required for successful mitosis in mammalian cells. Amplification of the centrosome leads to chromosomal missegregation and generation of aneuploidy, which are closely associated with cell transformation and tumorigenesis (Doxsey, S. J. (2001) Nat. Cell Biol. 3, E105-E108; Hinchcliffe, E. H., and Sluder, G. (2001) Genes Dev. 15, 1167-1181; Pihan, G. A., Purohit, A., Wallace, J., Malhotra, R., Liotta, L., and Doxsey, S. J. (2001) Cancer Res. 61, 2212-2219). However, there are currently limited insights into mechanism(s) for this critical biological event. Here we show that Gadd45a, a DNA damage-inducible protein that is regulated by tumor suppressors p53 and BRCA1, participates in the maintenance of centrosome stability. Mouse embryonic fibroblasts derived from gadd45a knock-out mice exhibit centrosome amplification (designated as increased centrosome numbers). Introduction of exogenous Gadd45a into mouse embryonic fibroblasts isolated from gadd45a-null mice substantially restored the normal centrosome profile. In contrast to p21waf1/cip1, which ensures coordinated initiation of centrosome, Gadd45a had no significant effect on centrosome duplication in S phase. Interestingly Gadd45a was found to physically associate with Aurora-A protein kinase, whose deregulated expression results in centrosome abnormality. Furthermore Gadd45a was demonstrated to strongly inhibit Aurora-A kinase activity and to antagonize Aurora-A-induced centrosome amplification. These findings identify a novel mechanism for Gadd45a in the maintenance of centrosome stability and broaden understandings of p53- and BRCA1-regulated signaling pathways in maintaining genomic fidelity.

Bile acids induce pancreatic acinar cell injury and pancreatitis by activating calcineurin

Background: Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca2+. Results: Pharmacologic and genetic inhibition of the Ca2+-activated phosphatase calcineurin dramatically reduces acinar cell injury and in vivo pancreatitis resulting from bile acid exposure. Conclusion: Acinar cell calcineurin mediates acinar cell injury and pancreatitis resulting from bile acid exposure. Significance: Calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis. Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca2+. Thus, it would be clinically relevant to know the targets of this aberrant Ca2+ signal. We hypothesized that the Ca2+-activated phosphatase calcineurin is such a Ca2+ target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca2+. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-κB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform Aβ (CnAβ) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca2+ generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.

Gadd45a regulates β -catenin distribution and maintains cell–cell adhesion/contact

Gadd45a, a growth arrest and DNA-damage gene, plays important roles in the control of cell cycle checkpoints, DNA repair and apoptosis. We show here that Gadd45a is involved in the control of cell contact inhibition and cell–cell adhesion. Gadd45a can serve as an adapter to enhance the interaction between β-catenin and Caveolin-1, and in turn induces β-catenin translocation to cell membrane for maintaining cell–cell adhesion/contact inhibition. This is coupled with reduction of β-catenin in cytoplasm and nucleus following Gadd45a induction, which is reflected by the downregulation of cyclin D1, one of the β-catenin targeted genes. Additionally, Gadd45a facilitates ultraviolet radiation-induced degradation of cytoplasmic and nuclear β-catenin in a p53-dependent manner via activation of p38 kinase. These findings define a novel link that connects Gadd45a to cell–cell adhesion and cell contact inhibition, which might contribute to the role of Gadd45a in inhibiting tumorigenesis.

The Mismatch Repair System Modulates Curcumin Sensitivity through Induction of DNA Strand Breaks and Activation of G2-M Checkpoint

The highly conserved mismatch (MMR) repair system corrects postreplicative errors and modulates cellular responses to genotoxic agents. Here, we show that the MMR system strongly influences cellular sensitivity to curcumin. Compared with MMR-proficient cells, isogenically matched MMR-deficient cells displayed enhanced sensitivity to curcumin. Similarly, cells suppressed for MLH1 or MSH2 expression by RNA interference displayed increased curcumin sensitivity. Curcumin treatment generated comparable levels of reactive oxygen species and the mutagenic adduct 8-oxo-guanine in MMR-proficient and MMR-deficient cells; however, accumulation of γH2AX foci, a marker for DNA double-strand breaks (DSB), occurred only in MMR-positive cells in response to curcumin treatment. Additionally, MMR-positive cells showed activation of Chk1 and induction of G2-M cell cycle checkpoint following curcumin treatment and inhibition of Chk1 by UCN-01 abrogated Chk1 activation and heightened apoptosis in MMR-proficient cells. These results indicate that curcumin triggers the accumulation of DNA DSB and induction of a checkpoint response through a MMR-dependent mechanism. Conversely, in MMR-compromised cells, curcumin-induced DSB is significantly blunted, and as a result, cells fail to undergo cell cycle arrest, enter mitosis, and die through mitotic catastrophe. The results have potential therapeutic value, especially in the treatment of tumors with compromised MMR function. Mol Cancer Ther; 9(3); 558–68