Aflatoxin genotoxicity is associated with a defective DNA damage response bypassing p53 activation

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths. Aflatoxins, which may play a causative role in 5–28% of HCCs worldwide, are activated in liver cells and induce principally G→T mutations, including the TP53 codon 249(G→T) hotspot mutation. The DNA damage checkpoint response acts as an antitumour mechanism against genotoxic agents, but its role in aflatoxin‐induced DNA damage is unknown. Aim: We studied the DNA damage checkpoint response of human cells to aflatoxin B1 (AFB1). Methods and results: The treatment of HepG2 hepatoma cells with mutation‐inducing doses (3–5 μmol/l) of AFB1 induced DNA adducts, 8‐hydroxyguanine lesions and DNA strand breaks that lasted several days. Persistent phospho‐H2AX and 53BP1 foci were also detected, but cell growth was not affected. AFB1‐exposed HepG2 cells formed phospho‐H2AX and 53BP1 foci, but failed to phosphorylate both Chk1 and Chk2. Huh7 hepatoma and HCT116 colorectal cancer cell lines also exhibited a similarly incomplete checkpoint response. p53 phosphorylation also failed, and AFB1‐exposed cells did not show p53‐dependent G1 arrest or a sustained G2/M arrest. These observations contrasted sharply with the fully functional DNA damage response of cells to Adriamycin. Cotreatment of cells with AFB1 did not inhibit p53 and p21^Cip1 accumulation induced by Adriamycin. Thus, the deficient checkpoint response to AFB1 was not due to an inhibitory effect, but could be explained by an inefficient activation. Conclusion: Genotoxic doses of AFB1 induce an incomplete and inefficient checkpoint response in human cells. This defective response may contribute to the mutagenic and carcinogenic potencies of aflatoxins.     

ATP-dependent activation of p21WAF1/CIP1-associated Cdk2 by Cdc6

When cells progressing in mid-S phase are damaged with a base-modifying chemical, they arrest in S phase long after the CHK1 checkpoint signal fades out, partly because of p53-mediated long-lasting induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). We have recently found that enforced expression of Cdc6, the assembler of prereplicative complexes, markedly advances recovery from the prolonged S-phase arrest and reactivation of Cdk2 despite the presence of a high level of induced p21. Here, we report that Cdc6 protein can activate p21-associated Cdk2 in an ATP-dependent manner in vitro. Consistently, Cdc6 mutated for ATPase or a putative cyclin binding motif is no longer able to activate the Cdk2 in vitro or promote reinitiation of S-phase progression and reactivation of Cdk2 in vivo. These results reveal the never anticipated function of Cdc6 and redefine its role in the control of S-phase progression in mammalian cells.     

AIMP2/p38, the scaffold for the multi-tRNA synthetase complex, responds to genotoxic stresses via p53

AIMP2/p38 is a scaffolding protein required for the assembly of the macromolecular tRNA synthetase complex. Here, we describe a previously unknown function for AIMP2 as a positive regulator of p53 in response to genotoxic stresses. Depletion of AIMP2 increased resistance to DNA damage-induced apoptosis, and introduction of AIMP2 into AIMP2-deficient cells restored the susceptibility to apoptosis. Upon DNA damage, AIMP2 was phosphorylated, dissociated from the multi-tRNA synthetase complex, and translocated into the nuclei of cells. AIMP2 directly interacts with p53, thereby preventing MDM2-mediated ubiquitination and degradation of p53. Mutations in AIMP2, affecting its interaction with p53, hampered its ability to activate p53. Nutlin-3 recovered the level of p53 and the susceptibility to UV-induced cell death in AIMP2-deficient cells. This work demonstrates that AIMP2, a component of the translational machinery, functions as proapoptotic factor via p53 in response to DNA damage.     

Dual inactivation of Hus1 and p53 in the mouse mammary gland results in accumulation of damaged cells and impaired tissue regeneration

In response to DNA damage, checkpoint proteins halt cell cycle progression and promote repair or apoptosis, thereby preventing mutation accumulation and suppressing tumor development. The DNA damage checkpoint protein Hus1 associates with Rad9 and Rad1 to form the 9-1-1 complex, which localizes to DNA lesions and promotes DNA damage signaling and repair. Because complete inactivation of mouse Hus1 results in embryonic lethality, we developed a system for regulated Hus1 inactivation in the mammary gland to examine roles for Hus1 in tissue homeostasis and tumor suppression. Hus1 inactivation in the mammary epithelium resulted in genome damage that induced apoptosis and led to depletion of Hus1-null cells from the mammary gland. Conditional Hus1 knockout females retained grossly normal mammary gland morphology, suggesting compensation by cells that failed to undergo Cre-mediated Hus1 deletion. p53-deficiency delayed the clearance of Hus1-null cells from conditional Hus1 knockout mice and caused the accumulation of damaged, dying cells in the mammary gland. Notably, compensatory responses were impaired following combined Hus1 and p53 loss, resulting in aberrant mammary gland morphology and lactation defects. Overall, these results establish a requirement for Hus1 in the survival and proliferation of mammary epithelium and identify a role for p53 in mammary gland tissue regeneration and homeostasis.     

Aurora B kinase phosphorylates and instigates degradation of p53

Aurora B is a mitotic checkpoint kinase that plays a pivotal role in the cell cycle, ensuring correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in many types of human cancers, which has made it an attractive target for cancer therapies. Tumor suppressor p53 is a genome guardian and important negative regulator of the cell cycle. Whether Aurora B and p53 are coordinately regulated during the cell cycle is not known. We report that Aurora B directly interacts with p53 at different subcellular localizations and during different phases of the cell cycle (for instance, at the nucleus in interphase and the centromeres in prometaphase of mitosis). We show that Aurora B phosphorylates p53 at S183, T211, and S215 to accelerate the degradation of p53 through the polyubiquitination-proteasome pathway, thus functionally suppressing the expression of p53 target genes involved in cell cycle inhibition and apoptosis (e.g., p21 and PUMA). Pharmacologic inhibition of Aurora B in cancer cells with WT p53 increased p53 protein level and expression of p53 target genes to inhibit tumor growth. Together, these results define a mechanism of p53 inactivation during the cell cycle and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise the tumor suppressor function of p53. We have elucidated the antineoplastic mechanism for Aurora B kinase inhibitors in cancer cells with WT p53.     

Recruitment of ataxia-telangiectasia mutated to the p21(waf1) promoter by ZBP-89 plays a role in mucosal protection

BACKGROUND & AIMS: Histone deacetylase inhibitors (HDACi) induce growth arrest, apoptosis, and differentiation, particularly in colon cancer cells where they are potential chemopreventive agents. HDACi induction of the cyclin-dependent kinase inhibitor p21(waf1) has been shown to require ataxia-telangiectasia mutated (ATM). Nevertheless, how ATM participates in p21(waf1) gene expression has not been defined. METHODS: In vivo protein complexes forming in response to butyrate were studied using co-immunoprecipitation and mass spectroscopy. DNA elements in the p21(waf1) promoter were analyzed in vivo by chromatin immunoprecipitation and in vitro DNA affinity precipitation assays. The expression of p21(waf1) was analyzed by immunoblots and reporter assays. RESULTS: Reduction of ZBP-89 or ATM with small interfering RNAs blocked HDACi-induced p21(waf1) expression. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that both ZBP-89 and ATM are recruited to the GC-rich DNA elements of the p21(waf1) promoter with HDACi treatment. Co-immunoprecipitation revealed that ATM associates with ZBP-89 in an HDACi-dependent manner. Serial deletions revealed that ATM interacts with both the N-terminal and DNA binding domains of ZBP-89. Moreover, we found that immunodepletion of ZBP-89 prevented recruitment of ATM to the p21(waf1) promoter in vitro. Silencing of ZBP-89 expression blocked HDACi-induced phosphorylation of ATM(Ser1981) and p53(Ser15). ATM(Ser1981) phosphorylation in the colons of mutant mice expressing an N-terminally truncated form of ZBP-89 was not observed after ingestion of dextran sodium sulfate and correlated with exacerbation of the mucosal injury. CONCLUSIONS: ZBP-89 interacts with ATM in a butyrate-dependent manner and is essential for colonic homeostasis in the setting of acute mucosal injury.     

大黄素通过p53途径抑制血管平滑肌细胞增殖的实验研究

目的探讨p53途径在大黄素抑制血管平滑肌细胞增殖作用中的地位。方法通过细胞计数、老化相关β-半乳糖苷酶染色、Annexin V标记等方法观察大黄素抑制血管平滑肌细胞增殖的特点。^3H-胸苷掺入法测定DNA合成、流式细胞仪了解细胞周期变化、Western blot检测p53蛋白表达变化、基因芯片观察mRNA表达水平。结果（1）1．6～3．1μg／ml大黄素延缓细胞生长,6．3～12．5μg／ml大黄素促进细胞老化,25．0μg／ml大黄素则可显著诱导细胞凋亡。（2）大黄素干预24h后,出现非计划性DNA合成现象,这是DNA损伤的敏感性标志。p53基因和蛋白表达水平呈大黄素浓度依赖性上调。除了细胞增殖基因表达下调,其他基因表达均上调,如细胞老化基因、细胞凋亡基因、DNA损伤修复基因。（3）大黄素能够迅速渗透进入细胞,在细胞内的分布具有明显的选择性,绝大多数以颗粒形态分布于细胞胞浆中,细胞核中也有少量分布。结论大黄素通过损伤DNA激活p53途径。随着大黄素浓度升高,p53途径激活程度也随之增强并产生多种细胞增殖抑制效应,即生长停滞、细胞老化和细胞凋亡。