Caffeine Suppresses Apoptosis of Bladder Cancer RT4 Cells in Response to Ionizing Radiation by Inhibiting Ataxia Telangiectasia Mutated-Chk2-p53 Axis

Background： Caffeine suppresses ataxia telangiectasia and Rad3 related and ataxia telangiectasia mutated （ATM） activities; ATM is the major kinase for DNA damage detection. This study aimed to investigate the effects of caffeine on DNA damage responses in cells from the bladder cancer cell line RT4 those were exposed to ionizing radiation （IR）. Methods： Immunofluorescent staining was performed to investigate changes in the proteins involved in DNA damage responses with or without caffeine. A mouse xenograft model was used to study the effects of caffeine on the DNA damage responses. Western blotting was used to investigate the effects of caffeine pretreatment on the ATM-Chk2-p53-Puma axis, while real-time polymerase chain reaction （RT-PCR） assessed changes in messenger RNA levels of p53 and downstream targets responding to IR. Finally, terminal deoxynucleotidyl transferase-dUTP nick end labeling assay. Western blotting and colony formation assay were used to measure the effects of caffeine on radiation-related apoptosis. All of the data were analyzed with a two-tailed Student＇s t-test. Results： lmmunofluorescent staining showed that caffeine pretreatment profoundly suppressed the formation ofyH2AXand p53-binding protein 1 foci in RT4 cells in response to irradiation. Cellular and animal experiments suggested that this suppression was mediated by suppression of the ATM-Chk2-p53-Puma DNA damage-signaling axis. RT-PCR indicated caffeine also attenuated transactivation of p53 and p53-inducible genes. The colony formation assay revealed that caffeine displayed radioprotective effects on RT4 cells in response to low-dose radiation compared to the radiosensitization effects on T24 cells. Conclusion： Caffeine may inhibit IR-related apoptosis of bladder cancer RT4 cells by suppressing activation of the ATM-Chk2-p53-Puma axis.

A Novel ATM Antisense Transcript ATM-AS Positively Regulates ATM Expression in Normal and Breast Cancer Cells

Objective:The ataxia telangiectasia mutated(ATM)gene is a master regulator in cellular DNA damage response.The dysregulation of ATM expression is frequent in breast cancer,and is known to be involved in the carcinogenesis and prognosis of cancer.However,the underlying mechanism remains unclear.The bioinformatic analysis predicted a potential antisense transcript ATM-antisense(AS)from the opposite strand of the ATM gene.The purpose of this study was to identify ATM-AS and investigate the possible effect of ATM-AS on the ATM gene regulation.Methods:Single strand-specific RT-PCR was performed to verify the predicted antisense transcript ATM-AS within the ATM gene locus.qRT-PCR and Western blotting were used to detect the expression levels of ATM-AS and ATM in normal and breast cancer cell lines as well as in tissue samples.Luciferase reporter gene assays,biological mass spectrometry,ChIP-qPCR and RIP were used to explore the function of ATM-AS in regulating the ATM expression.Immunofluorescence and host-cell reactivation(HCR)assay were performed to evaluate the biological significance of ATM-AS in ATM-mediated DNA damage repair.Breast cancer tissue samples were used for evaluating the correlation of the ATM-AS level with the ATM expression as well as prognosis of the patients.Results:The ATM-AS significantly upregulated the ATM gene activity by recruiting KAT5 histone acetyltransferase to the gene promoter.The reduced ATM-AS level led to the abnormal downregulation of ATM expression,and impaired the ATM-mediated DNA damage repair in normal breast cells in vitro.The ATM-AS level was positively correlated with the ATM expression in the examined breast cancer tissue samples,and the patient prognosis.Conclusion:The present study demonstrated that ATM-AS,an antisense transcript located within the ATM gene body,is an essential positive regulator of ATM expression,and functions by mediating the binding of KAT5 to the ATM promoter.These findings uncover the novel mechanism underlying the dysregulation of the ATM gene in breast cancer,and enrich our understanding of how an antisense transcript regulates its host gene.

PI3K-like Kinases Restrain Pim Gene Expression in Endothelial Cells

Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.We hypothesized that DNA repair gene would regulate Pim ex-pression in ECs.Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium.The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining.The siRNA fragments were synthesized and transfected by using Lipofectamine LTX.The total cellular RNA was extracted from the cells by using Trizol reagent.cDNAs were quantified by semi-quantity PCR.The effects of LY294002 and wortmannin on RNA stability in ECs were also ex-amined.Our data showed that LY294002 and wortmannin,phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors,increased Pim mRNA expression in ECs without altering the mRNA stabil-ity.RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1,respectively.Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs.But etoposide,a nucleoside analogue,which could activate DNA-PKcs and ATM,increased Pim expression in ECs.Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.

ATM单核苷酸多态性与放射性肺损伤的研究现状

放射性肺损伤是胸部肿瘤患者放疗后常见的毒副反应。研究发现,放射性肺损伤除了与放疗剂量等临床因素相关外,还与患者遗传因素密切相关。ATM(ataxia telangiectasia mutated,ATM)是重要的双链DNA修复蛋白,在放射线损伤细胞DNA双链时参与同源重组修复。ATM基因在人群中的遗传多态性与放疗后发生放射性肺损伤易感性有密切相关性。本文总结国内外最新研究进展,就ATM基因单核苷酸多态性与放射性肺损伤发生风险之间的关联性研究进行综述,为放射性肺损伤易感基因筛查及今后个体化放疗的实施提供参考。

DNA Damage-Induced Transcription of Transposable Elements and Long Non-coding RNAs in Arabidopsis Is Rare and ATM-Dependent

Induction and mobilization of transposable elements （TEs） following DNA damage or other stresses has been reported in prokaryotes and eukaryotes. Recently it was discovered that eukaryotic TEs are frequently associated with long non-coding RNAs （IncRNAs）, many of which are also upregulated by stress. Yet, it is unknown whether DNA damage-induced transcriptional activation of TEs and IncRNAs occurs sporadically or is a synchronized, genome-wide response. Here we investigated the transcriptome of Arabidopsis wild- type （WT） and ataxia telangiectasia mutated （atm） mutant plants 3 h after induction of DNA damage. In WT, expression of 5.2% of the protein-coding genes is 〉 2-fold changed, whereas in atm plants, only 2.6% of these genes are regulated, and the response of genes associated with DNA repair, replication, and cell cy- cle is largely lost. In contrast, only less than 0.6% of TEs and IncRNAs respond to DNA damage in WT plants, and the regulation of 〉95% of them is ATM-dependent. The ATM-downstream factors BRCA1, DRM1, JMJ30, AGO2, and the ATM-independent AGO4 participate in the regulation of individual TEs and IncRNAs. Remarkably, protein-coding genes located adjacent to DNA damage-responsive TEs and IncRNAs are frequently coexpressed, which is consistent with the hypothesis that TEs and IncRNAs located close to genes commonly function as controlling elements.

Correlativity study between expression of DNA double-strand break repair protein and radiosensitivity of tumor cells

DNA double-strand break(DSB)is generally regarded as the most lethal of all DNA lesions after radiation.Ku80,DNA-PK catalytic subunit(DNA-PKcs)and ataxia telangiectasia mutated(ATM)proteins are major DSB repair proteins.In this study,survival fraction at 2Gy(SF2)values of eight human tumor cell lines(including four human cervical carcinoma cell lines HeLa,SiHa,C33A,Caski,three human breast carcinoma cell lines MCF-7,MDA-MB-231,MDA-MB-453,and one human lung carcinoma cell line A549)were acquired by clone formation assay,and western blot was applied to detect the expressions of Ku80,DNA-PKcs and ATM protein.The correlativity of protein expression with SF2 value was analyzed by Pearson linear correlation analysis.We found that the expression of the same protein in different cell lines and the expression of three proteins in the same cell line had a significant difference.The SF2 values were also different in eight tumor cell lines and there was a positive correlativity between the expression of DNA-PKcs and SF2(r=0.723,P=0.043),but Ku80 and ATM expression had no correlation with SF2(P>0.05).Thesefindings suggest that the expression level of DNA-PKcs protein can be an indicator for predicting the radiosensitivity of tumor cells.