Regulation Of DNA Repair Genes Research Articles

TAp63γ enhances nucleotide excision repair through transcriptional regulation of DNA repair genes

p63 and p73, two p53 family members, play crucial roles in development and tumor suppression. p63 and p73 have multiple isoforms, which have similar or distinct biological functions. Transactivation (TA) isoforms of p63 and p73 have high similarity with p53 and often have biological functions similar to p53. p53 plays an important role in nucleotide excision repair (NER) through transcriptional regulation of target genes involved in NER, including DDB2, XPC and GADD45. To investigate whether TAp63 and TAp73 play a similar role in NER, Saos2 cells with inducible expression of specific isoforms of TAp63 and TAp73, including TAp63α/β/γ and TAp73α/β/γ isoforms, were employed. Overexpression of TAp63γ significantly enhances NER of ultraviolet (UV)-induced DNA damage, including cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts, and enhances cell survival after UV irradiation in Soas2 cells. The enhancement of NER of UV-induced DNA damage by TAp63γ was also confirmed in H1299 cells with overexpression of TAp63γ. Consistently, knockdown of endogenous TAp63 decreases NER of UV-induced DNA damage in H1299 cells. TAp63α/β and TAp73α/β/γ isoforms do not have a clear effect on NER in Saos2 or H1299 cells. TAp63γ overexpression clearly induces the expression of DDB2, XPC and GADD45 at both RNA and protein levels. Furthermore, luciferase reporter assays show that TAp63γ transcriptionally activates DDB2, XPC and GADD45 genes through the regulation of the p53 binding elements in these genes. These results demonstrate that TAp63γ enhances NER to remove UV-induced DNA damage and maintain genomic stability through transcriptional induction of a set of NER proteins, which provides an additional important mechanism that contributes to the function of TAp63 in tumor suppression.

Abstract 3991: miR-31 modulates tumor sensitivity to radiation in esophageal cancer by regulation of DNA repair genes

Abstract Introduction: Radiation therapy is fundamental to the treatment of esophageal cancer. However, radioresistance is a significant clinical problem, with only ∼ 30% of patients achieving a complete pathological response to neoadjuvant chemoradiotherapy (CRT). The elucidation of biomarkers and molecular mechanisms underlying this radioresistance would be of substantial clinical benefit. Methods: An isogenic model of radioresistance in esophageal adenocarcinoma (EAC) was established by chronically irradiating OE33 cells with fractionated, clinically-relevant doses of 2 Gy X-ray radiation (cumulative dose, 50 Gy). Radiosensitivity was assessed by clonogenic assay. DNA damage and repair was assessed by immunofluorescence and high content analysis. Global microRNA (miRNA) expression was analyzed using miRNA arrays. Overexpression of miR-31 was performed using synthetic Pre-miRs. miR-31-regulated gene targets were investigated using qPCR-based gene arrays. miR-31 and gene target expression were measured in 37 and 29 esophageal diagnostic tumor biopsies, respectively, by qPCR. Expression levels were analyzed across tumor regression grade, lymph node metastasis and overall survival. Results: Chronic exposure of OE33 cells to fractionated doses of radiation resulted in a radioresistant subline, OE33 R. Characterization of this model revealed that, relative to the age- and passage-matched parent control (OE33 P), the radioresistant cells had a significantly enhanced DNA repair efficiency and a significantly altered miRNA profile, including a downregulation of miR-31. Overexpression of miR-31 re-sensitized the radioresistant esophageal cancer cells to radiation. miR-31 overexpression altered the mRNA expression levels of 13 DNA repair genes, suggesting miR-31-mediated regulation of DNA repair in this model. In vivo, decreased miR-31 expression was significantly associated with a poor response to neoadjuvant CRT. In parallel, increased expression of the miR-31 gene targets SMUG1, MLH1, RAD51L3 and MMS19 was significantly associated with a poor response to neoadjuvant CRT. Increased SMUG1 expression was also significantly associated with the presence of lymph node metastasis. Conclusion: To our knowledge we have developed the first isogenic model of radioresistance in EAC. Characterization of this model revealed alterations in DNA repair efficiency and miRNA expression. We demonstrate for the first time a functional role for miR-31 in modulating the cellular response to radiation. Reduced miR-31 expression and increased expression of gene targets SMUG1, MLH1, RAD51L3 and MMS19, in tumor tissue of poor responders suggests a mechanism for resistance to neoadjuvant CRT in vivo, possibly via enhanced DNA repair. Our data imply a role for miR-31 as a biomarker of response to neoadjuvant CRT in esophageal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3991. doi:10.1158/1538-7445.AM2011-3991

頭頸部癌におけるシスプラチン耐性の分子機序

Cisplatin is a potent and pivotal anticancer drug in the treatment of various solid tumors including head and neck cancers. Since the beginning of its clinical use in the early 1980s, cisplatin has been one of the most prevailing and reliable anticancer drugs. Its major limitation, however, is the development of resistance during the courses of chemotherapy. The mode of action of cisplatin is mediated by the formation of DNA adducts, which activate several signal transduction pathways, eventually leading to cellular apoptosis. Cisplatin resistance depends on a number of factors that reduce drug uptake, promote cytoplasmic detoxification, and increase DNA repair activity. We herein review a novel mechanism of cisplatin resistance involving the transcriptional regulation of DNA repair genes by ZNF143. ZNF143 is overexpressed in cisplatin-resistant cells, and its knockdown increases sensitivity to cisplatin but not to oxaliplatin, etoposide, or vincristine. We show that ZNF143 is associated with p73, but not with p53. p73 enhances the action of ZNF143 by facilitating the binding of ZNF143 to ZNF143 binding sites and cisplatin-modified DNA. We further present direct evidence that DNA repair genes, Rad51 and FEN-1, are targeted by ZNF143 and overexpressed in cisplatin-resistant cells. These lines of evidence indicate that the interplay among ZNF143, p73, and ZNF143 target DNA repair genes is responsible for cisplatin resistance. Prediction of cisplatin sensitivity and molecular targeted therapy for ZNF143 remain to be conducted in future basic and clinical studies for managing cisplatin-resitant cancers.

Expression of DNA repair and metabolic genes in response to a flavonoid-rich diet

A diet rich in fruit and vegetables can be effective in the reduction of oxidative stress, through the antioxidant effects of phytochemicals and other mechanisms. Protection against the carcinogenic effects of chemicals may also be exerted by an enhancement of detoxification and DNA damage repair mechanisms. To investigate a putative effect of flavonoids, a class of polyphenols, on the regulation of the gene expression of DNA repair and metabolic genes, a 1-month flavonoid-rich diet was administered to thirty healthy male smokers, nine of whom underwent gene expression analysis. We postulated that tobacco smoke is a powerful source of reactive oxygen species. The expression level of twelve genes (APEX, ERCC1, ERCC2, ERCC4, MGMT, OGG1, XPA, XPC, XRCC1, XRCC3, AHR, CYP1A1) was investigated. We found a significant increase (P < 0.001) in flavonoid intake. Urinary phenolic content and anti-mutagenicity did not significantly change after diet, nor was a correlation found between flavonoid intake and urinary phenolic levels or anti-mutagenicity. Phenolic levels showed a significant positive correlation with urinary anti-mutagenicity. AHR levels were significantly reduced after the diet (P = 0.038), whereas the other genes showed a generalized up regulation, significant for XRCC3 gene (P = 0.038). Also in the context of a generalized up regulation of DNA repair genes, we found a non-significant negative correlation between flavonoid intake and the expression of all the DNA repair genes. Larger studies are needed to clarify the possible effects of flavonoids in vivo; our preliminary results could help to better plan new studies on gene expression and diet.

ZNF143 interacts with p73 and is involved in cisplatin resistance through the transcriptional regulation of DNA repair genes

Zinc-finger protein 143 (ZNF143) is a human homolog of Xenopus transcriptional activator staf that is involved in selenocystyl tRNA transcription. We previously showed that ZNF143 expression is induced by treatment with DNA-damaging agents and that it preferentially binds to cisplatin-modified DNA. In this study, the potential function of ZNF143 was investigated. ZNF143 was overexpressed in cisplatin-resistant cells. ZNF143 knockdown in prostate cancer caused increased sensitivity for cisplatin, but not for oxaliplatin, etoposide and vincristine. We also showed that ZNF143 is associated with tumor suppressor gene product p73 but not with p53. p73 could stimulate the binding of ZNF143 to both ZNF143 binding site and cisplatin-modified DNA, and modulate the function of ZNF143. We provide a direct evidence that both Rad51 and flap endonuclease-1 are target genes of ZNF143 and overexpressed in cisplatin-resistant cells. Taken together, these experiments demonstrate that an interplay of ZNF143, p73 and ZNF143 target genes is involved in DNA repair gene expression and cisplatin resistance.

Differential regulation of expression of the mammalian DNA repair genes by growth stimulation.

During DNA replication, DNA becomes more vulnerable to certain DNA damages. DNA repair genes involved in repair of the damages may be induced by growth stimulation. However, regulation of DNA repair genes by growth stimulation has not been analysed in detail. In this report, we analysed the regulation of expression of mammalian MSH2, MSH3 and MLH1 genes involved in mismatch repair, and Rad51 and Rad50 genes involved in homologous recombination repair, in relation to cell growth. Unexpectedly, we found a clear difference in regulation of these repair gene expression by growth stimulation even in the same repair system. The expression of MSH2, MLH1 and Rad51 genes was clearly growth regulated, whereas MSH3 and Rad50 genes were constitutively expressed, suggesting differential requirement of the repair gene products for cell proliferation. MSH3 gene is located in a bidirectionally divergent manner with DHFR gene that is regulated by growth stimulation, indicating that bidirectionally divergent promoters are not necessarily coordinately regulated. Promoter analysis showed that the growth-regulated expression of MLH1 and Rad51 genes was mainly mediated by E2F that plays crucial roles in regulation of DNA replication, suggesting close relation between some of the repair genes and DNA replication.