DNA repair pathways : effects of SNPs on their functions and their role in drug resistance

Abstract

DNA repair is important in maintaining genome integrity. Failure of DNA repair systems may lead to genomic instability and hence carcinogenesis. This study aimed to investigate (1) how the genetic variations affect the functions of DNA repair proteins; and (2) how DNA repair mechanisms are involved in cancer drug resistance. The human DNA repair gene human nth endonuclease III-like 1 (E. coli) (NTHL1) is involved in base excision repair, and was chosen for investigating the effect of sequence variations on DNA repair activity. Humans and yeast have similar DNA repair mechanisms, and the NTG2 gene is the yeast homologue of NTHL1. Therefore, NTG2-knockout Saccharomyces cerevisiae (S. cerevisiae) was used as the study model for examining the influence of sequence variations on NTHL1. Yeast expression vectors were constructed to express the wildtype and two mutant human NTHL1 proteins, and the mutant protein carried a frameshift mutation at 105 amino acid position (FS105) or a missense mutation at 239 amino acid position (D239Y). Yeast cells expressing the wildtype or mutant human NTHL1 protein did not demonstrate increased cytotoxicity to DNA damaging reagents. However, treatment with methyl methanesulphonate arrested the cell cycle at S phase in yeast cells carrying no recombinant plasmid or plasmid with the frameshift construct, but not in yeast cells expressing the wildtype NTHL1 protein or the D239Y mutant protein. This indicates that functional NTHL1 protein can prevent S-phase cell cycle arrest after genotoxic treatment. This finding may aid in the development of a simple protein functional assay using the S. cerevisiae model to detect the effects of other SNPs' on proteins, such as apoptotic proteins that are drug targets. In another part of the study, the involvement of DNA repair pathways in drug resistance was investigated. The comet assay was used to evaluate the relationship between DNA damage, DNA repair and Photofrin-mediated photodynamic therapy (Ph-PDT) in U87 glioma cells. Results showed that Ph-PDT prominently induced DNA damage, followed by repair being observed 24 hours after treatment with Ph-PDT in glioma cells. Quantitative reverse transcription-polymerase chain reaction, Western blotting and gene knockdown assays demonstrated that alkB, alkylation repair homolog 2 (E. coli) or ALKBH2 of the DNA damage reversal pathway was significantly increased at both mRNA and protein levels from 30 minutes to 48 hours post-treatment with Ph-PDT in relatively resistant glioma cells. Conversely, down-regulating ALKBH2 expression enhances Ph-PDT efficiency. Chromatin immunoprecipitation assay confirmed that TP53 may play a role in promoting the transcription of ALKBH2 after Ph-PDT. TP53 and ALKBH2-related proteins were further investigated to explore their involvement in regulating ALKBH2 expression after Ph-PDT. Quantitative RT-PCR and Western blotting showed that the expression of c-Jun N-terminal kinases (JNKs) and nucleophosmin (nucleolar phosphoprotein B23, numatrin) or NPM1 increased rapidly after Ph-PDT. Gene knockdown and mRNA stability assays demonstrated that the increase in NPM1 was also involved in Ph-PDT resistance by affecting TP53 mRNA stability and thus ALKBH2 expression. The identification of ALKBH2 involvement via the regulation of TP53 mRNA stability by NPM1 contributing to Ph-PDT resistance may provide insight into the development of more effective therapies for glioblastoma.