PARP-2 depletion results in lower radiation cell survival but cell line-specific differences in poly(ADP-ribose) levels.

Poly(ADP-ribose) polymerase-2 (PARP-2) activity contributes to a cells' poly(ADP-ribosyl)ating potential and like PARP-1, has been implicated in several DNA repair pathways including base excision repair and DNA single strand break repair. Here the consequences of its stable depletion in HeLa, U20S, and AS3WT2 cells were examined. All three PARP-2 depleted models showed increased sensitivity to the cell killing effects on ionizing radiation as reported in PARP-2 depleted mouse embryonic fibroblasts providing further evidence for a role in DNA strand break repair. The PARP-2 depleted HeLa cells also showed both higher constitutive and DNA damage-induced levels of polymers of ADP-ribose (PAR) associated with unchanged PARP-1 protein levels, but higher PARP activity and a concomitant lower PARG protein levels and activity. These changes were accompanied by a reduced maximal recruitment of PARP-1, XRCC1, PCNA, and PARG to DNA damage sites. This PAR-associated phenotype could be reversed in HeLa cells on re-expression of PARP-2 and was not seen in U20S and AS3WT2 cells. These results highlight the complexity of the relationship between different members of the PARP family on PAR metabolism and suggest that cell model dependent phenotypes associated with the absence of PARP-2 exist within a common background of radiation sensitivity.

The impact of single-nucleotide polymorphisms (SNPs) in OGG1 and XPC on the age at onset of Huntington disease.

The age at onset of Huntington disease (HD) shows a strong, negative correlation with the number of CAG repeats within the huntingtin (HTT) gene. However, this does not account for all the inter-individual variability seen among patients. In order to assess whether single-nucleotide polymorphisms (SNPs) in the OGG1 and XPC genes, both implicated in responses to oxidative stress, are associated with the age of onset of HD, 9 SNPs have been genotyped in 299 individuals with HD and 582 controls. After correction for multiple testing, two OGG1/XPC haplotypes were found to be associated with younger age at onset independently of the number of CAG repeats within the HTT gene. Both haplotypes contain XPC coding variants that would be expected to impact on protein function and/or variants in the 3'UTR that could result in altered protein levels via allele-specific mIR binding. One haplotype also contains the OGG1-326Cys (rs1052133) allele that has been associated with a lower 8-oxoG repair activity and is particularly sensitive to the cellular redox status. These results highlight the potential role of oxidative stress in determining the age at onset of HD.

The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation.

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5(KD) cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5(KD) cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5(KD) compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5(KD) cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5(KD) cells is due to a role of Cdk5 in other pathways or the altered polymer levels.

The role of microRNA-binding site polymorphisms in DNA repair genes as risk factors for bladder cancer and breast cancer and their impact on radiotherapy outcomes.

MicroRNAs (miRNAs) are involved in post-transcriptional regulation of gene expression through binding to messenger RNAs (mRNA) thereby promoting mRNA degradation or altered translation. A single-nucleotide polymorphism (SNP) located within a miRNA-binding site could thus alter mRNA translation and influence cancer risk and treatment response. The common SNPs located within the 3'-untranslated regions of 20 DNA repair genes were analysed for putative miRNA-binding sites using bioinformatics algorithms, calculating the difference in Gibbs free binding energy (ΔΔG) for each wild-type versus variant allele. Seven SNPs were selected to be genotyped in germ line DNAs both from a bladder cancer case-control series (752 cases and 704 controls) and 202 muscle-invasive bladder cancer radiotherapy cases. The PARP-1 SNP rs8679 was also genotyped in a breast cancer case-control series (257 cases and 512 controls). Without adjustment for multiple testing, multivariate analysis demonstrated an association with increased bladder cancer risk with PARP1 rs8679 (P(trend) = 0.05) while variant homozygotes of PARP1 rs8679 were also noted to have an increased breast cancer risk (P = 0.03). In the radiotherapy cases, carriers of the RAD51 rs7180135 minor allele had improved cancer-specific survival (hazard ratio 0.52, 95% confidence interval 0.31-0.87, P = 0.01). This is the first report of associations between DNA repair gene miRNA-binding site SNPs with bladder and breast cancer risk and radiotherapy outcomes. If validated, these findings may give further insight into the biology of bladder carcinogenesis, allow testing of the RAD51 SNP as a potential predictive biomarker and also reveal potential targets for new cancer treatments.

G-quadruplex structures in TP53 intron 3: role in alternative splicing and in production of p53 mRNA isoforms.

The tumor suppressor gene TP53, encoding p53, is expressed as several transcripts. The fully spliced p53 (FSp53) transcript encodes the canonical p53 protein. The alternatively spliced p53I2 transcript retains intron 2 and encodes Δ40p53 (or ΔNp53), an isoform lacking first 39 N-terminal residues corresponding to the main transactivation domain. We demonstrate the formation of G-quadruplex structures (G4) in a GC-rich region of intron 3 that modulates the splicing of intron 2. First, we show the formation of G4 in synthetic RNAs encompassing intron 3 sequences by ultraviolet melting, thermal difference spectra and circular dichroism spectroscopy. These observations are confirmed by detection of G4-induced reverse transcriptase elongation stops in synthetic RNA of intron 3. In this region, p53 pre-messenger RNA (mRNA) contains a succession of short exons (exons 2 and 3) and introns (introns 2 and 4) covering a total of 333 bp. Site-directed mutagenesis of G-tracts putatively involved in G4 formation decreased by ~30% the excision of intron 2 in a green fluorescent protein-reporter splicing assay. Moreover, treatment of lymphoblastoid cells with 360A, a synthetic ligand that binds to single-strand G4 structures, increases the formation of FSp53 mRNA and decreases p53I2 mRNA expression. These results indicate that G4 structures in intron 3 regulate the splicing of intron 2, leading to differential expression of transcripts encoding distinct p53 isoforms.

Functional assays to determine the significance of two common XPC 3'UTR variants found in bladder cancer patients.

BACKGROUND: XPC is involved in the nucleotide excision repair of DNA damaged by carcinogens known to cause bladder cancer. Individuals homozygous for the variant allele of XPC c.1496C > T (p.Ala499Val) were shown in a large pooled analysis to have an increased bladder cancer risk, and we found two 3'UTR variants, *611T > A and c.*618A > G, to be in strong linkage disequilibrium with c.1496T. Here we determined if these two 3'UTR variants can affect mRNA stability and assessed the impact of all three variants on mRNA and protein expression. METHODS: In vitro mRNA stability assays were performed and mRNA and protein expression measured both in plasmid-based assays and in lymphocytes and lymphoblastoid cell lines from bladder and breast cancer patients. RESULTS: The two 3'UTR variants were associated with reduced protein and mRNA expression in plasmid-based assays, suggesting an effect on mRNA stability and/or transcription/translation. A near-significant reduction in XPC protein expression (p = 0.058) was detected in lymphoblastoid cell lines homozygous for these alleles but no differences in mRNA stability in these lines was found or in mRNA or protein levels in lymphocytes heterozygous for these alleles. CONCLUSION: The two 3'UTR variants may be the variants underlying the association of c.1496C > T and bladder cancer risk acting via a mechanism modulating protein expression.

The associations of sequence variants in DNA-repair and cell-cycle genes with cancer risk: genotype-phenotype correlations.

DNA-repair systems maintain the integrity of the human genome, and cell-cycle checkpoints are a critical component of the cellular response to DNA damage. Thus the presence of sequence variants in genes involved in these pathways that modulate their activity might have an impact on cancer risk. Many molecular epidemiological studies have investigated the association between sequence variants, particularly SNPs (single nucleotide polymorphisms), and cancer risk. For instance, ATM (ataxia telangiectasia mutated) SNPs have been associated with increased risk of breast, prostate, leukaemia, colon and early-onset lung cancer, and the intron 3 16-bp repeat in TP53 (tumour protein 53) is associated with an increased risk of lung cancer. In contrast, the variant allele of the rare CHEK2 (checkpoint kinase 2 checkpoint homologue) missense variant (accession number rs17879961) was significantly associated with a lower incidence of lung and upper aerodigestive cancers. For some sequence variants, a strong gene-environment interaction has also been noted. For instance, a greater absolute risk reduction of lung and upper aerodigestive cancers in smokers than in non-smokers carrying the I157T CHEK2 variant has been observed, as has an interaction between TP53 intron 3 16-bp repeats and multiple X-ray exposures on lung cancer risk. The challenge now is to understand the molecular mechanisms underlying these associations.

Oxidation status of human OGG1-S326C polymorphic variant determines cellular DNA repair capacity.

The hOGG1 gene encodes the DNA glycosylase that removes the mutagenic lesion 7,8-dihyro-8-oxoguanine (8-oxoG) from DNA. A frequently found polymorphism resulting in a serine to cysteine substitution at position 326 of the OGG1 protein has been associated in several molecular epidemiologic studies with cancer development. To investigate whether the variant allele encodes a protein with altered OGG1 function, we compared the 8-oxoG repair activity, both in vivo and in cell extracts, of lymphoblastoid cell lines established from individuals carrying either Ser/Ser or Cys/Cys genotypes. We show that cells homozygous for the Cys variant display increased genetic instability and reduced in vivo 8-oxoG repair rates. Consistently, their extracts have an almost 2-fold lower basal 8-oxoG DNA glycosylase activity when compared with the Ser variant. Treatment with reducing agents of either the Cys variant cells directly or of protein extracts from these cells increases the repair capacity to the level of the Ser variant, whereas it does not affect the activity in cells or extracts from the latter. Furthermore, the DNA glycosylase activity of cells carrying the Cys/Cys alleles is more sensitive to inactivation by oxidizing agents when compared with that of the Ser/Ser cells. Analysis of the redox status of the OGG1 protein in the cells confirms that the lower activity of OGG1-Cys326 is associated with the oxidation of Cys326 to form a disulfide bond. Our findings support the idea that individuals homozygous for the OGG1-Cys variant could more readily accumulate mutations under conditions of oxidative stress.