Isabel Mellon

Transcription-Coupled Repair Deficiency and Mutations in Human Mismatch Repair Genes

Deficiencies in mismatch repair have been linked to a common cancer predisposition syndrome in humans, hereditary nonpolyposis colorectal cancer (HNPCC), and a subset of sporadic cancers. Here, several mismatch repair-deficient tumor cell lines and HNPCC-derived lymphoblastoid cell lines were found to be deficient in an additional DNA repair process termed transcription-coupled repair (TCR). The TCR defect was corrected in a mutant cell line whose mismatch repair deficiency had been corrected by chromosome transfer. Thus, the connection between excision repair and mismatch repair previously described in Escherichia coli extends to humans. These results imply that deficiencies in TCR and exposure to carcinogens present in the environment may contribute to the etiology of tumors associated with genetic defects in mismatch repair.

Polymorphisms in the human xeroderma pigmentosum group A gene and their impact on cell survival and nucleotide excision repair.

Polymorphisms in DNA repair genes may contribute to defects in DNA repair and increased susceptibility to cancer. The xeroderma pigmentosum group A (XPA) gene is required for nucleotide excision repair (NER) and mutations in XPA highly predispose humans to skin cancer. We examined DNA samples from 189 individuals for polymorphisms in the XPA gene. First, SSCP analysis was used to examine each of the six exons and their intron boundaries. One frequent single nucleotide polymorphism (SNP) in the untranslated region of exon 1 and two rare SNPs which produce the changes Arg228Gln and Val234Leu in the coding region of exon 6 were identified. Quite surprisingly, no sequence variants were found within the coding regions or the adjacent intron boundaries of exons 1-5. Ecdysone-inducible expression vectors containing wild type XPA cDNA or cDNAs representing the two polymorphisms that we identified in exon 6 were created and independently introduced into the XPA deficient cell line XP12RO-SV. Transcription-coupled repair (TCR), global genome repair (GGR) and cell survival following UV irradiation were studied in each cell line in the absence or presence of the ecdysone hormone analog, ponasterone A. No substantial difference in repair or cell survival was found in cells complemented with wild type or polymorphic alleles of XPA. A 10-fold increase in the expression of XPA by addition of ponasterone A resulted in faster removal of 6-4 photoproducts from the total genomes of cells complemented with wild type or polymorphic alleles of XPA but had no significant impact on TCR or global genome repair of cyclobutane pyrimidine dimers (CPDs). Since our SSCP analysis failed to detect significant numbers of polymorphisms we directly sequenced exons 4-6 in a subset of our samples. One additional rare SNP, which produces the change Leu252Val was found in exon 6 and four rare SNPs and one rare single nucleotide deletion were found in intron 4. Hence, the XPA gene appears to be a cold spot for genetic variation and rare polymorphisms in the coding region of the gene do not reduce NER or cell survival after UV irradiation.

Spontaneous and ionizing radiation induced mutations involve large events when selecting for loss of an autosomal locus

The mouse P19H22 embryonal carcinoma cell line contains two distinct chromosome 8 homologs, one derived from Mus musculus domesticus (M. domesticus) and the other derived from Mus musculus musculus (M. musculus). It also contains a deletion for the M. musculus aprt allele, which is located on chromosome 8. In this study, cells with spontaneous or induced aprt deficiencies were isolated from P19H22 and examined to determine the nature of the mutational events that had occurred. Ultraviolet radiation (UV), ethyl methanesulfonate (EMS), and two forms of ionizing radiation, 137Cs and 252Cf, were used for mutation induction. DNA preparations from the aprt deficient cells were initially screened with a Southern blot analysis and separated into two broad classes: those that had lost the M. domesticus aprt allele and those that had retained it. The overwhelming majority (> 95%) of the spontaneous and ionizing radiation-induced mutants exhibited aprt gene loss, indicating that relatively large events had occurred and that homozygosity for the deleted region was not a lethal event. Loss of heterozygosity for syntenic markers was found to be a common event in cells exhibiting aprt gene loss. In contrast, a majority of the UV-induced mutants (61%) and a substantial minority of the EMS-induced mutants (38%) retained the aprt gene. A sequence analysis confirmed that base-pair substitutions were responsible for this class of mutation. Gene inactivation associated with hypermethylation of the promoter region was found to be a rare event and was not induced by any of the mutagenic agents tested. The results demonstrate the suitability of the P19H22 cell line for mutational studies, particularly those that are large in nature.

Multiple mechanisms underlie metastasis suppressor function of NM23-H1 in melanoma

Abstractnm23-h1 was the first metastasis suppressor gene to be identified in humans, with early studies demonstrating its ability to inhibit the metastatic potential of breast carcinoma and melanoma cell lines. This report outlines recent findings from our laboratory indicating that the metastasis suppressor function of NM23-H1 in human melanoma involves a spectrum of molecular mechanisms. Analysis of NM23-H1-dependent profiles of gene expression in human melanoma cell lines has identified a host of target genes that appear to mediate suppression of directional motility. Of particular interest is a subset of motility-suppressing genes whose regulation by NM23-H1 is independent of its known kinase and 3′–5′ exonuclease activities. In parallel, we have recently observed that NM23-H1 expression appears to be required for genomic stability and for optimal repair of DNA damage produced by ultraviolet radiation and other agents. Thus, NM23-H1 might oppose not only the motile and invasive characteristics of metastatic cells but also the acquisition of mutations that drive malignant progression to the metastatic phenotype itself.

Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway.

Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER) pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC), a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6–4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6–4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke.

Relationships Between DNA Repair and Transcription in Defined DNA Sequences in Mammalian Cells

Certain types of damage to DNA pose blocks to the process of transcription. In particular, the presence of cyclobutane pyrimidine dimers has been shown to result in termination of transcription at the sites of the lesions (Sauerbier and Hercules, 1978). A predictable consequence of this fact is that the persistence of one or more pyrimidine dimers in the transcribed DNA strand of all copies of an essential, active gene will most certainly result in death of the cell. Thus, it might be considered a good strategy for cells to selectively repair the transcription blocking damage in their active genes and, in fact, to focus specifically upon the DNA strands that are being transcribed.

Inorganic arsenic inhibits the nucleotide excision repair pathway and reduces the expression of XPC

Chronic exposure to arsenic, most often through contaminated drinking water, has been linked to several types of cancer in humans, including skin and lung cancer. However, the mechanisms underlying its role in causing cancer are not well understood. There is evidence that exposure to arsenic can enhance the carcinogenicity of UV light in inducing skin cancers and may enhance the carcinogenicity of tobacco smoke in inducing lung cancers. The nucleotide excision repair (NER) pathway removes different types of DNA damage including those produced by UV light and components of tobacco smoke. The aim of the present study was to investigate the effect of sodium arsenite on the NER pathway in human lung fibroblasts (IMR-90 cells) and primary mouse keratinocytes. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts (6-4 PP) and cyclobutane pyrimidine dimers (CPDs). We find a concentration-dependent inhibition of the removal of 6-4 PPs and CPDs in both cell types treated with arsenite. Treatment of both cell types with arsenite resulted in a significant reduction in the abundance of XPC, a protein that is critical for DNA damage recognition in NER. The abundance of RNA expressed from several key NER genes was also significantly reduced by treatment of IMR-90 cells with arsenite. Finally, treatment of IMR-90 cells with MG-132 abrogated the reduction in XPC protein, suggesting an involvement of the proteasome in the reduction of XPC protein produced by treatment of cells with arsenic. The inhibition of NER by arsenic may reflect one mechanism underlying the role of arsenic exposure in enhancing cigarette smoke-induced lung carcinogenesis and UV light-induced skin cancer, and it may provide some insights into the emergence of arsenic trioxide as a chemotherapeutic agent.

Abstract 1445: The metastasis suppressor NM23-H1 promotes genomic stability through its 3’-5’ exonuclease and nucleoside diphosphate kinase activities following UV irradiation

NM23-H1 is a metastasis suppressor whose reduced expression is associated with aggressive forms of melanoma, hepatoma, and carcinomas of the breast, stomach and colon. The current study has identified NM23-H1 (termed H1 isoform in human, M1 in mouse) and two of its attendant enzymatic activities, the 3’-5’ exonuclease and nucleoside diphosphate kinase (NDPK), as novel participants in the response to UV-induced DNA damage. Kinetics of repair for total DNA polymerase-blocking lesions and nucleotide excision pathway-mediated repair of 6-4 photoproducts were significantly compromised in different cellular settings of NM23-H1-deficiency. These included the human melanoma cell line WM793 and embryo fibroblasts (MEFs) derived from mouse strains rendered deficient in either NM23-M1 alone or both the M1 and M2 isoforms in tandem. The NDPK activity of NM23-H1 was critical for early repair of both polychromatic UVB/UVA (275-400 nm)- and UVC (254 nm)-induced DNA damage. Elevated rates of spontaneous and UV-induced mutations were observed in WM793 cells and NM23-deficient MEFs. The mutational spectra reflected aberrant repair of 6-4 photoproducts and oxidatively-induced DNA damage, with the 3’-5’ exonuclease being the principal enzymatic activity required to reduce UV-induced mutagenesis. This study has provided the first evidence for an essential role of mammalian NM23 isoforms in maintaining genomic stability. This novel anti-mutator function appears relevant not only to the metastasis suppressor activity of NM23-H1, but also possibly resistance to UV-induced carcinogenesis. 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 1445. doi:10.1158/1538-7445.AM2011-1445

Selective DNA Repair in the Transcribed Strands of Active Genes in Mammalian Cells

Cyclobutane pyrimidine dimers are preferentially removed from transcriptionally active genes: (i) In Chinese hamster cells which remove only a small fraction of dimers from their total DNA, dimers are efficiently removed from the transcriptionally active dihydrofolate reductase (DHFR) gene and poorly removed from a sequence near the gene (Bohr et al., 1985). (ii) In mouse cells which also remove only a small fraction of dimers from their total DNA, dimers are proficiently removed from the transcriptionally active c-abl gene and poorly removed from the inactive c-mos gene (Madhani et al., 1986). (iii) In repair proficient human cells, dimers are removed more rapidly from the active DHFR gene than from bulk DNA or from the nontranscribed repetitive alpha sequences (Mellon et al., 1986). This work was recently reviewed by Smith (1987).