Molecular Mechanism of Nucleotide Excision Repair Deficiency in Novel Breast Cancer Cell Lines

[摘要] Deficiency of DNA repair has been shown to lead to cancer in multiple human disease states. We therefore hypothesized that deficiency of Nucleotide Excision Repair is involved in the etiology of breast cancer. This work was carried out using a novel in vitro culture system for both normal and cancerous breast tissue developed in the Latimer laboratory. Primary cultures of non-diseased and tumor tissue were utilized in functional studies, and cell lines generated from these primary cultures were used for expression analysis. Cell lines were generated from the Latimer primary culture system without the use of exogenously transforming agents. Using the Unscheduled DNA Synthesis Assay, we have shown that stage I and stage II breast tumors are deficient in functional NER capacity as compared to non-diseased controls. Analysis of the steady-state mRNA expression levels of 20 genes in the NER pathway using an RNase Protection kit showed that 7 genes (CSB, XPA, XPB, TFIIHp52, TFIIHp44, TFIIHp34, and Cdk7) also showed a loss of expression in early stage tumor cell lines. Microarray analyses corroborated these trends and protein expression was also lost in early stage tumor cell lines for the XPA and CSB genes. In contrast to the loss of DNA repair capacity manifested in early stage tumors, stage III tumors showed an increase in repair capacity. Gain of mRNA expression and protein expression was seen for our candidate genes in late stage tumors. Because loss of expression was reversible and occurred in multiple genes and cell lines, we hypothesized that the mechanism responsible was epigenetic regulation of gene expression. Two early stage cell lines that demonstrated loss of expression of these candidate genes were treated with the demethylating agent 5-aza-2'deoxycytidine, resulting in the re-expression of 3 genes: TFIIHp52, TFIIHp34, and Cdk7. In contrast, two of the candidate genes that were not reactivated by azacytidine treatment (CSB and XPA) showed a 7-element transcription factor framework that may begin to explain their coordinate regulation. The functional loss of NER capacity in early stage tumors is consistent with a transcriptional regulatory mechanism including both methylation and other transcription factor based functions.

[发布机构] the University of Pittsburgh

[效力级别] DNA repair