UC Riverside

Breast cancer is the second leading cause of cancer deaths among women in the United States. Research has linked genetic and lifestyle factors to increased risks of developing this disease. However, in spite of recent advances, there continue to be significant gaps in our understanding of the causes, susceptibility factors and mechanisms underlying the etiology of breast cancer. Environmental factors also contribute and it is believed that unintentional exposures to various physical and chemical agents play a major role in the etiology of this and other types of cancer. !Studies have reported that women with increased size variability of the constitutive heterochromatin regions of chromosomes 1 and 9 in normal peripheral blood cells are at increased risk of developing breast cancer. Previous work in our laboratory using a novel technique integrating fluorescence in-situ hybridization (FISH) with probes specific for the classical satellite regions located at the heterochromatin regions of chromosomes 1 and 9 was applied to lymphoblastoid cells lines derived from breast cancer patients and age-, ethnicity- and sex-matched controls. These results corroborated early studies reporting that the paracentromeric heterochromatin regions of chromosomes 1 and 9 were significantly more variable in size than those from the matched controls. The mechanisms underlying the increase in variability are unknown. The objectives of this research were to first, confirm and extend the earlier results from our laboratory, and secondly, to investigate the hypothesis that the increased variability observed is due to inefficient DNA repair of DNA inter-strand crosslinks. In comparing the results of the new and the earlier studies, similar results were seen, and as a result, the data were combined to enlarge the sample size and increase the accuracy of the results. The combined analyses confirmed that the size of the constitutive heterochromatin was significantly more variable in most cells obtained from the breast cancer patients when compared with the matched controls; the constitutive heterochromatin region was significantly more variable in 7 of the 10 breast cancer patient-derived cell lines when examining both chromosomes 1 and 9.

To assess the role of DNA damage, we exposed six breast cancer patient cell lines to the alkylating agents, mitomycin C, melphalan and 2-chloroethylamine to investigate changes in the heterochromatin regions that occurred following treatment. Two of the six cell lines showed variability in the heterochromatin region of chromosome 9 that was strikingly greater following treatment than that seen in the matched controls. Interestingly, a similar trend was not seen with chromosome 1. The increase in heterochromatin variability was seen following treatment with both bifunctional and monofunctional alkylating agents indicating that the increase was not dependent upon the formation of DNA crosslinks. Of note, the two particularly sensitive cell lines did not exhibit major increases in alkylating agent-induced chromosome breakage in the micronucleus assay indicating that variability is not likely to be due to inefficient repair of genome-wide DNA breaks. Lastly, in a time-course experiment, the increases in heterochromatin variability in the two sensitive cell lines were seen at both 24 and 48 hr after treatment with mitomycin C, a potent cross linking agent. Interestingly, similar but only transient increases (at 24 hr. only) were also seen with DMSO treatment in the sensitive cells, but not controls, suggesting that the observed increases are not due to changes in DNA sequence but are more likely due to epigenetic changes occurring in the sensitive cell lines.