The Effect Of DNA Damage Agent On Toxicity Injury Of Breast Cancer Cell Line Carrying Ring And BRCT Functional Region Mutation In BRCA1

Objective:To compare the susceptibility of breast cancer cell lines(HCC1937)with different functional mutation sites of RING and BRCT domains in breast cancer susceptibility gene 1(BRCA1)in response to different types of DNA damage agents.Methods:two tumor associated-relative mutant sites,C16G of RING domain and P1749R of BRCT domain in BRCA1,were selected to investigate the response of RING and BRCT functional region to DNA damage.By BRCA1 deficient HCC1937 cell line,the cell lines expressing C16G,P1749R,wild-type BRCA1(wtBRCAl)or empty vector blank control vector(vector)were established respectively.The expression of BRCA1 protein in four cell lines was detected by Western-blot.Cells were treated with ionizing radiation(IR,the doses of 2,4,6 Gy),mitomycin C(MMC,incubation with 0.2,1,5 ?g/ml for 1 h),ultraviolet(UV,the doses of 2 4,6 J/m2),respectively,each treatment were set the untreated group.The changes of 3H-TdR incorporation rate and cell survival rate were studied by 3H-TdR incorporation test,MTT assay and cell clonogenic formation assay respectively.Results:HCC1937 cell line expressing C16G,P1749R,wtBRCA1 or vector were successfully established,and the amount of C16G,P1749R and wtBRCAl were the same in the cells,indicating that the results in this study was reliable.The incorporation rate of 3H-TdA was decreased in four kinds of cell lines after different doses of IR,MMC or UV treatment,and the difference was significant(P<0.05)compared with the untreated group.3H-TdR incorporation was negatively correlated with exposure dose of IR,MMC or UV.Compared with wtBRCAl,the incorporation rate and cell viability of the cell lines expressing C64G,P1749R and vector were significantly lower(P<0.05),and the changes of the incorporation rate and cell survival in the cell lines expressing C64G,P1749R and vector were basically the same.By clonogeic formation and cell viability,the sensitivity of cell line expressing C64G to was higher than the cells expressing P1749R in response to IR,MMC or UV,and the difference was statistically significant(P<0.05).Conclusion:The mutations in RING and BRCT functional regions of BRCA1 can lead to increased sensitivity of cells to DNA damage induced by IR,MMC or UV,and the degree of injury is dose-dependent.Among them,in response to a variety of DNA damage,the cells with C64G mutation was more sensitive than the cells expressing P1749R mutation.