In comparison with MCF cells, V cells and HBL cells, HCC cells were significantly more radiationsensitive.Radiation sensitivity seemed to correlate with the genetic status of BRCA.All other mutants tested, however, destroyed the growth suppressive effect of BRCA and could therefore be used in the cell growth and survival assay. The HCC cells contain an endogenous BRCA mutation that eliminates the BRCT domain but leaves the transactivation domain intact.Because the HCC cells are radiationsensitive, this indicates that the BRCT domain is necessary for radiation resistance.Mutants that deleted both the BRCT domain and the transactivation domain could not rescue radiation sensitivity. In contrast to these findings, two deletion mutants. These two mutants can eliminate the role of BRCA as a growth suppressor but can reverse radiation sensitivity, showing that these functions can be separated.To study the repair capacity of HCC cells lacking BRCA and those containing a form of BRCA that reverses radiation sensitivity, stably transfected cell lines were generated.HCC cells were transfected with BRCAD on a vector also containing the neomycin resistance gene.After weeks of selection in G, approximately <a href="http://inhibit09.online/archives/221"></a>
colonies were selected and propagated.These antibodies recognize the far C terminus of BRCA.HCC cells contain a mutant form of BRCA that lacks this epitope.These two clones were then tested for radiation sensitivity in the colony forming assay.Thus by both transient and stable transfection BRCAD reverses the radiation sensitivity of the HCC cells.The ability to generate HCC cells containing stably transfected BRCAD allowed us to compare the DNA repair capabilities of parental HCC cells and HCC cells containing BRCAD. Under the electrophoresis conditions chosen, damaged DNA runs as a discrete band below the undamaged or repaired DNA. HCC BRCAD cells also repaired most of the damaged DNA within h. These cells showed slightly faster repair than parental HCC cells, but the difference is too small to account for the log difference in radiation sensitivity between these two cell lines.Note that virtually no signal is present in the bound sample at time zero because insufficient time has passed for any repair to have occurred.Upper panels, HCC cells show little difference in the transcribed DNA either or hafter repair. The same result is seen in the nontranscribed strand.Lower panels, HCC cells stably transfected with BRCAD. As expected, the nontranscribed strand shows little repair.Each experiment was performed three times with similar results each time.Because only a handful of cell lines have been shown to be as radiationsensitive as the XRVB cell line, this finding indicates that the sensitivity of the HCC cell line is likely to be a real effect.We used a recently described method to measure cell survival after transient transfection and irradiation to determine whether reintroduction of BRCA into the HCC cells could reverse the radiation sensitivity of these cells.Surprisingly, we were unable to demonstrate that reintroduction of fulllength BRCA into HCC reverses radiosensitivity because this assay depends on the continued growth of the cells after transfection.Thus, because BRCA is such a potent suppressor of cell growth, transfection of BRCA stops cell growth and does not allow us to accurately measure cell survival following irradiation.