Figure 3
Genotypic and phenotypic alterations of c-MYC in irradiated huMECs and MCF-10A cells. c-MYC interphase FISH copy number analysis of parental and irradiated MCF-10A populations. Three main cell populations were identified by FISH: cells with two copies of chromosome 8 centromere (green probe) and three copies of c-MYC (red probe), cells with two copies of chromosome 8 and four copies of c-MYC and cells with two copies of chromosome 8 and over four copies of c-MYC (a). The proportion of 100 scored nuclei with these three c-MYC genotypes was combined and determined for each population. The proportion of nuclei with ⩾4 copies of c-MYC and therefore any cell population with a c-MYC copy number gain was determined (b). Aliquots of 10 μg of protein extracted from parental and irradiated MCF-10A cell populations were electrophoresed on polyacrylamide gels and analysed for c-MYC and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression by western transfer analysis as described in the Materials and methods (c). c-MYC expression was quantified for each cell population by densitometric analysis of western blots from three independent protein samples (d). c-MYC expression is expressed as a percentage of c-MYC expression in parental MCF-10A, which is set at 100% expression. Expression of c-MYC was significantly higher in the 60 Gy population than parental MCF-10A (Turkey's test; P<0.05). c-MYC interphase FISH copy number analysis of parental and irradiated HuMECs (e). Four main cell populations were identified by FISH: diploid cells (two copies of chromosome 8 centromere (aqua probe), two copies of IGH (green probe) and two copies of c-MYC (red probe)); triploid cells (three copies of each locus); tetraploid cells (four copies of each locus); cells with amplification of c-MYC. At least 70 interphase cells were counted at each radiation dose (mock-treated, 2, 3 and 4 Gy) and example images are shown for diploid, tetraploid and c-MYC-amplified cells.
