The CCND1 gene, localized to chromosome band 11q13, is amplified in approximately 15% of human primary breast tumors. From 30 to 40% of the tumors presenting this amplification show concomitant amplification at the FGFR1 locus in 8p12. Similarly, MDA-MB-134 breast cancer cells bear CCND1 and FGFR1 coamplified, resulting in the formation of a hybrid intrachromosomal amplification assembling 11q13 and 8p12 sequences. To learn whether similar amplified structures arise in breast tumors, we used a two-color FISH approach on interphase nuclei. A cohort of 225 breast tumors was analyzed by Southern blotting and a subset of 12 tumors presenting the 11q13-8p12 coamplification was selected for further study by interphase FISH. In 6/12 tumors the FISH signals for 11q13 and 8p12 probes formed colocalizing clusters of green and red spots in the nuclei. The FISH patterns were identical to those observed on MDA-MB-134 interphase nuclei hybridized with 11q13 and 8p12. These data, suggesting the formation in these tumors of a hybrid amplification domain in which 11q13 and 8p12 sequences are joined, were reinforced by dual-color FISH on extended chromatin showing that the said were sequentially aligned in these tumors. Furthermore, 3/6 nuclei with colocalized 11q13 and 8p12 amplifications showed fusion of centromeric sequences from chromosomes 8 and 11. Our data strongly suggest the occurrence, in approximately 3% of primary breast tumors, of a recurrent rearrangement involving the proximal portions of 8p and 11q and resulting in the formation of a hybrid amplified structure composed of 11q13 and 8p12 sequences.
The role of the p53 protein in mediating G1 and G2 cell-cycle arrests after genotoxic insult has been clearly and reproducibly established in primary diploid fibroblasts, but data obtained from p53 wild-type (wt) cancer cell lines are inconsistent. Furthermore, a large proportion of human tumors have p53 wt genotypes but present genetic aberrations that may result from defective cell-cycle checkpoints. We therefore investigated the integrity of G1/S and G2/M cell-cycle arrests in p53 wt cancer cell lines. In the study presented here, we showed that in most cancer cells tested, G1 arrest was relaxed or absent in comparison with arrest in normal diploid fibroblasts, despite seemingly normal p53 and p21 responses. Two cell lines (MCF7 and HCT116) were synchronized in G0/G1 by leucine starvation and subjected to genotoxic stress to determine more precisely the relative proportion of cells arresting in G1 and G2. Whereas the MCF7 cells showed consistent G1 arrest, the HCT116 cells showed none at all. Furthermore, cell-cycle arrests in G1 and G2 in response to gamma irradiation and bleomycin treatment were transient, as the cells resumed cycling after 48-72 h. The cells resuming proliferation suffered massive apoptosis, but a proportion of the cells were rescued and showed normal doubling times. These cells retained a p53 wt genotype but presented gross chromosomal aberrations in 15-20% of the analyzed metaphases. The aberrations were not clonal. These data show that p53 wt cancer cells have relaxed cell-cycle controls after genotoxic insult and tolerate unrepaired chromosomal damage, despite normal p53 function.
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