PROHIBITIN IN BREAST-CANCER CELL-LINES - LOSS OF ANTIPROLIFERATIVE ACTIVITY IS LINKED TO 3'-UNTRANSLATED REGION MUTATIONS

The evolutionarily conserved prohibitin gene is located on human chrom osome 17q21, and two alleles have been identified. Our previous studie s characterizing prohibitin in immortalized cells, classified into fou r complementation groups (A-D) based on the ability of whole-cell hybr ids to become senescent, have suggested that it has tumor suppressor a ctivity in group B cells, Only the cell lines assigned to group B are sensitive to the antiproliferative activity of prohibitin, and all are homozygous for an allele designated a because of its exclusive associ ation with this group. Prohibitin genotyping of 22 breast cancer cell lines identified 17 homozygous for the a allele, 5 homozygous for the non-a allele, and no heterozygotes. Four of these cell lines were chos en for further characterization of prohibitin. In cell proliferation a ssays, the homozygous B breast cancer cell lines (BT-20, SK-BR-3, and MCF7) are all inhibited from traversing the cell cycle following the i ntroduction of wild-type prohibitin transcripts. The cell line homozyg ous for the alternative non-a allele (BT-549) is not inhibited by tran scripts. All of the breast cancer cell lines overexpress the longer fo rm of the prohibitin mRNA (1.9 kb) and the protein, Mutational analysi s of the protein-coding region detected no mutations in any of the lin es. However, BT-20, SK-BR-3, and MCF7 cells are all mutated in the fin al 200 bases of the 3' untranslated region (3'UTR) exclusive to the 1. 9-kb transcript, but BT-549 cells had no alterations in this region of the 3'UTR. Functional mapping experiments performed in the mutated SK -BR-3 line showed that the wild-type 3'UTR alone is sufficient to inhi bit cell cycle progression, indicating that the antiproliferative acti vity of the prohibitin transcript is localized to this region. Overall , our results show that most (80%) of the cell lines derived from brea st tumors have a common prohibitin genotype, suggesting that they belo ng to the same group of immortalized cells, group B. The results also show that the prohibitin 3'UTR exhibits the characteristics of a trans -acting regulatory RNA (riboregulator), the tumor suppressor activity of which is inactivated by mutation in group B immortalized cells.