Evidence for the transforming activity of a truncated Int6 gene, in vitro

Int6/eIF3-p48 was first identified as a common integration site for MMTV in mouse mammary tumors. In all cases, the MMTV integration event resulted in an interruption of the normal Int6 transcript from one allele leaving the second allele intact and operative. We hypothesize that insertion of MMTV i nto Int6 results in a mutated allele that encodes a shortened Int6 mRNA and protein (Int6sh), which either modifies normal Int6 function or possesses a new independent function. To confirm the transforming potential of the mu tation and its dominant function, we transfected two mammary epithelial cel l lines, MCF10A (human), and HC11 (mouse), with Int6sh under the control of the elongation factor-la (eEF1A) promoter. Expression of Int6sh in MCF10A and HC11 mammary epithelial cells leads to anchorage-independent growth in soft agar indicative of a transformed phenotype. Colonies selected from aga r exhibited high levels of mutated Int6sh and wild type Int6 RNA transcript s by RT-PCR and Northern blot analysis. In addition, Int6sh transformed MCF 10A and HC11 cells formed nodular growths, in vivo, in immune compromised h osts. NIH3T3 cells, mouse embryo fibroblasts, were also transformed to anch orage-independent growth in vitro by Int6sh expression. These observations provide direct evidence that the Int6 mutations observed in MMTV-induced tu mors and hyperplasia contribute to the malignant transformation of the mamm ary epithelial cells.