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 into 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 mutation and its dominant function, we transfected two mammary epithelial cell lines, MCF10A (human), and HC11 (mouse), with Int6sh under the control of the elongation factor-1alpha (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 agar exhibited high levels of mutated Int6sh and wild type Int6 RNA transcripts by RT-PCR and Northern blot analysis. In addition, Int6sh transformed MCF10A and HC11 cells formed nodular growths, in vivo, in immune compromised hosts. NIH3T3 cells, mouse embryo fibroblasts, were also transformed to anchorage-independent growth in vitro by Int6sh expression. These observations provide direct evidence that the Int6 mutations observed in MMTV-induced tumors and hyperplasia contribute to the malignant transformation of the mammary epithelial cells.
Rasmussen
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National Institutes of Health, National Cancer Institute, Laboratory of Tumor Immunology and Biology, 10 Center Drive Room 8B07, Bethesda, Maryland, MD 20892-1750, USA.
Kordon
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Callahan
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Smith
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D016475:3T3 Cells; D000362:Agar / Q000494:pharmacology; D000483:Alleles; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D001940:Breast / Q000473:pathology; D002455:Cell Division; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000378:metabolism; D039621:Eukaryotic Initiation Factor-3; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D039621:Eukaryotic Initiation Factor-3; D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D000362:Agar