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Evidence for the transforming activity of a truncated Int6 gene, in vitro
S B Rasmussen1, E Kordon, R Callahan
1National Institutes of Health, National Cancer Institute, Laboratory of Tumor Immunology and Biology, 10 Center Drive Room 8B07, Bethesda, Maryland, MD 20892-1750, USA.
Abstract:
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.
Insights
Mouse mammary tumor virus (MMTV) integration into Int6 creates a shortened Int6 protein (Int6sh). This Int6sh drives mammary epithelial cell transformation and tumor formation, indicating its role in malignant progression.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Int6/eIF3-p48 is a common integration site for Mouse Mammary Tumor Virus (MMTV) in mouse mammary tumors.
- MMTV integration interrupts the normal Int6 transcript, creating a mutated allele producing a shortened Int6 mRNA and protein (Int6sh).
Purpose of the Study:
- To investigate the transforming potential and dominant function of the mutated Int6sh protein.
- To provide direct evidence for the contribution of Int6 mutations to mammary epithelial cell malignant transformation.
Main Methods:
- Transfection of human (MCF10A) and mouse (HC11) mammary epithelial cells with Int6sh.
- Assessing anchorage-independent growth in soft agar to identify transformed phenotypes.
- RT-PCR and Northern blot analysis to quantify Int6sh and wild-type Int6 RNA transcripts.
- In vivo studies using immune-compromised hosts to evaluate tumor formation.
Main Results:
- Expression of Int6sh induced anchorage-independent growth in MCF10A and HC11 cells, indicating transformation.
- Transformed cells exhibited high levels of both mutated Int6sh and wild-type Int6 RNA.
- Int6sh-transformed cells formed nodular growths in vivo.
- NIH3T3 mouse embryo fibroblasts were also transformed by Int6sh expression.
Conclusions:
- The mutated Int6sh protein possesses transforming potential and a dominant function.
- Int6 mutations are directly implicated in the malignant transformation of mammary epithelial cells.
- Int6sh contributes to tumor development in MMTV-induced mammary tumors and hyperplasia.