Characterization of cells carrying human mammary tumor virus (HMTV).

MELANA SM; VEPKHIA PILAURI; NEPOMNASCHY I; FERNADEZ-COBO M; SAKALIAN M; HASA J; ROJOWSKY H; DJOUGARIANM A; HOLLAND J; POGO B

Mystic, Connecticut, USA.

Workshop; •Workshop on Endogenous Retroviruses & Retroelements.; 2006

Cancer Research Institute, University of California

The association of human breast cancer with sequences similar to the mouse mammary tumor virus (MMTV) has been shown in different populations (Wang et al, Cancer Research 1995, 55: 5173; Etkind P et al, Clin Cancer Res, 2000, 6: 1273-8; Ford CE et al, 2003, Clin Cancer Res, 9: 1118-20; Zammarchi et al, 2006, J.Pathol.209: 436-44). The env gene sequences have been found in human tumor breast cells but not in the normal breast of the same individual (Melana et al, 2001 Clin Cancer Res, 7: 283-4). The complete 9.9Kb proviral sequence of an MMTV-like agent was amplified, sequenced in two breast cancers, and designated human mammary tumor virus (HMTV). This provirus was 95% homologous to MMTV and revealed features of a replication competent virus (Liu, B. et al, Cancer Res 2001, 61:1754-9). HMTV particles were isolated and characterized from primary cultures of human breast cancer (MSSM cells)  (Melana et al, submitted). Co-cultures experiments demonstrated that HMTV from MSSM cells was able to infect human mammary epithelial cells (HMEC), MCF10F, T lymphocytes (Jurkat), B lymphocytes (Ramos) and peripheral blood mononuclear cells (PBMC). Presence of env gene was confirmed by PCR. MCF10F cells have been remained infected after a year in culture. We have now investigated the expression pattern of MSSM cells, HMTV-infected human cells and two sublines of MCF7 cells using a panel of different antibodies against viral proteins by western blot, FACS analysis and immunofluorescence. Expression of ENV protein was found in all MSSM cells studied, in infected MCF10F and Ramos cells by western blot. Approximately 10% of the cells expressed Env proteins as shown by FACS analysis and immunofluorescence. We have also explored the expression of cellular proteins involved in epithelial messenchymal transition and signaling (cytokeratin 8,18, 19, vimentin, e-cadherin and Syk) in infected, uninfected MCF10F and MSSM cells. The results indicated that protein pattern of infected MCF10F cells is more similar to that of MSSM cells than that of the parental MCF10F, suggesting that this change was induced by the presence of HMTV. We have also performed cDNA arrays to compare the transcriptional profile of HMTV-expressing cells. For that purpose, we have used two sublines of MCF7cells, one containing and expressing env sequences, MCF7 env (+), and the other that lacks these, MCF7 env (-). MCF7 env (+) display a profile enriched in genes involved in infection/inflammatory processes. These results are significant because they were obtained by comparing cells derived from the same cell line that have similar genetic background and minimal expressing differences. Finally, we have found HMTV insertion sites in human infected cells. The results showed that there were several insertion sites, some of which may have important functional implications in pathogenesis.