Stella M. Melana

Increasing evidence for a human breast carcinoma virus with geographic differences

An early immunologic study suggesting that a virus similar to the mouse mammary tumor virus (MMTV) was associated highly with breast carcinoma in Tunisian patients, compared with patients in the United States, led the authors to examine different breast carcinoma populations by using more current molecular techniques.

Characterization of viral particles isolated from primary cultures of human breast cancer cells.

The association of human breast cancer with sequences similar to the mouse mammary tumor virus (MMTV) has been shown, but convincing evidence for the presence of viral particles in breast tumors has been lacking. We have described the complete proviral structure of a retrovirus in human breast cancer. This provirus, designated as human mammary tumor virus (HMTV), was 95% homologous to MMTV and revealed features of a replication-competent virus. We have therefore investigated the production of viral particles in primary cultures of human breast cancer (MSSM). Cells isolated from ascites or pleural effusions of patients with metastatic breast cancer contained viral sequences in their DNA, expressed Env protein, and showed retroviral particles by electron microscopy. Viral particles from culture media exhibited morphologic features of beta-retroviruses sedimenting at buoyant densities of 1.12 to 1.18 g/mL in sucrose gradients and showed reverse transcriptase activity. cDNA sequences from virion RNA were synthesized, amplified, and sequenced and all the virion genes were detected and 70% of the virion RNA was sequenced. The sequence homologies were, respectively, 85% to 95% compared with the MMTV and HMTV proviruses we have previously described. These results clearly show that breast cancer cells in primary cultures produced HMTV viral particles that are similar to the mouse virus and which may play a role in human breast cancer pathogenesis.

MMTV-like env gene sequences in human breast cancer.

Summary. We have previously detected an MMTV env gene-like 660 bp sequence in 38% of human breast cancers, but not in normal tissues or other tumors. In this communication we report the sequences from eleven tumors and three breast cancer cell lines, and compare them to four strains of MMTV and to the known endogenous retroviral sequences. The breast cancer sequences were highly homogenous to the MMTV’s, but not to the endogenous sequences suggesting an exogenous origin.

High prevalence of MMTV-like env gene sequences in gestational breast cancer.

Gestational breast cancer (BC) is generally associated with rapid growth and increased mortality. Because the presence of MMTV-like sequences in BC has been associated with laminin receptor expression, a marker of poor prognosis, gestational BCs were analyzed for MMTV env gene-like sequences to explore whether MMTV-like sequences were also associated with its adverse outcome. Whereas 30–38% of sporadic BC have the sequences, in gestational BC the prevalence is 62%. We suggest that hormonal response elements present in the MMTV-like LTR may play a role in promoting cell growth, as they do in the mouse system.

Detection of human mammary tumor virus proteins in human breast cancer cells.

Mouse mammary tumor virus (MMTV) has been proven to induce mammary cancer in mice. MMTV-like env gene sequences have been detected in one-third of the human breast tumors studied. The whole proviral structure with 95% homology to MMTV was found in two human breast tumors and was designated as human mammary tumor virus (HMTV). HMTV viral particles with betaretroviral features have been isolated. In addition, a retrovirus called human betaretrovirus (HBRV), homologous to the mentioned retroviruses, has been isolated from tissues of patients with primary biliary cirrhosis. In this report, the expression of HMTV envelope (Env) and capsid (Ca) was detected in 10 primary cultures of human breast cancer containing HMTV sequences (MSSM) by Western blot and fluorescence activated cell sorting (FACS), using a panel of antibodies against HMTV Env, HBRV Env and Ca and the MMTV Env Gp36 and Ca P27 proteins. By contrast, HMTV proteins did not react with antibody against the MMTV Env Gp52 protein. All the antibodies detected MMTV proteins with exception of two out of four monoclonal antibodies against HMTV Env. Approximately 13% of the MSSM cells showed HMTV protein expression by FACS analysis. This report shows the expression of HMTV proteins for the first time in human breast cancer cells using a panel of antibodies against HMTV, HBRV and MMTV proteins. This should be taken into consideration when MMTV antibodies are used to detect HMTV proteins in human tissues.

Human Mammary Tumor Virus (HMTV) sequences in human milk

BackgroundRetroviral sequences 90-95% homologous to the mouse mammary tumor virus (MMTV) were present in 38% of the breast cancers studied from American women and were not detectable in non-tumor breast tissue from the same patient. The entire proviral structure was described and viral particles were isolated from primary cultures of human breast cancer. This virus was designated as human mammary tumor virus (HMTV). Hormone response elements present in the HMTV Long-Terminal-Repeat (LTR) suggest a mechanism for association of HMTV with hormonally responding tissues. In fact, the incidence of HMTV sequences is higher in gestational breast cancers, which are associated with hormonal changes. Milk epithelial cells are also under hormonal regulation and therefore are excellent specimens for HMTV sequence detection.MethodsThe HMTV sequence was studied in milk samples from lactating women recruited with increased risk of breast cancer because they had undergone breast biopsies (Biopsy-Group) and lactating women without breast biopsies (Reference-Group).ResultsHMTV-env sequences were detected by PCR in milk of 7.61% of 92 women of the Reference-Group and in 20.55% of 73 women of the Biopsy-Group (p: 0.015). The sequences were 94-98% homologous to MMTV. HMTV-env and HMTV-env/LTR junction sequences were detected in high-speed pellet RNA, implying the presence of HMTV viral particles. PCR assays to detect the murine mitochondrial cytochrome oxidase gene and intracisternal-A-type particle sequences were performed to rule out mouse mitochondrial or genomic DNA contamination. Eight women of the 73 Biopsy-Group participants had breast cancer and the milk of only one of these eight women had HMTV-env sequences. In the remaining 65 women of the Biopsy-Group, under enough clinical suspicion to lead to biopsy, HMTV was detected in 14, nearly three times the number of milks as compared to the Reference-Group (21.54% versus 7.61%; p: 0.016).ConclusionThe significance of HMTV in milk from the Reference-Group, the greater frequency in the milk of women who had undergone a breast biopsy and its possible infectivity for infants are important questions under study. The similarity of HMTV to MMTV is striking and suggests one possible avenue for viral transmission in humans.

Transcription profile of a human breast cancer cell line expressing MMTV-like sequences

BackgroundIt has been postulated that inflammation caused by certain viruses might result in cancer. Recently, it was shown that childhood lymphoblastic leukemia, breast and ovarian cancers express an interferon-related signature, providing support for this notion. We have previously shown that 38% of the sporadic breast cancers contain MMTV-like env gene sequences. To find out if the presence and expression of MMTV-like sequences correlated with an inflammatory phenotype, we have compared the expression profile of two sublines of MCF-7 cells, one containing the MMTV-like sequences (env+), the other one lacking them (env-).ResultsThe results indicated that there were 47 differentially expressed genes between the two sublines. Among 27 upregulated genes in the env+ cells there were 7 interferon-related genes, 5 TNF-connected genes and 2 TGFβ-related genes.ConclusionThese results suggest that the env+ cells were most likely responding to an infectious agent, and support the hypothesis that a viral infection may play a role in breast cancer pathogenesis.

Poxvirus infection and apoptosis

The following excellent reviews have been published on poxviruses and apoptosis during the last few years: P.C. Turner and R.W. Moyer, Semin. Virology, 8: 453–469, 1998; J.L. Shisler and B. Moss, Semin. Immunol., 13: 67–72, 2001; and H. Everett and G. McFadden, Curr. Opin. Microbiol., 5: 395–402, 2002. These articles dealt with the viral products and the mechanisms by which they interfere with apoptosis. In this review, we summarize new and old information and also introduce a new approach to explore interactions between the host cell and the replicating virus.

Human mammary tumor virus (HMTV) in endometrial carcinoma.

Objective Human mammary tumor virus (HMTV) is 90% to 98% homologous to mouse mammary tumor virus, the etiological agent of mammary tumors in mice. Human mammary tumor virus sequences were found in 40% of the breast cancers studied in both American and Australian women. In addition, 10% of endometrial carcinomas studied in Australian women also contained HMTV sequences. We have explored the possibility that endometrial cancer of American women may also contain HMTV. Methods/Materials Nested polymerase chain reactions, radioactive internal probing, and sequencing were used to establish the presence of unique nucleotide sequences of HMTV in human genomic DNA. The genomic DNAs were tested to guarantee that they were free of murine DNA. Immunohistochemistry with a monoclonal antibody specific for HMTV envelope protein demonstrated that HMTV sequences were translated. Results Thirteen (23.2%) of 56 of the endometrial cancers studied contained HMTV sequences and proteins. Human mammary tumor virus sequences and protein were not detected in the 33 normal endometria studied. Conclusion Human mammary tumor virus, an agent with high homology to mouse mammary tumor virus, was found in 23.2% of the endometrial cancers studied, thus opening the possibility of a pathogenic role.

Presence of MMTV-like env gene sequences in human breast cancer

Park et al. 2010 (No evidence of MMTV-like env sequences in specimens from the Australian Breast Cancer Family Study, Breast Cancer Research and Treatment online first) have reported lack of evidence for MMTV-like env gene sequences in breast cancer specimens from Australia. We have recently addressed the question of differences in published results [1, 2] concerning MMTV-like env sequences in breast cancers. Besides obvious technical differences such as the ones considered here, there are major geographical variations in the incidence of breast cancer and in the frequency of viral sequences in the breast cancers. Ford et al. [3] reported that 40% of Australian women’s breast cancers contained MMTV-like viral sequences but none could be detected in specimens from Vietnam. We have also found 30–40% positivity in breast cancer specimens from four countries in the Americas and three in Europe, but a sharply lower frequency in breast cancer specimens from Iran, Japan, and China where breast cancer is less common [unpublished]. This coordinate epidemiology of breast cancer and viral presence suggests a relationship that may turn out to be causal. We have thus compared on the same American breast cancer specimens results obtained using the methodology of Park et al. with those analyzed under the experimental conditions we have previously described [1]. The results are shown in Fig. 1. The PCR product of MMTV-like env gene amplification following our conditions is shown (Fig. 1a). Although two bands in the gel are faint, three bands of molecular weight around 250 bp can be seen after hybridization with a labeled probe. The same three DNAs were used for amplification using the conditions of Park et al. In only one instance a 123 bp band in the gel and one hybridization band is seen (Fig. 1b). The amplified DNA was sequenced and shown to be 90% homologous to MMTV env gene. Park et al. stated that their methodology can amplify five copies of a plasmid containing env gene sequences diluted in 10 ng of negative human DNA. This does not establish that the conditions reliably amplify viral sequences in human tumor DNA. Our technology can detect one copy of a plasmid containing 660 bp of MMTV-like env gene in 100 ng of genomic DNA. It also includes hybridization with a labeled oligonucleotide internal probe, which allows conclusive detection and identification of the specific band product. In addition, we usually sequenced the PCR products.