Mouse Mammary Tumor Virus Env Research Articles

Abstract 1149: Decitabine inhibits breast cancer cell proliferation through inhibition of endogenous retroviruses in vitro

Abstract The purpose of the study is to elucidate the antineoplastic mechanisms of decitabine, a DNA methyltransferase inhibitor. Previous literature suggests that decitabine activates human endogenous retroviruses which subsequently enhances expression of interferons, thereby suppressing tumor cell proliferation. Contrary to this hypothesis, our in vitro study with human and mouse breast cancer cell lines showed a positive correlation between the antiproliferative effect of decitabine and its suppressive effect on two endogenous retroviruses, the human endogenous retrovirus type K (HERV-K) and the mouse mammary tumor virus (MMTV). Decitabine stimulated proliferation of the T47D human breast cancer cell line at lower doses but inhibited cell proliferation at higher doses. Correspondingly, HERV-K transcripts (env and pol) increased at lower doses and decreased at higher doses. Interestingly, expression of the E-cadherin gene, an indicator of epithelial differentiation, followed an opposite trend. Decitabine inhibited proliferation of the mouse 4T1 breast cancer cell line and its expression of MMTV in a dose-dependent manner, while enhancing expression of the mouse E-cadherin. 4T1 cells overexpressing MMTV env became more resistant to decitabine, so was a human breast cancer cell line (BT-20) overexpressing HERV-K env, while T47D cells with HERV-K knockdown became more susceptible to the drug. Meanwhile, in vivo studies showed that decitabine successfully suppressed tumor development in BALB/c mice inoculated with 4T1 cells. Surprisingly, expression of MMTV env was elevated in tumors of mice treated with decitabine, along with enhanced E-cadherin expression. These data suggest decitabine inhibits cancer cell proliferation (at least partially) through inhibition of endogenous retroviruses in vitro, but its mechanisms of action in vivo are different. Further studies of the interaction between decitabine and endogenous retroviruses using engineered 4T1 cells in BALB/c mice are currently underway. Citation Format: Jiayi Li, John Lin, Leo Andrada, Bryce Grohol, Serratt Nong, Yingguang Liu. Decitabine inhibits breast cancer cell proliferation through inhibition of endogenous retroviruses in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1149.

Association of Mouse Mammary Tumor Virus With Human Breast Cancer: Histology, Immunohistochemistry and Polymerase Chain Reaction Analyses.

The purpose of this study is to determine whether mouse mammary tumor virus (MMTV)-associated human breast cancer has the same or similar histology to MMTV-associated mouse mammary tumors. Such associations may indicate a role for MMTV in human breast cancer. Immunohistochemical techniques (using antibodies directed against the signal peptide p14 of the envelope precursor protein of MMTV) and polymerase chain reaction (PCR) analyses were used to identify MMTV proteins and MMTV-like envelope gene sequences in a series of breast cancers from Australian women. The histological characteristics of these human breast cancer specimens were compared with MMTV positive mouse mammary tumors. The same methods were used to study benign breast tissues which 1-11 years later developed into breast cancer. MMTV p14 proteins were identified in 27 (54%) of 50 human breast cancers. MMTV env gene sequences were identified by PCR in 12 (27%) of 45 human breast cancers. There was a significant correlation between the presence of MMTV (identified by p14 immunohistochemistry) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.001). There was a non-significant correlation between the presence of MMTV env gene sequences (identified by PCR) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.290). MMTV p14 proteins were identified in 7 (54%) of 13 benign breast specimens that later developed into human breast cancers. MMTV by PCR was identified in two benign specimens one of whom later developed MMTV positive breast cancer. These observations offer evidence that MMTV may be associated with characteristic human breast cancer histology. p14-based immunohistochemistry appears to be a more reliable technique than PCR for the identification of MMTV in human breast cancer. Identification of MMTV-associated p14 proteins in benign breast tissues confirms prior PCR-based studies that MMTV infection occurs before the development of MMTV positive breast cancer. Many MMTV positive human breast cancers have similar histology to MMTV positive mouse mammary tumors. MMTV infection identified in benign breast tissues precedes development of MMTV positive human breast cancer. When considered in the context of prior studies, these observations indicate a likely role for MMTV in human breast cancer.

Prevalence of Mouse Mammary Tumor Virus (MMTV)-like sequences in human breast cancer tissues and adjacent normal breast tissues in Saudi Arabia

BackgroundBreast cancer is considered the most common cancer in women worldwide and is the leading cause of cancer mortality. Sequences similar to Mouse Mammary Tumor Virus (MMTV) were detected in human breast cancer in several studies from different geographical areas. However, the role played by this virus in breast cancer tumorigenesis is not completely understood. These MMTV-like sequences were found to be associated with breast cancer of more malignant types. The aim of this study is to determine the prevalence of MMTV-like envelope gene (env) positivity in breast cancer and non-cancerous breast tissue from Saudi Arabia.MethodsDetection of MMTV-like env proviral sequences was done using newly designed primers for conventional polymerase chain reaction (PCR). One hundred nighty four samples were collected from 103 females with breast cancer in addition to 51 control breast tissue obtained from individuals without cancer. We additionally investigated the association of proviral positivity with age of the patients, grade of breast cancer and presence of lymph node metastasis. The results were confirmed by sequencing.ResultsThe prevalence of MMTV-like env proviral positivity was 8.7% (9/103). MMTV env proviral sequences were detected in 5.9% (6/101) of breast cancer tissues and 9.7% (9/93) of non-cancerous adjacent tissues obtained from the same patients. None of the 51 control sample showed positive result for the MMTV env gene. No significant association was found between detection of the virus and the age of the patient, grade of the cancer or presence of metastasis.ConclusionWe document the presence of low frequency of MMTV env provirus sequence among breast cancer patients from Saudi Arabia. Further studies are needed to explore the role of the MMTV in breast cancer.

No Evidence of Mammary Tumor Virus env Gene-Like Sequences among Iranian Women with Breast Cancer

Objective: The mouse mammary tumor virus (MMTV) is the well-established etiological agent of mammary tumors in mice. A series of studies have implicated that a human murine mammary tumor virus-like virus occurs in human breast cancer, but it is unclear whether it has any causal role. Methods: The aim of the present study was to investigate the presence of MMTV env gene-like sequences in a group of Iranian women with or without breast cancer. A total of 65 breast cancer and 65 noncancerous breast specimens from the Department of Pathology of Tabriz University in East Azerbaijan, Iran, were analyzed by nested PCR. Results: All breast cancer and benign breast samples were negative for MMTV env gene-like DNA. Conclusion: These results indicate that the MMTV env gene-like virus may not play a significant role in the etiology of breast cancer among Iranian women.

Genomic DNA of MCF-7 breast cancer cells not an ideal choice as positive control for PCR amplification based detection of Mouse Mammary Tumor Virus-Like Sequences

The identification of the etiology of breast cancer is a crucial research issue for the development of an effective preventive and treatment strategies. Researchers are exploring the possible involvement of Mouse Mammary Tumor Virus (MMTV) in causing human breast cancer. Hence, it becomes very important to use a consistent positive control agent in PCR amplification based detection of MMTV-Like Sequence (MMTV-LS) in human breast cancer for accurate and reproducible results. This study was done to investigate the feasibility of using genomic DNA of MCF-7 breast cancer cells to detect MMTV-LS using PCR amplification based detection. MMTV env and SAG gene located at the 3′ long terminal repeat (LTR) sequences were targeted for the PCR based detection. No amplification was observed in case of the genomic DNA of MCF-7 breast cancer cells. However, the 2.7kb DNA fragment comprising MMTV env and SAG LTR sequences yielded the products of desired size. From these results it can be concluded that Genomic DNA of MCF-7 cell is not a suitable choice as positive control for PCR or RT-PCR based detection of MMTV-LS. It is also suggested that plasmids containing the cloned genes or sequences of MMTV be used as positive control for detection of MMTV-LS.

Mouse Mammary Tumor Virus Suppresses Apoptosis of Mammary Epithelial Cells through ITAM-Mediated Signaling

Many receptors in hematopoietic cells use a common signaling pathway that relies on a highly conserved immunoreceptor tyrosine-based activation motif (ITAM), which signals through Src family tyrosine kinases. ITAM-bearing proteins are also found in many oncogenic viruses, including the mouse mammary tumor virus (MMTV) envelope (Env). We previously showed that MMTV Env expression transformed normal mammary epithelial cells and that Src kinases were important mediators in this transformation. To study how ITAM signaling affects mammary cell transformation, we utilized mammary cell lines expressing two different ITAM-containing proteins, one encoding a MMTV provirus and the other a B cell receptor fusion protein. ITAM-expressing cells were resistant to both serum starvation- and chemotherapeutic drug-induced apoptosis, whereas cells transduced with these molecules bearing ITAM mutations were indistinguishable from untransduced cells in their sensitivity to these treatments. We also found that Src kinase was activated in the MMTV-expressing cells and that MMTV-induced apoptosis resistance was completely restored by the Src inhibitor PP2. In vivo, MMTV infection delayed involution-induced apoptosis in the mouse mammary gland. Our results show that MMTV suppresses apoptosis through ITAM-mediated Src tyrosine kinase signaling. These studies could lead to the development of effective treatment of nonhematopoietic cell cancers in which ITAM-mediated signaling plays a role.

Abstract 5751: Detection of HMTV in breast and endometrium carcinomas

Abstract Retroviral sequences homologous to the betaretrovirus mouse mammary tumor virus (MMTV), the etiological agent of mammary tumor in mice, are present in 40% of American women's breast cancers. A 660 bp sequence homologous to MMTV env gene with no significant homology to any other viral or human sequence reported in the Gen Bank is found in breast cancer, but not in adjacent normal breast tissues, indicating its exogenous origin. A complete provirus structure with 95% homology to MMTV has been isolated from two human breast tumors and named human mammary tumor virus (HMTV). Betaretroviral particles from primary cultures of metastatic breast cancer cells (MSSM) have been isolated and characterized. HMTV virion RNA is more than 90% homologous to MMTV RNA and to the HMTV proviral DNA. HMTV is able to infect a variety of cells bringing about striking molecular changes, as seen by co-culture experiments between MSSM cells and normal human epithelial breast cells, B and T human lymphocytes and human dendritic cells. Protein expression was only observed in 10-15% of the infected cells by FACS analysis, suggesting the presence of innate resistance in human cells after HMTV infection, as well as in breast cancer cells. The human retroviral restriction factors APOBEC F and G and the TRIM proteins are all highly expressed in HMTV infected cells as well as in breast cancer cells as measured by quantitative RT-PCR. Disruption of the cytoskeleton, evidence for epithelial mesenchymal transition (EMT) and increase in invasiveness are additional molecular changes seen in HMTV infected cells. Analysis of the sequencing data showed that HMTV also contains hormone response elements (HRE) homologous to MMTV HRE. This result encouraged us to investigate if HMTV were present in hormonal responsive tissues other than breast. Endometrium is a classical example of such tissue. Endometrial cancers and normal endometrium were analyzed for HMTV env sequences. HMTV env sequences were present in 0% of 29 normal tissues and 23.2% of 56 tumors analyzed. Johal et al. recently reported that MMTV-like sequence was found in 10% of the endometrial cancers (J. Med. Virology; 82: 1044-50). We describe here the detection of HMTV proteins by immunohistochemistry (IHC) in formalin fixed paraffin embedded (FFPE) slides obtained from breast and endometrium carcinomas but not in their respective normal tissues. These results are compared with the results obtained by PCR. PCR is a very sensitive technique able to detect a single DNA copy number but potential contaminations can result in false positives. In addition, IHC is accessible to pathology laboratories since no hybridization step with radioactive material is necessary as in the HMTV PCR. In conclusion, we have developed a contamination resistant method like IHC using monoclonal antibodies against HMTV that correlates with the results we have obtained by western blot and by PCR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5751. doi:1538-7445.AM2012-5751

The 5′ leader sequence of mouse mammary tumor virus enhances expression of the envelope and reporter genes

Mouse mammary tumor virus (MMTV) is a complex betaretrovirus, which utilizes a Rev-like auxiliary protein Rem to export the unspliced viral RNA from the nucleus. MMTV env mRNA appears to be exported via a distinct, Rem-independent, mechanism. Here, we analysed the effect of an extensively folded region coinciding with the 5' leader sequence on env gene expression. We found that the presence of the 5' leader stimulates expression of the envelope protein. Enhanced Env production was accompanied by increased cytoplasmic levels of env mRNA. The 5' leader promotes nucleocytoplasmic translocation and increases stability of env mRNA. The region responsible for this effect was mapped to the distal part of the 5' leader. Furthermore, the 5' leader inserted in the sense orientation into a heterologous luciferase expression construct increased luciferase activity.

Abstract 2723: Host response to HMTV

Abstract Work from our laboratory demonstrated the presence of retroviral sequences homologous to the mouse mammary tumor virus (MMTV) in a large proportion of human breast cancers. These observations have been confirmed by others. A 660 bp sequence of the MMTV env gene, with no significant homology to any viral or human sequence reported in the GenBank, has been found in 40% of the U.S. human breast cancers studied. This sequence is expressed in most of the env positive tumors, and is not detected in the normal breast tissue of patients with env positive carcinomas, indicating its exogenous origin. A complete provirus structure with 95% homology to MMTV has been described and designated as human mammary tumor virus (HMTV). Betaretroviral particles from primary cultures of breast cancer cells (MSSM) have been isolated and characterized. Virion RNA is more than 90% homologous to MMTV RNA and to the HMTV proviral DNA previously identified. To find out if these particles were infectious, co-culture experiments between MSSM cells and normal human epithelial breast cells, B and T human lymphocytes and human dendritic cells were performed. The results demonstrated that the recipient human cells became infected with HMTV. However, protein expression was only observed in 10-15% of the infected cells by FACS analysis suggesting the presence of an innate resistance to HMTV in human cells. We have then investigated whether human APOBEC and TRIM proteins are implicated in resistance to HMTV expression. The results indicated that APOBEC G and F, TRIM 5, 21 and 25 are all highly expressed in HMTV cells as measured by quantitative RT-PCR. Additional molecular changes seen in infected cells are disruption of the cytoskeleton and evidence for epithelial-mesenchymal transition (EMT). Since expression of an endogenous retrovirus (HERV-K) env gene has been reported to be upregulated in human breast cancers we have also investigated its expression in HMTV-infected cells, primary MSSM cells and breast tumors by quantitative RT PCR. The results indicated that in all instances where HMTV was present, expression of HERV-K (HML-2) was less than the control counterpart. In conclusion, HMTV is able to infect a variety of cells bringing about striking molecular changes. Whether these changes play a role in tumorigenesis remains to be shown. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2723. doi:10.1158/1538-7445.AM2011-2723

Mouse mammary tumor virus in anti-mitochondrial antibody producing mouse models

A human betaretrovirus resembling the mouse mammary tumor virus (MMTV) has been characterized in primary biliary cirrhosis (PBC) and associated with aberrant pyruvate dehydrogenase complex (PDC)-E2-like expression. As MMTV is prevalent in mice as either an exogenous or endogenous infection, we tested the hypothesis that MMTV is linked with anti-mitochondrial antibody (AMA) production in models with severe immune dysfunction. Evidence for MMTV was assessed in the liver and spleen of mice by PCR and immunochemistry and PDC-E2-like protein by immunochemistry. ELISA and Western blot were used to investigate AMA and anti-MMTV antibody production. Increased MMTV gag or env expression was detected in the livers of AMA producing mice including NOD.c3c4, CD4 directed dominant negative TGF-β receptor II and IL-2 receptor α knockout mice as well as the NOD parental strain when compared to healthy strains and biliary disease control mice. The NOD.c3c4 mice expressed MMTV surface and capsid proteins and aberrant PDC-E2-like protein in the bile ducts, whereas IL-2 receptor α knockout mice, NOD.c3c4 and the NOD mice expressed MMTV proteins and aberrant PDC-E2-like protein in the spleen. A significant correlation between anti-MMTV antibody production and AMA production was observed in the sera of NOD and NOD.c3c4 mice (p<0.0001). The association of betaretroviral protein production and aberrant PDC-E2-like protein expression in the NOD.c3c4, NOD, and the IL-2 receptor α knockout mice is comparable to observations in patients with PBC. The correlation of AMA and anti-MMTV suggests the hypothesis that MMTV infection may trigger the production of AMA.

Increased Detection of Breast Cancer Virus Sequences in Inflammatory Breast Cancer

Earlier studies have suggested an association between breast cancer aggressiveness and the presence of viral sequences resembling mouse mammary tumor virus (MMTV). This study was to determine if inflammatory breast cancer (IBC) in the United States had a higher proportion of cases with these viral sequences than non-IBC patients and if specific risk factors for the sequences could be identified. Biospecimens from 67 patients in the North American IBC Registry were selected for sequencing of MMTV env- like sequences. The presence or absence of the viral sequences was compared to progression free survival (PFS), risk factors including exogenous hormones, and tumor markers. Of the 67 cases, 44 were positive for viral sequences (VSP), 17 were negative (VSN) and six were excluded from analysis because of insufficient DNA to perform replicates. The 72% of VSP cases was significantly more than the 40% in non-IBC U.S. breast cancer patients (p < 0.0001). Non-significant trends suggested that VSP patients were more likely to be HER-2 neu positive and ER negative, have a stronger exposure to exogenous hormones, and have a shorter PFS than VSN patients. MMTV-related sequences appear to be related to the aggressiveness of breast cancer with a higher incidence in North American IBC than in non-IBC breast cancer.

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.

No association of mouse mammary tumor virus‐related retrovirus with Japanese cases of breast cancer

Mouse mammary tumor virus (MMTV) is the causative agent of breast tumors in mice. Recently, DNA sequences homologous or closely related to MMTV env gene have been specifically detected in breast cancer tissue from significant numbers of American, Australian, and Tunisian women, suggesting a viral etiology for at least a part of human breast cancer. However, the viral sequences have not been detected from any of breast cancer samples in several subsequent studies. Thus, whether MMTV-related retrovirus is a causative agent of human breast cancer remains controversial. To demonstrate if MMTV-related retrovirus is involved in Japanese cases of breast cancer, breast tissue specimens from 46 breast cancer patients and 3 patients with benign mammary tumors were investigated. Extensive analysis using PCR and Southern blot hybridization, however, could not detect the MMTV env gene-like sequence in any of the samples tested as well as in MCF7 cells that has previously been described as a positive control. Thus, MMTV itself or MMTV-related retrovirus is not associated with breast carcinogenesis in Japanese women, and it is unclear whether this conclusion is merely a reflection of regional differences in its epidemics.

Mouse mammary tumor virus‐like env sequences in human breast cancer

Mouse mammary tumor virus (MMTV) has long been known as a causal agent of breast cancer in mice. To date, varied MMTV-like envelope gene (env) sequences have been identified in up to 74% of human breast cancers. However, the role and origin of these MMTV-like sequences in human breast cancer remain uncertain. Our study was initiated to study the integration of MMTV-like env sequences in human breast cancer. PCR screening has identified 28 (56%) Australian breast cancer specimens and 7 (87.5%) human breast cancer cell lines to be positive for MMTV-like env sequence. In the MCF-7 genome, a fragment containing an MMTV-like env sequence of approximately 1.9 kb plus a downstream rodent-like sequence of approximately 200 bp was found to be integrated into a bacterial-like beta-lactamase sequence by insertional mutagenesis. The identified MMTV-rodent fragment is present in some MCF-7 sublines but absent in the screened specimens and other cell lines. Sporadic mutations found in this fragment indicate it has multiple copies in the MCF-7 genome. Sequence analysis has identified a novel ORF of approximately 1.6 kb which is 94-99% identical to MMTV env genes. RT-PCR was performed on the MCF-7 cDNA but no MMTV-like env transcript was detected. This is the first report to reveal the locus of MMTV-like env sequence in human cells. The MMTV-like env sequence was shown to be distinct from the human endogenous retroviral sequences and is closely related to rodents.

Mouse mammary tumor virus (MMTV)-like DNA sequences in the breast tumors of father, mother, and daughter

BackgroundThe diagnosis of late onset breast cancer in a father, mother, and daughter living in the same house for decades suggested the possibility of an environmental agent as a common etiological factor. Both molecular and epidemiological data have indicated a possible role for the mouse mammary tumor virus (MMTV), the etiological agent of breast cancer in mice, in a certain percentage of human breast tumors. The aim of this study was to determine if MMTV might be involved in the breast cancer of this cluster of three family members.ResultsMMTV-like envelope (env) and long terminal repeat (LTR) sequences containing the MMTV superantigen gene (sag) were detected in the malignant tissues of all three family members. The amplified env gene sequences were 98.0%–99.6% homologous to the MMTV env sequences found in the GR, C3H, and BR6 mouse strains. The amplified LTR sequences containing sag sequences segregated to specific branches of the MMTV phylogenetic tree and did not form a distinct branch of their own.ConclusionThe presence of MMTV-like DNA sequences in the malignant tissues of all three family members suggests the possibility of MMTV as an etiological agent. Phylogenetic data suggest that the MMTV-like DNA sequences are mouse and not human derived and that the ultimate reservoir of MMTV is most likely the mouse. Although the route by which these family members came to be infected with MMTV is unknown, the possibility exists that such infection may have resulted from a shared exposure to mice.

PREVALENCE OF MOUSE MAMMARY TUMOR VIRUS (MMTV) IN WILD HOUSE MICE (MUS MUSCULIS) IN SOUTHEASTERN AUSTRALIA

We determined the prevalence of mouse mammary tumor virus (MMTV) in introduced, free-roaming, wild house mice (Mus musculus domesticus) [corrected] and compared envelope (env) and long terminal repeat (LTR) nucleotide sequences of viruses from wild mice and other sources. Mice were trapped on two occasions, in October (spring) and the following May (autumn) of 2003-2004 in the Mallee region of northwestern Victoria, Australia. Animals were assigned to three cohorts (subadult, young, and old adults) based on their body length. The DNA from salivary glands (62 of 62 mice) and mammary glands (19 of 32 female mice) was screened for the MMTV envelope (env) gene, and the long terminal repeat (LTR) region including the superantigen (SAg) sequence was amplified from a subset. Positive polymerase chain reaction (PCR) results for the MMTV env PCR were detected from salivary gland tissues from 60 of 62 (97%) mice and from mammary gland tissues from 19 of 19 (100%) female mice. All but two mice were positive for MMTV env across both sexes and the three cohorts. Similarity of the SAg carboxy-terminal nucleotide sequence between free-roaming wild house mice varied from 64% to 99%, although most of this variation was due to DNA sequences from two mice (M4 and M5). Phylogenetic analysis of the LTR region did not result in distinct grouping of sequences derived from mice when comparisons were made among sequences from mice in the US, Europe, and Australia, and MMTV-like virus (MMTV-LV) env sequences derived from human hosts. We report a high prevalence of the MMTV env sequence during a sampling period when peak mouse density was low. This indicates that MMTV is an enzootic virus in a population of wild, free-ranging mice in northwestern Victoria, in Australia. Phylogenetic analysis, based upon env and LTR sequence data, indicated minor variation among all isolates. This represents the first report on the prevalence of MMTV in mouse populations in Australia.

Human, rhesus macaque, and feline sequences highly similar to mouse mammary tumor virus sequences

Sequences highly similar (>95%) to the mouse mammary tumor virus (MMTV) env gene have been amplified from human DNA samples, including DNA samples from patients with breast cancer (BC) and persons who did not have BC. The sequences from human DNA were distinct from the MMTV sequences used as controls in these PCR reactions, indicating that these results are not simply due to contamination. In addition to both, mouse and human-related sequences were also amplified from some monkey and cat genomic DNA samples. These products were shown to be distinct from, but highly related to, the MMTV env gene, whereas, testing of other sources (lambda phage, snake, cockroach, sea urchin, chicken, or dog) demonstrated no specific amplification. A sequence 90% similar to the MMTV group antigen gene (gag) was amplified from cat DNA. These results indicate that DNA from vertebrate species other than rodents, including some but not all humans, monkeys, and cats, can contain sequences closely related to MMTV.

Mouse Mammary Tumor Virus Env–Derived Peptide Associates with Nucleolar Targets in Lymphoma, Mammary Carcinoma, and Human Breast Cancer

We have previously shown that the leader peptide (p14) of the Env-precursor of mouse mammary tumor virus is translocated into the nucleoli of murine T cell lymphomas that harbor this virus. Using a polyclonal antibody against recombinant p14, we show here that p14 is also localized to the nucleoli of murine mammary carcinomas and some human breast cancer samples. Affinity purification studies define a number of proteins, mostly nucleolar, that bind p14. Taken together, these findings point towards a more general involvement of p14 in lymphomagenesis and mammary carcinogenesis.

Regulation of mouse mammary tumor virus env transcriptional activator initiated mammary tumor virus superantigen transcripts in lymphomas of SJL/J mice: role of Ikaros, demethylation, and chromatin structural change in the transcriptional activation of mammary tumor virus superantigen.

Mammary tumor virus (Mtv29)-encoded superantigen expressed by SJL/J mouse B cell lymphomas stimulates CD4+V16+ T cells and thereby acquires T cell help necessary for lymphoma growth. Mtv29 mouse mammary tumor virus env transcriptional activator (META) env-controlled Mtv29 superantigen (vSAg29) mRNA transcripts (1.8 kb) are not expressed in normal B or other somatic cells. Real-time PCR-based assays with DNA from normal SJL liver and vSAg29- lymphoma (cNJ101), digested with methylation-sensitive enzymes, showed hypermethylation at AvaI, FspI, HpaII, ThaI, and the distal HgaI sites of the META env, but vSAg29+ lymphoma cells showed significant demethylation at AvaI, HpaII, and the distal HgaI sites. The distal HgaI site that is adjacent to an Ikaros binding site is significantly demethylated in the META env DNA from primary lymphomas. Gel shift assays showed binding of Ikaros to a sequence representing this region in the META env. SJL lymphomas expressed the Ikaros isoform Ik6 that was absent in normal B cells. vSAg29+ cells exhibited increased DNaseI accessibility to chromatin at the vSAg29 initiation site. Treatment of cNJ101 cells with a demethylating agent, 5-azacytidine, and a histone deacetylase inhibitor, trichostatin A, caused hypomethylation at AvaI, HpaII, and distal HgaI sites and led to chromatin structural change at the vSAg29 initiation site, accompanied by the expression of vSAg29 transcripts. This enabled cNJ101 cells to stimulate SJL lymphoma-responsive CD4+V16+ T hybridoma cells. Thus, demethylation at the distal HgaI site of the Mtv29 META env permits vSAg29 expression, which may have an impact on the development of germinal center-derived B cell lymphomas of SJL/J mice.

Possibilities of a viral etiology for human breast cancer. A review.

Previous studies related mouse mammary tumor virus (MMTV) to human breast cancer. However, the presence of human endogenous retroviruses (HERs) confounded these results. We selected a 660-bp sequence of the MMTV env gene with low homology to HER (or any other known gene) and searched for a sequence homologous to it, using the polymerase chain reaction (PCR). The 660-bp sequence was detected in 131 (39%) of 335 unselected breast cancers, in 2 (6.9%) of 29 fibroadenomas, and in 2 (1.65%) of 121 normal breast specimens. The sequence was not present in normal tissues, or in other human cancers or cell lines. Cloning and sequencing of the 660-bp sequence revealed that it is 95-98% homologous to MMTV env gene, but not the known HERs or other viral or human gene. Southern blot hybridization using labeled cloned sequences demonstrated that the 660-bp sequence was present in very low copy number as a 6-8 kb EcoRI fragment only in breast cancer samples and in some of the human breast cancer cell lines that were positive by PCR. Preliminary experiments using reverse transcriptase (RT)-PCR indicated that expression of the 660-bp sequence can be detected in 65% of the positive tumors. We were also able to identify in breast cancer DNA a segment of 1.6 kb comprising LTR and env gene sequences, which are homologous to MMTV, but not to the HERs. The origin of the MMTV-like sequences in tumor DNA could be the result of integrated MMTV-like sequences derived from a human mammary virus.