Mouse mammary tumor virus-like ENV gene sequences in human breast tumors and in a lymphoma of a breast cancer patient.

DNA sequences with very high similarity (95-98%) to the mouse mammary tumor virus (MMTV) ENV gene have been amplified by PCR in 38.5% of human breast tumors and in <2% of normal breast tissue (Wang et al., Cancer Res., 55: 5173-5179, 1995). Intrigued by these findings, which suggested an exogenous viral etiology for a certain percentage of human breast tumors, we have screened a panel of human breast tumors and normal breast tissue for the presence of MMTV-like DNA sequences. Using similar PCR procedures and stringent hybridization techniques, we have detected the presence of MMTV-like ENV gene sequences in 37% of the human breast tumors that we have analyzed. DNA sequencing has shown these sequences to be 99% homologous to the BR6 strain of MMTV and 100% homologous to the GR and C3H strains of MMTV. We have not detected these MMTV-like sequences in normal breast tissue. However, we have detected these sequences by PCR and stringent hybridization in a T-cell lymphoma of a breast cancer patient who was simultaneously diagnosed with both diseases. Our results support the possibility of an exogenous retroviral etiology for a certain percentage of human breast tumors. Our results also suggest that a similar exogenous retroviral etiology may exist for certain human T-cell lymphomas. In many inbred strains of mice, both breast cancer and T-cell lymphoma are caused by MMTV, hence, in a certain percentage of humans, one or both of these diseases may be caused by an MMTV-like retroviral entity.     

Clonal isolation of different strains of mouse mammary tumor virus-like DNA sequences from both the breast tumors and non-Hodgkin's lymphomas of individual patients diagnosed with both malignancies.

PURPOSE: In a previous study, we had detected the presence of mouse mammary tumor virus (MMTV)-like envelope (ENV) gene sequences in both the breast tumors and non-Hodgkin's lymphoma tissue of two of our breast tumor patients who had been diagnosed simultaneously with both malignancies. The aim of this study was to determine if MMTV-like DNA sequences are present in the breast tumors and non-Hodgkin's lymphomas of additional patients suffering from both malignancies and if so to characterize these sequences in detail. EXPERIMENTAL DESIGN: DNA was extracted from formalin-fixed, paraffin-embedded tissue sample blocks of breast tumors and non-Hodgkin's lymphomas from patients suffering from both malignancies. A 250-bp region of the MMTV ENV gene and a 630-bp region of the MMTV long terminal repeat (LTR) open reading frame (ORF) that encodes the MMTV superantigen (sag) gene were amplified by PCR from the isolated DNA. Amplified products were analyzed by Southern blotting, cloned, and sequenced. RESULTS: MMTV-like ENV and LTR sequences were detected in both the breast tumors and non-Hodgkin's lymphomas of 6 of 12 patients suffering from both malignancies. A novel mutant of the MMTV ENV gene was identified in these patients. Characterization of the MMTV-like LTR highly variable sag sequences revealed total or nearly total identity to three distinct MMTV proviruses from two different branches of the MMTV phylogenetic tree. CONCLUSIONS: The presence of MMTV-like ENV and LTR sequences in both the breast tumors and non-Hodgkin's lymphomas of 6 additional patients suggests a possible involvement of these sequences in these two malignancies. MMTV-like LTR sequence homology to different MMTV proviruses revealed the presence of more than one strain of MMTV-like sequences in each individual suggesting the possibility of multiple infections in these patients.     

Evidence of separate pathways for viral and chemical carcinogenesis in c3h/stwi mouse mammary glands

Mammary tumors in mice may arise as the result of exogenous infection with the mouse mammary tumor virus [MMTV(S)], usually via the milk; by the action of endogenous MMTV genes which are transmitted genetically and are sometimes expressed as infectious virus [MMTV(L)]; or by the action of chemical carcinogens. We have examined the etiological relationship between chemical and virus in the induction of mammary cancer in C3H/StWi mice. Mammary tumors were induced with 7.12-dimethylbenz(a)anthracene (DMBA) in virgin C3H/StWi mice infected with exogenous mouse mammary tumor virus (C3H/StMTV) and in uninfected C3H/StWi females. The percent tumor risk in females whose glands were infected with exogenous mouse mammary tumor virus [MMTV(S)] was not different from that of MMTV(S)-negative, C3H/StWi mice following treatment with DMBA. This result suggested that there was no synergistic effect between the two carcinogens, exogenous MMTV and Dmba. All the tumors arising in DMBA-treated C3H/StMTV virgins were positive for MMTV env gene product, gp52 and MMTV gag gene product, p²⁷ by radioimmune competition assay. MMTV(S)-induced hyperplastic alveolar nodules (HAN) were observed in 62 % of the untreated C3H/StMTV glands at 8 months of age. Therefore, MMTV(S) was present and active in the mammary gland during the experimental period but had no enhancing influence on the sensitivity of the gland to carcinogenesis by DMBA. Tumors appearing in DMBA-treated C3H/StWi virgin females were also tested for MMTV gp⁵² and p²⁷ antigens to determine the presence of endogenous MMTV gene activity. Only occasional C3H/StWi tumors were positive and in most of these, p²⁷, but not gp⁵² was detected, suggesting non-coordinate expression of these endogenous MMTV gene products. Both alveolar (HAN) and ductal hyperplasia (DH) were found in DMBA-treated C3H/StMTV glands, whereas only DH were found in DMBA-treated C3H/StWi mice. Nevertheless, the DMBA-induced C3H/StMTV tumor histopathology was remarkably indistinguishable from that in tumors produced by DMBA in C3H/StWi mice, implying that in both groups, tumors arose primarily from the chemically-induced mammary dysplasias. These data taken together appear to support the conclusion that DMBA and Mmtv follow separate pathways to the induction of cancer in the mouse mammary gland.     

Hormones,chemicals and proviral gene expression as contributing factors during mammary carcinogenesis in C3H/StWi mice

C3H/StWi mice spontaneously lost their exogenous MMTV (MMTV-S) in 1958 and became a low mammary cancer subline. They do not ordinarily express their endogenous MMTV provirus as virions. Virigin C3H/StWi females were exposed to chemical carcinogens, 7-12 dimethyl(α)benzanthracene (2,6 mg) and urethane (200 mg) in the presence or absence of chronic hormonal stimulation of the mammary gland by pituitary isografts. By 10 months of age, mammary tumors developed in 40% of the females given DMBA, and in 59% of those given DMBA and carrying pituitary isografts. With urethane treatment alone, 14% of the mice developed mammary cancer during a 12-months period; however, 74% of the mice bore mammary tumors when pituitary isografts were present. None of the females given pituitary isografts alone developed mammary cancer during the experimental period. Mammary tumors from each group were evaluated by radioimmune competition assay, immunoperoxidase and electron microscopy to determine the extent of endogenous MMTV gene expression. In addition, the mammary glands of some of the non-tumor-bearing mice were studied by whole mount and by histology to determine the type, extent, and number of mammary dysplasias present in each group. In general, there was no correlation between tumor incidence and the presence of mammary tumor virus antigens. Of 32 mammary tumors tested in all groups, 10 were positive at low levels for MMTV antigens. In the positive tumors, the internal MMTV gag gene antigen, p28, was prevalent. Immunoperoxidase studies on these same tumors gave quantitatively similar results. It is apparent from these observations that chemical carcinogenesis of mouse mammary glands does not require or even favor the complete expression of endogenous MMTV genes. In this study, hormonal stimulation of the glands during and after exposure to the carcinogen increases the rate of tumorigenesis, but does not seem to favor MMTV gene expression. We conclude that C3H/StWi mice are susceptible to DMBA and urethane induction of mammary cancer and to an extent this process is positively influenced by the presence of hormonal stimulation. Our results support the view that a qualitative or quantitative shift in the expression of MMTV proviral sequences does not occur during tumor development.     

A biochemical approach to the study of the transmission of mouse mammary tumor viruses in mouse strains RIII and C3H.

Mouse mammary tumor virus (MMTV) proviral sequences were detected in the cellular DNA of mammary tumors and livers of RIII and C3H mice by molecular hybridization with radioactively labelled MMTV 60-70S RNA or tritiated MMTV complementary DNA (cDNA). By means of DNA:DNA reassociation kinetics, the DNA of the mammary tumor cells of these two mouse strains were found to contain more MMTV proviral sequences than the DNA of liver cells of these same tumor-bearing mice. Evidence is also presented that the DNA of the liver cells lacks a part (approximately 25%) of the MMTV proviral sequences found in the mammary tumor cells of these mouse strains. The relationship of the extra MMTV proviral sequences found in mammary tumor cells to the early mammary tumor-igenesis seen in these mouse strains is discussed.     

Tissue Distribution of Mouse Mammary Tumor Virus (MMTV) Antigens and Endogenous MMTV Loci in Japanese Laboratory Mouse Strains

The distribution of mouse mammary tumor virus (MMTV) antigens was studied by the immunoper-oxidase method in the II-TES and I-TES mouse strains as well as their progenitors, CS and DBA/2 strains. In the II-TES, I-TES and CS strains, and BALB/c mice foster-nursed with these strains, MMTV antigens were found not only in epithelial cells of the mammary glands but also in those of other tissues including the seminal vesicle, vas deferens, epididymis, prostate, parotid, submandibular, lacrimal, sebaceous, and urethral glands. In DBA/2 and BALB/cfDBA/2 mice, however, the MMTV antigens were found only in the mammary glands. Electron microscopic examination showed MMTV particles in these organs. When we examined the presence of Mtv- 1 and 2 proviruses, which are known to be responsible for MMTV expression, in the genomes of the II-TES, I-TES, CS and DBA/2 strains by Southern blotting, Mtv- 2 was not found in any of the mice and Mtv- 1 was found in the II-TES and DBA/2 mice but not in the I-TES and CS mice. Instead, four new endogenous MMTV loci, which have never previously been reported in laboratory mouse strains, were detected in the genomes of the II-TES, I-TES and CS strains. One (designated Mtv -42) was common in the three strains and the other three (designated Mtv- 43, 44 and 45) were common in the II-TES and I-TES strains or the II-TES and CS strains. These results thus suggest that new endogenous MMTV loci may be responsible for MMTV expression in a variety of tissues of these three strains.     

Tissue distribution of mouse mammary tumor virus (MMTV) antigens and new endogenous MMTV loci in Japanese laboratory mouse strains.

The distribution of mouse mammary tumor virus (MMTV) antigens was studied by the immunoperoxidase method in the II-TES and I-TES mouse strains as well as their progenitors, CS and DBA/2 strains. In the II-TES, I-TES and CS strains, and BALB/c mice foster-nursed with these strains, MMTV antigens were found not only in epithelial cells of the mammary glands but also in those of other tissues including the seminal vesicle, vas deferens, epididymis, prostate, parotid, submandibular, lacrimal, sebaceous, and urethral glands. In DBA/2 and BALB/cfDBA/2 mice, however, the MMTV antigens were found only in the mammary glands. Electron microscopic examination showed MMTV particles in these organs. When we examined the presence of Mtv-1 and 2 proviruses, which are known to be responsible for MMTV expression, in the genomes of the II-TES, I-TES, CS and DBA/2 strains by Southern blotting, Mtv-2 was not found in any of the mice and Mtv-1 was found in the II-TES and DBA/2 mice but not in the I-TES and CS mice. Instead, four new endogenous MMTV loci, which have never previously been reported in laboratory mouse strains, were detected in the genomes of the II-TES, I-TES and CS strains. One (designated Mtv-42) was common in the three strains and the other three (designated Mtv-43, 44 and 45) were common in the II-TEX and I-TES strains or the II-TES and CS strains. These results thus suggest that new endogenous MMTV loci may be responsible for MMTV expression in a variety of tissues of these three strains.     

A congenic line of the BALB/c mouse strain with the endogenous mouse mammary tumor virus proviral gene Mtv-3: tissue-specific expression and correlation with resistance to mouse mammary tumor virus infection and tumorigenesis

Mouse mammary tumor virus (MMTV) expression and MMTV-induced tumorigenesis were studied in a congenic line of the BALB/cHeA strain, termed BALB/c-Mtv-3+, that carries the Mtv-3 proviral gene. BALB/c-Mtv-3+ mice were free of milk-transmitted MMTV and did not spontaneously develop mammary tumors. A specific Mtv-3 expression was observed in the mammary gland and spleen, but not in other lymphoid tissues, such as thymus and bone marrow. This expression was hormone dependent, as shown by the increase of MMTV mRNA during pregnancy. At the protein level, large amounts of p28, but only traces of gp52, the main MMTV core and envelope antigens, respectively, were observed, in agreement with the already described "partial" expression of the Mtv-3 gene products. The presence of the 24S (3.8 kilobases) mRNA encoding the MMTV env antigens in the spleen and the low gp52 reactivity in lactating mammary glands showed that this noncoordinate expression was probably due to a defect in translation or posttranslational processing of env proteins. The susceptibility of BALB/c-Mtv-3+ to experimental MMTV infection was studied. The presence of Mtv-3 conferred to BALB/c mice resistance to MMTV infection, as shown by measuring viral antigens released in the milk of infected mice and by recording the incidence of early mammary tumors. The presence of a nontumorigenic endogenous MMTV gene was therefore protective against exogenous MMTV infection.     

Quantitation of viral antigens released into plasma and culture fluids by murine mammary tumor cells

Radioimmunoassay capable of measuring mouse mammary tumor virus (MMTV) 52,000 M.W. envelope glycoprotein (gp52) and 27,000 M.W. protein (p27) have been used to quantitatively compare plasma concentrations of these viral antigens in mice bearing spontaneous mammary tumors. Although gp52 was detected in the plasma of all tumor-bearing mice tested, p27 was detected in only a portion of tumor-bearing animals. In p27-positive animals, gp52 was detected in higher concentrations than p27. These findings demonstrate that gp52 has preferential utility as a plasma marker for the presence of mammary tumors in MMTV-infected hybrid (BALB/c x DBA/8 F1), Paris RIII, and C3H/HeJ mice. In addition, cultures of MMTV-producing cells [GR-MMTV and MMTV(C3H)Fel I] were used as models to study the release of viral antigens in the absence of serum antibody or additional host factors. Comparisons of extracellular soluble and particulate antigen concentrations demonstrated that gp52 and p27 were present in substantially higher concentrations as soluble than virion-associated antigens. The mean ratio of non-virion-associated gp52 to virion-associated gp52 was 12.5:1 for GR-MMTV cells and 37.3:1 for MMTV(C3H)Fel I cells. The marked stability of MMTV in culture fluids suggested that virion breakdown was not responsible for the accumulation of soluble viral antigens in culture. The information obtained suggests that abundant virus-free antigens may be of greater use than virion-associated antigens as a source of viral antigen to evaluate mammary tumor status.     

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

Background

The 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.

Results

MMTV-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.

Conclusion

The 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.     

Mouse mammary tumor virus proviral integration in the DD/Tbr mice

The patterns of mouse mammary tumor virus (MMTV) integration in the DNA of spontaneous-mammary tumors, salivary glands and livers of DD/Tbr mice were examined using MMTV env, int -1c and int-2c probes. The MMTV env probe revealed 1 to 7 new proviral insertions in all mammary tumors. MMTV integration into int-1 was observed in 10 of 18 mammary tumors, whereas that into int-2 was seen in only 2 of 18 tumors. Of the 13 salivary glands examined, only 3 showed new MMTV proviral integrations, but rearrangement in int-1 or int-2 loci by MMTV was not observed. Immuno-collidal gold electron microscopy revealed the presence of MMTV particles both in mammary tumors and in salivary glands, but no tumors were found to be developed in salivary glands. Taken together these results suggest that salivary glands support MMTV replication, but the virions thus produced may not lead to salivary gland tumorigenesis. It is suggested that the salivary gland is the source of horizontally transmitted MMTV in DD/Tbr mice.     

Progression from normal breast pathology to breast cancer is associated with increasing prevalence of mouse mammary tumor virus-like sequences in men and women

Mouse mammary tumor virus (MMTV)-like sequences have been found in up to 40% of breast cancer samples but in <2% of normal breast tissue samples from Australian women studied by our group. Screening of a larger and more diverse cohort of female breast cancer samples has now shown a correlation of MMTV-like sequences with the severity (grade) of breast cancer. Thirty-two percent (43 of 136) of female breast cancer samples were positive for MMTV-like sequences when screened using PCR. A significant gradient of MMTV positivity was observed with increasing severity of cancer from 23% of infiltrating ductal carcinoma (IDC) grade I tumors to 34% of IDC grade II tumors (P = 0.00034) and 38% of IDC grade III tumors (P = 0.00002). We also report for the first time the detection of MMTV-like sequences in 62% (8 of 13) of male breast cancer samples and 19% (10 of 52) of male gynecomastia samples screened. MMTV-like sequences were demonstrated in various premalignant breast lesions of females, including fibroadenoma (20%) and fibrocystic disease (28%) samples, at a significantly higher prevalence than that seen in normal breast tissue (1.8%; P = 0.00001). Study of a longitudinal cohort of female breast cancer patients indicated that MMTV was co-incident with tumor but was not present when tumor was absent on histology. These results support the association of MMTV-like sequences with development of breast tumors in men and women and suggest association of MMTV with increasing severity of cancer.     

Expression of mammary tumor virus proteins in preneoplastic outgrowth lines and mammary tumors of BALB/cV mice

A subline of BALB/c mice, designated BALB/cV, has been segregated which exhibits an intermediate mammary tumor incidence and which harbors a unique milk-transmitted virus. Six stable hyperplastic alveolar nodule outgrowth lines were established from chemical carcinogen-treated and hormonally stimulated mice. Tumor incidences exhibited by the individual preneoplastic lines ranged from 22 to 95%; no differences in tumor-producing capability were observed when lines were transplanted in virus-negative (BALB/c) or virus-positive (BALB/cV) animals. Viral antigen expression was monitored using antisera prepared against C3H mouse mammary tumor virus (MMTV) proteins. The preneoplastic lines exhibited more virus antigenpositive cells in a peroxidase-antiperoxidase immunocytochemical assay than did primary tumors which arose from the hyperplastic alveolar nodule transplants. Analysis of BALB/cV preneoplastic and tumor tissue by metabolic labeling and by protein electroblotting methodologies revealed that viral precursor and structural proteins were expressed in both types of tissue; the polypeptides were similar in molecular weight to those encoded by exogenous MMTVs. These studies demonstrate that the coding capacity of the BALB/cV isolate of MMTV is similar to that of known MMTV isolates and that each of the BALB/cV structural polypeptides shares group-specific antigenic determinants with the analogous protein encoded by MMTV from the C3H mouse. The hyperplastic outgrowth lines established in the BALB/cV subline provide an additional system for the study of mammary tumorigenesis.     

Activation of endogenous MMTV proviruses in murine mammary cancer induced by chemical carcinogen

A study was undertaken to determine whether activation of expression of silent endogenous mouse mammary tumor virus (MMTV) proviruses may occur during tumor induction by a chemical carcinogen. A series of transplantable mammary tumors induced in BALB/c mice by treatment with dimethylbenz(alpha)anthracene (DMBA), pituitary isograft, or both was examined. The results obtained suggest that chemical carcinogens may induce mammary tumors through more than one pathway. Two of 9 tumor lines produced virus-specific products at levels above those observed during the course of normal mammary gland development. One tumor contained high levels of MMTV-specific envelope [3.8 kilobase (kb)] and genomic length (8.9 kb) RNAs. This tumor expressed core- and envelope-related proteins detectable by immunoblotting (including p28, gp52, and gp36), displayed an acquired provirus with a restriction map different from those of described exogenous MMTV strains, and contained abundant virus particles. The other tumor that expressed high levels of MMTV gene products contained envelope-specific (3.8 kb) and long-terminal-repeat-specific (1.6 kb) messages but no full-length RNA. It exhibited an aberrant 39 kDa, envelope-related protein, but no virus particles. Methylation data implicated the usually silent endogenous Mtv-8 provirus as the source of the abnormal envelope protein. None of the tumors expressed RNA from the putative mammary oncogenes, int-1 or int-2. We propose that chemical carcinogens may activate different cellular genes by mutation and that, in a subset of DMBA-induced mammary tumors, the target genes include endogenous MMTV proviruses that are normally not expressed. The effect on provirus expression varies from tumor to tumor, but is stable over passage of a given tumor. MMTV may be of etiological importance in the genesis of those DMBA-induced tumors which contain high levels of MMTV-specific products, but its action in the BALB/c system is not mediated through enhanced expression of the int-1 or int-2 preferred integration regions.     

Detection and identification of mouse mammary tumor virus-like DNA sequences in blood and breast tissues of breast cancer patients

Mouse mammary tumor virus (MMTV) is a well-known cause of mammary tumors in mice transmitted as endogenous proviruses or exogenously as infectious virions. The hypothesis that a retrovirus homologous to MMTV is involved in human breast cancers has resulted in renewed interest in the etiology of human breast cancer. Therefore, the detection of MMTV-like exogenous sequences in 30–40 % of invasive breast cancer has increased attention towards this hypothesis. To detect the prevalence of MMTV in Pakistani population, 666-bp-long MMTV envelop and 630-bp LTR sequences were amplified from breast cancer patient samples (tissue biopsies and peripheral blood) using mouse with mammary tumor as control. MMTV-like virus env and LTR DNA sequences were detected in 20 and 26 % of breast tumor samples, respectively, from the total of 80 breast cancer patients’ blood and tissue samples. No significant association was observed between age, grade of disease, and lymph node involvement with the prevalence of MMTV-like sequences. Our data add to the growing number of studies implicating MMTV-like virus in human breast cancer, but still clear causal association of MMTV to breast cancer remains to be reputable.