Mouse Mammary Tumor Virus Antigens Research Articles

Quantitative analysis of mouse mammary tumor virus in milk in two sublines of RIII/AnOk mice with low and high mammary tumor incidence.

Mammary tumors have ceased to develop in descendants of one female of the RIII/AnOk mouse strain which was brought from the Mouse Colony of Okayama University Medical School in 1975, while descendants of another female of this strain have maintained a high tumor incidence. The former and latter descendants were separated out as RIII/AnOk/2 and RIII/AnOk/1 sublines, respectively. The amount of mouse mammary tumor virus (MMTV) in the milk of individual females in these two sublines was determined by means of a sensitive enzyme immunoassay. No MMTV antigens were detected in 59 milk samples of RIII/AnOk/2 females collected during the 1st to 7th lactation periods. On the other hand, all the milk samples of RIII/AnOk/1 females contained MMTV antigens with concentrations ranging from 9 to 400 micrograms/ml. When RIII/AnOk/2 mice were foster-nursed by RIII/AnOk/1 mothers, the infected RIII/AnOk/2 females produced MMTV in the milk to almost the same degree as did the RIII/AnOk/1 females. No genetic differences between the two sublines were observed by the use of reciprocal skin grafting, tumor transplantation, and analysis of biochemical genetic markers. These results indicate that the arrest of mammary tumor development in the RIII/AnOk/2 subline was due to the disappearance of MMTV in the milk.

Amplification of mouse mammary tumor virus genomes in non-mammary tumor cells

Extra proviral copies of mouse mammary tumor virus (MMTV) are known to be present in the genomes of certain T-cell lymphomas of mice. Analysis of additional non-mammary tumor cell types known to express MMTV transcripts and antigens revealed the presence of extra acquired MMTV proviruses in a pituitary tumor cell line, a macrophage line, and Leydig testicular tumor cells. The nature of the amplified MMTV proviruses in these various tumor cell types differed with regard to copy number and presence of alterations in the long terminal repeat region.

Isolation of a pathogenic clone of mouse mammary tumor virus

Exogenous mouse mammary tumor virus (MMTV) was cloned from a GR mammary tumor. Clone lambda GRT39 contained a full-length integrated MMTV(GR) provirus and both 5' and 3' host flanking DNA. The lambda GRT39 provirus had no apparent structural changes associated with cloning and retained the exogenous MMTV gag gene poison sequence. When introduced into rat mammary adenocarcinoma LA7 cells, the lambda GRT39 provirus was fully expressed. lambda GRT39-transfected LA7 cells made MMTV RNA, had gp52 SU protein on the cell surface, and produced B-type retrovirus particles characteristic of MMTV. Mammary tumors developed in hormone-stimulated BALB/c females injected with MMTV from lambda GRT39-transfected LA7 cells [MMTV (lambda GRT39)]. The tumors had new, clonally integrated copies of the MMTV(lambda GRT39) provirus and were expressing MMTV antigen. These data indicate that the lambda GRT39 provirus is biologically active and pathogenic.

Towards an idiotype vaccine against mammary tumors. Induction of an immune response to breast cancer-associated antigens by anti-idiotypic antibodies.

Previously we have reported on the production of two sets of human monoclonal antibodies reacting with mouse mammary tumor virus (MMTV) and human mammary tumor virus (HuMTV) cross-reacting antigens. B11 is a monoclonal IgG generated by the human-human hybridoma technique using axillary lymph node of breast cancer patients. 4.6/6 is a monoclonal IgG established by the mouse-human hybridoma procedure using peripheral blood lymphocytes of a healthy investigator working with the breast cancer cell line T47D which secretes HuMTV antigens. Two anti-idiotypic (Id) antibodies were produced by immunizing rabbits with B11 or 4.6/6. Following exhaustive adsorption on unrelated human-Ig, fetal calf serum and purification on B11 or 4.6/6 affinity columns, the anti-Id antibodies were shown to react specifically with their respective Id. The binding of these anti-Id antibodies to the respective Id was specifically inhibited by prior incubation of the Id with MMTV and/or HuMTV antigens. Rabbit anti-B11 and rabbit anti-4.6/6 anti-Id antibodies were employed to immunize female C3Heb mice. Following immunizations, humoral and cellular immune responses to MMTV-related antigens could be demonstrated. The sera of the mice contained anti-MMTV antibodies and delayed-type hypersensitivity was specifically expressed when irradiated cells of the Mm5MT line (which carry surface MMTV antigens) were injected. Our results support the notion that anti-Id antibodies harboring the internal image can immunize animals against tumor cells bearing on their surface viral-associated antigens.

Mouse lymphoma cell variants with genetically dominant alterations in the maturation of viral glycoproteins.

The appearance of Mouse Mammary Tumor Virus (MMTV) glycoproteins on the surface of infected mouse lymphoma cells is regulated by glucocorticoid hormones. The hormone stimulates transcription of the proviral genes and is also required for maturation of viral proteins. We have reported earlier the isolation of variant cell lines with altered patterns of cell-surface expression of the MMTV antigens. One type of variant expresses high levels of viral glycoproteins on the cell surface in a constitutive or hormone-independent manner. Another type of variant expresses reduced levels of viral glycoproteins on the cell surface compared with wild-type cells, both in the presence and in the absence of glucocorticoids. In this report pulse-chase studies demonstrate that the variant phenotypes are caused by changes in the efficiency of processing the envelope polyprotein precursor into the mature glycoproteins. In addition, both variant phenotypes are dominant over the wild-type phenotype in stable hybrid cell lines produced by cell fusion.

Horizontal transmission of the mouse mammary tumor virus in cage mates of the same and opposite sex of low and high mammary cancer strain mice.

Horizontal transmission of mouse mammary tumor virus (MTV) was investigated in cage mates of the same and opposite sex of low (BALB/c) and high mammary cancer strains (DD/Tbr, SHN and GR). By MTVp27 and MTVgp52 radioimmunoassay, MTV antigen expression was found in the salivary glands, mammary glands and secondary male genital organs of the MTV-free BALB/c strain. Infectivity and oncogenicity were also found in DDf or BALB/c mice by inoculating extracts of salivary gland and/or seminal vesicle in high mammary cancer strains. It is suggested that the primary source of the infectious agent in cases of caged animals of the same sex is saliva, while the primary source in cases of caged animals of the opposite sex is the seminal fluid, although additional infection through saliva cannot be ruled out in the latter case.

Characterization and chromosomal location of endogenous mouse mammary tumor virus loci in GR, NFS, and DBA mice

Endogenous mouse mammary tumor virus (MMTV) proviral copies were characterized in three genetically dissimilar mouse strains: GR, a high-tumor-incidence strain bred in Europe that carries an MMTV proviral copy associated with early mammary tumors; DBA, a high-tumor-incidence laboratory strain bred in the USA with an endogenous copy that is associated with MMTV antigen expression in the milk; and NFS, a recently inbred line of the low-tumor-incidence NIH Swiss mouse. MMTV proviral loci were studied using restriction endonuclease analysis and the Southern transfer procedure in genetic crosses and in somatic cell hybrids. By studying the segregation of MMTV-specific EcoRI, BamHI, and PstI fragments, the organization of these fragments into MMTV proviral loci was determined and it was shown that (1) many homologous proviral loci are present in these three mouse strains, (2) these MMTV proviruses differ in their pattern of internal restriction sites, and (3) the MMTV loci are distributed on multiple chromosomes including 1 and 7.

Radioimmunolocalization of murine mammary tumor virus proteins in gels

A procedure for the immunological detection of antigens on sodium dodecyl sulfate-polyacrylamide gels using iodinated antibodies combined with an immunoconcentration step to enrich for the desired antigen(s) is described. This selective enrichment is achieved by separation of the antigen-antibody complexes with formalin-treated cells of Staphylococcus aureus and is followed by radioimmunolocalization on gels. This method is particularly useful for the characterization of antigens present at low concentrations in complex biological fluids. Its usefulness is exemplified here by the successful identification of mouse mammary tumor virus antigens in extracts of murine mammary tumors and in plasmas of tumor-bearing animals. In particular, the procedure permits the ready determination that the gp52 protein detected in the plasmas of tumor-bearing animals is in fact present as a 52,000-dalton species and not as a higher molecular weight precursor.

Localization of a gene for expression of mouse mammary tumor virus antigens in the GR/Mtv-2- mouse strain.

The GR/Mtv-2- mouse strain is congenic to the GR strain but lacks the Mtv-2 gene for high amounts of mouse mammary tumor virus (MMTV) virion particles in the milk and early mammary tumors. With a sensitive competition radioimmunoassay for individual viral proteins of MMTV, substantial amounts of the gag proteins p27 and p10 could still be detected in extracts of the mammary glands of GR/Mtv-2- mice, but essentially no viral envelope antigens. The genetic transmission of the MMTV gag expression in the GR/Mtv-2- strain was investigated. In a cross with the virus-negative BALB/c strain, the MMTV p27 expression behaved as a dominant feature. Double backcross analysis proved that the p27 expression was governed by a single gene located on chromosome 11, cloe to the Es-3 locus. The gene was thereby not allelic to any of the previously described MMTV induction genes, Mtv-1 and Mtv-2, and is therefore called Mtv-3. It is concluded that the total MMTV expression in the GR strain is under control of two separate loci, Mtv-2 on chromosome 18, inducing high levels of complete virus particles and also early mammary tumors; and Mtv-3 on chromosome 11, coding for partial MMTV expression.

Prevalence and Distribution of Murine Mammary Tumor Virus Antigen Detectable by Immunocytochemistry in Spontaneous Breast Tumors of Wild Mice<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

Mouse mammary tumor virus (MuMTV) antigens were detected by immunoperoxidase cytochemistry in spontaneous breast tumors of wild mice from two widely separated areas of southern California. Eleven of 25 (44%) tumors from Lake Casitas (LC) mice and 4 of 5 tumors from Bouquet Canyon mice were positive. Included among the tumors lacking detectable MuMTV antigen were well-differentiated type A and type B carcinomas as well as tumors with an atypical pattern. In the antigen-positive tumors the distribution of staining was patchy and extremely variable in extent (less than 1-70% stained cells). The intensity and extent of staining were generally greater in breast tumors from hybrids of LC wild mice and C5LBL/10Sn or AKR inbred mice. A good correlation was found in the same tumors between immunoperoxidase staining, detection of MuMTV gp52 antigen by radioimmunoassay, and detection of type B particles by electron microscopy. All of the breast tumors in LC mice were positive for type C virus particles.

Mouse mammary tumor virus (MMTV) antigen(s) are present on B lymphocytes of BALB/c mice.

Immunofluorescence studies using a polyvalent anti-MMTV serum revealed the presence of MMTV antigen(s) on the surface of spleen cells from the low mammary tumor incidence mouse strain BALB/cCrgl. Upon separation of the splenocytes on nylon-wool columns the populations of cells exhibiting positive fluorescence were nylon-adherent. These cells were considered to be B lymphocytes by the following criteria: presence of surface immunoglobulin (Slg), absence of Thy-1 antigens, enhanced responses to B-cell mitogens and decreased reactivity to T-cell mitogens. In order to ascertain the specificity of the cell surface immunofluorescence reaction, blocking studies were performed. Immunofluorescence was blocked by preincubation of the antiserum or with purified MMTV, but not by R-MuLV. Non-specific binding of the anti-MMTV to Fc receptors present on B lymphocytes was ruled out by the use of Fab'2 fragments of IgG from the various sera. Double label immunofluorescent studies with rhodamine-conjugated anti-mouse Slg Fab'2 fragments and rabbit anti-MMTV Fab'2 with fluorescein-conjugated goat anti-rabbit Fab'2 showed the simultaneous appearance of both labels in the nylon-adherent splenocytes. These results suggest the expression of viral polypeptide(s) encoded by an endogenous MMTV on the surface of B-type splenocytes in mice devoid of intact viral particles.

Immunocytochemical Distribution of Mouse Mammary Tumor Virus Antigens in BALB/cfC3H Mammary Epithelium2

The distribution of mouse mammary tumor virus (MuMTV) antigens was studied in normal, preneoplastic, and neoplastic mammary epithelia from female BALB/cfC3H mice with the use of a polyvalent anti-MuMTV serum and indirect immunoperoxidase techniques. The MuMTV antigens were on the apical surface or in focal cytoplasmic aggregates or diffused throughout the infected cells. As much as 70% of all cells in adenocarcinomas and as much as 100% of all cells in preneoplastic hyperplastic alveolar nodules contained MMTV antigens. Comparable percentages of cells from mammary glands of multiparous mice were MuMTV-positive. Some mammary tissues of nullparous and primiparous mice did not contain detectable MuMTV antigen. The MuMTV antigen-containing cells in lactating mammary glands tended to be in discrete lobuloalveolar clusters surrounded by antigen-negative alveoli. The percentage of MuMTV-positive cells in a given gland was proportional to the amount of virus found in the animal's milk.

Interferon-mediated inhibition of mouse mammary tumor virus expression in cultured cells

The effect of interferon on glucocorticoid-stimulated mouse mammary tumor virus expression in a murine mammary tumor cell line, Mm5mt/c 1, was investigated. Interferon at a concentration of 10–100 IU/ml causes a 5- to 10-fold inhibition of virus production. The effect has the typical time course of interferon action, is dose dependent and reversible, and is not associated with depression of cellular synthesis of macromolecules. The mechanism appears complex. In contrast to the interferon-mediated inhibition of murine leukemia virus, cellular RNA-dependent DNA polymerase (RDDP) is significantly reduced. The ratio of cellular to extracellular RDDP is, however, increased, and expression of mouse mammary tumor virus antigens on the cell membrane is not inhibited. The evidence therefore suggests effects of interferon on translation or transcription of some viral macromolecules as well as on viral assembly and release.

Genetic Analysis of Mammary Tumor Induction and Expression of Mammary Tumor Virus Antigen in Hormone-Treated Ovariectomized GR Mice 2

Early stages of mammary tumors (EMT) were induced with a combined treatment of progesterone (P) and estrone (E) in ovariectomized adult GRS/A (GR) mice, a strain of European origin and with a high incidence of mammary cancer. The mammary tumors were comparable to the pregnancy-dependent tumors of breeding females of this strain. The hormone treatment did not lead to EMT in a variety of other strains and only occasionally in the RIII an C3H strains. Treatment with P or E alone di not lead to EMT in GR mice, but treatment with the steroid compound 17 alpha-ethynyl-19-nortestosterone (ANT) did mimic the combined effe(t of P and E. Since EMT could be induced by ANT in all ovariectomized adult GR mice within 3 weeks, this tumor-induction method was suitable for analysis of the gene responsible for palpable, pregnancy-dependent mammary tumors of this strain. Another argument for the usefulness of this test for genetic analysis was the fact that, though mouse mammary tumor virus (MuMTV) of the GR strain was introduced into BALB/c and tmas mice by foster-nursing, ANT treatment did not lead to EMT. First and second backcross analyses showed that one gene was responsible for EMT induction. There was a strong (orrelation between the presence of EMT and MuMTV antigens in the mammary glands and milk of several first backcross populations between GR and other strains such as C57BL, BALB/c, DBAf, and C3Hf. This suggested that the expression of MuMTV antigens was also controlled by the EMT gene. Two types of resistance phenomena were observed. Neither type could prevent EMT after hormone treatment; however, they could delay EMT development. One resistance factor for EMT induction was noticeable and dominant in reciprocal hybrids of the GR and DBAf strains, whereas another resistance factor was detected in the backcross population only [i.e., in the C57BL X (C57BL X Gr) ba(kcross] and not in hybrids; therefore, this factor was recessive. Until now, linkage experiments with 18 markers to locate the gene for EMT induction in the map of the mouse were unsuccessful.

Quantitative estimation of mouse mammary tumor virus (MTV) antigens by radioimmunoassay;.

A radioimmunoassay for the antigens of the mouse mammary tumour virus has been developed. -125Iodine-labelling of intact virus [derived from mammary tumours of (C3H TIMES 020)F1 mice] followed by ether extraction and separation of the ether and water layers resulted in a separation of the labelled material into two main groups of antigenic components. The intact labelled material from each group was separated from viral debris and other possible contaminants by affinity chromatography. The antigens of the ether phase were proved to belong mainly to the viral nucleoid whereas the water phase contained mainly envelope antigens. No type-specific antigens could be demonstrated in either of the phases. Radioimmunological assessment of plasma revealed that in the plasma of MTV-S- and MTV-P-positive animals viral antigens were only measurable when palpable mammary tumours were present, whereas in MTV-L-positive tumour-bearing animals some negative samples were found. In milk of individual nontumour-bearing mice a close correlation was found between the expression of viral antigens and the generally accepted presence of virus in the strain of mice. In milk, viral antigen levels tend to increase with parity with a possible decrease after a finite number of pregnancies. In a pilot study the presence of MTV antigens could also be demonstrated in epididymis extracts of male GRS/A mice. Genetical analysis of the low-virulent MTV-L by radioimmunoassay of milk is in progress.

Characteristics of the structural components of the mouse mammary tumor virus: II. Viral proteins and antigens

Mild Tween 80-ether treatment of mouse mammary tumor virus (MTV) results in disruption of the viral membrane and release of subviral components. Prolonged lipid extraction, however, results in virtually complete solubilization of the virion. Soluble proteins have been separated by Sephadex G-200 chromatography and used for immunization of rats These antisera identify five viral antigens (designated MTV- s1, s2, s3, s4, and s5). The s1 and s2 antigens are group-specific, and are the antigens of the viral nucleoid. MTV- s3 antigen is a structural protein of the viral membrane. Poly-acrylamide gel electrophoresis of MTV shows five major polypeptides in the virion; the molecular weights of these are p1 (90,000), p2 (70,000), p3 (52,000), p4 (33,000), and p5 (23,000). The major protein of the virus is p3; it is found in the nucleoid and carries the s1 antigen. Protein p5 is the structural protein of the membrane, and it carries the s3 antigen. Antisera prepared against isolated MTV proteins were used also in immunofluorescence tests to determine the intracellular deposition of MTV antigens. These studies show that all MTV structural proteins are synthesized cytoplasmically. Screening of a variety of neoplasms by immunofluorescence and immunodiffusion tests demonstrates MTV infection in only three types of neoplastic diseases, mammary tumors, ML+ leukemias, and Leydig cell tumors of MTV-infected mice. Mammary tumors synthesize all viral structural proteins, while the synthesis of MTV proteins in the ML+ leukemias and Leydig cell tumor is limited to the s1 and s2 antigens. Electron microscopic examination of these tissues confirms the synthesis and accumulation of viral nucleocapsids (A particles) without the production of budding virions.

Detection of antigens of the mouse mammary tumor (MTV) and murine leukemia virus (MuLV) in cells of cultures derived from mammary tumors of mice of several strains

A number of tissue cultures of mouse mammary tumors were studied for the presence of the mouse mammary tumor virus (MTV) and of the murine leukemia virus (MuLV) antigens. Mixed hemadsorption reaction (MHA) was used for detection on the cell surface and indirect immunofluorescence (IF) on fixed cells for detection of antigens of the two viruses within the cells. Three rabbit anti-MTV sera, three rat anti-MuLV sera, and one rabbit anti-FeLV (feline leukemia virus) serum were used in these studies. The same cells of a 4-year-old cell line derived from a spontaneous mammary tumor of an RIII/Dm mouse strain, designated as Dmochowski-Williams line (D-W line), were found positive at the same time for the antigens of the two oncogenic RNA viruses (MTV and MuLV) in the cytoplasm (IF) and on the cell surface (MHA). Several cell lines derived from induced BALB/c/Dm mammary tumors were found negative on the cell surface (by MHA) and in the cytoplasm (by IF) for MTV antigens. Some of the BALB/c/Dm mammary tumor cell lines negative for MTV antigens were found positive for MuLV antigens (by both MHA and IF). The antigens of MTV and MuLV were found by IF in the cytoplasm of the tumor cells. No nuclear fluorescence was observed. The shared antigen of mammalian leukemia-sarcoma viruses (MuLV-gs3) was found by IF in the cytoplasm of mammary tumor cells producing type C virus particles. MuLV-gs1 and MuLV-gs3 antigens were not found in mammary tumor cell lines shown to be positive only for MTV. Mammary tumor cell lines found positive by IF for MTV and/or MuLV antigens present in the cytoplasm were also positive by MHA for MTV and/or MuLV antigens present on the cell surface. The anti-MuLV-gs1 and anti-MuLV-gs3 sera gave positive IF test (cytoplasmic antigens) and negative MHA test (cell surface antigens) with cell lines shown to contain type C virus particles. The same cell lines were positive by both MHA and IF tests with a polyvalent anti-MuLV serum.