Murine Mammary Tumor Virus Research Articles

Antibodies reactive with murine mammary tumor virus in sera of patients with breast cancer: geographic and family studies.

Sera of patients with breast cancer, of healthy women from the United States, East India, East Africa, and China, and of healthy women of American and Parsi families in which breast cancer occurred in several family members were assayed for levels of antibody reactive with the murine mammary tumor virus (MuMTV) by an enzyme-linked immunosorbent assay. Increased levels of antibody to MuMTV (absorbance greater than or equal to 0.4) were found in sera of 18.6% of American patients with breast cancer and of 2.8% of healthy American women and in 38% of patients from India and 61.9% from East Africa (healthy, 26.9%). In contrast, antibody reactive with MuMTV was found in less than 5 of women with breast cancer from mainland China (healthy Chinese, 5.0%). Differences in serum MuMTV antibody levels between breast cancer patients in the four groups were found to be significant (P less than 0.0001). Studies of two families from the United States and of one Parsi family from India with genetic propensity to breast cancer showed that high levels of antibody to MuMTV were found in 33%, 71%, and 23% of healthy family members, respectively. The antibody to MuMTV was readily absorbed with purified MuMTV and gp52. In contrast, fetal calf serum, murine type c retroviruses, or erythrocytes from various species failed to absorb the antibody.

Virus-specific cytotoxic activity to mammary tumor cells of sera from normal and tumor-bearing mice with inhibition at low dilutions.

Sera from normal mice and mice bearing murine mammary tumor virus (MTV)-induced tumors showed cytotoxic reactions to the MTV-producing Mm5mt/c1 cell line. The reaction could be blocked by the addition of MTV, but not of purified gp52 and p28. In sera from tumor bearers, cytotoxic responses ranged from 15 to 66%; reactivity was generally highest when the serum was diluted 32 to 128 times. The cytotoxic sera from the normal animals showed a much lower activity; again, there was a lack of cytotoxic response at lower serum dilutions. Low dilutions (1 to 64) of sera from tumor-bearing and normal mice were found to inhibit the MTV-specific cytotoxic activity of rabbit anti-MTV serum on the Mm5mt/c1 cell line. Specificity of this phenomenon was deduced from the fact that there was less inhibition by the mouse sera of anti-Rauscher leukemia virus-specific cytotoxic activity to Rauscher leukemia virus-infected cells and of anti-vaccinia virus serum to vaccinia virus-infected cells. Absorption of the mouse sera with rabbit anti-gp52 serum almost completely abolished the inhibition; after absorption with MTV, inhibition was somewhat reduced. The effect of free gp52 and MTV/anti-MTV immune complexes on the anti-MTV immune response is discussed.

Absence of antigens related to murine mammary tumour virus polypeptides in rat mammary tumours

Fifty-two rat mammary tumours were tested by radioimmunoassay for the presence of antigens related to the envelope glycoprotein gp 52 and core protein p 28 of the murine mammary tumour virus and found to be negative. The tumours assayed were from WAG/Rij ( 16 cases), BN/BiRij ( 16 cases) and Sprague—Dawley ( 20 cases) rats. Thirty of the rats had been irradiated with fast neutrons, 16 with X-rays, one had been implanted with a 17-β-oestradiol pellet and 5 were untreated. The tumours studied included 22 fibroadenomas, 2 adenomas, 19 adenocarcinomas, 4 sarcomas, 1 carcinosarcoma, and 4 of undetermined type.

Precursor product relationship between intracytoplasmic a particle and murine mammary tumor virus core proteins established by tryptic peptide analysis

Abstract The polypeptide pattern of intracytoplasmic A particles, isolated from mammary tumors of ICRC mice, displayed a prominant doublet of about 75,000 molecular weight (Ap75) and a lesser band of 42,000 molecular weight (Ap42). A particle proteins were analyzed by tryptic fingerprint mapping and compared to similarly treated core proteins (p10, p14, p23, and p28) of murine mammary tumor virus (MuMTV) purified from the milk of ICRC mice. Comparison of the peptide maps showed that almost all the major spots of the MuMTV core proteins could be located within the map of Ap75, and that Ap42 contained peptides of only p28 and p14. Since tryptic peptide maps of MuMTV core proteins have been shown to be strain-specific markers, we conclude that the A particles present in mammary tumors of ICRC mice are coded for by the same genes that code for the virions present in the milk of these mice.

Development, Morphology, and Progression of Mammary Tumors During and After Fertile Life in BALB/cfRIII Mice<xref ref-type="fn" rid="fn2">2</xref>

A study was done on 368 BALB/cfRIII mammary tumor-bearing breeding female mice that are of the BALB/c genotype and carry milk-transmitted RIII murine mammary tumor virus (MuMTV) infection initiated by foster-nursing. All tumors were characterized by chronologic, biologic, and morphologic analyses. Mammary tumors occurring during pregnancy-lactation periods were mostly pregnancy-dependent plaques showing organoid tubular structure, sudden growth during late pregnancy, regression after delivery, and slow progression. The frequency of pregnancy-dependent tumors per 100 mice at risk increased steadily from first (1 tumor) to sixth (38 tumors) pregnancy-lactation period. Mammary tumors occurring after fertile life were mostly characterized by an irregular, slow growth and a type B morphology. The frequency of these tumors per 100 mice at risk per 100 days of survival also increased regularly in females undergoing from one (73 tumors) to six (117 tumors) pregnancy-lactation periods. Data were discussed in the light of the pattern of MuMTV release through BALB/cfRIII milk.

Induction of type-C retrovirus by the tumor promotor TPA.

The tumor promotor 12-0-tetradecanoyl-phorbol-13-acetate (TPA) induced endogenous murine xenotropic type-C retrovirus from A1-2 cells, derived from the BALB/c mouse, as determined by infectious center focus-forming assay on permissive normal rat kidney (NRK) cells. Kinetic dose-response studies showed that the number of cells induced to release virus was dependent on TPA concentration and the time of assay following TPA exposure. Maximal induction occurred when cells were treated with 80 ng/ml of TPA for 24 h and assayed at 24 or 48 h. A 30-min pulsed TPA exposure and 200 ng/ml induced virus levels approximating those observed after a continuous 24-h exposure to 80 ng/ml. The combination of TPA at concentrations of 10 and 20 ng/ml and a suboptimal level of 5-iodo-2-deoxyuridine (IdUrd) enhanced retrovirus induction threefold above that seen by the optimum IdUrd concentration alone. The protease inhibitors, antipain and leupeptin, decreased virus induction by TPA, a protease inducer. The capacity of TPA to induce type-C retrovirus complements results demonstrating the enhancement of Epstein-Barr virus (EBV) and murine mammary tumor virus (MMTV) synthesis by TPA.

Kinetics of Responses to Tumor Antigens and Mouse Mammary Tumor Virus in BALB/cCrgl and BALB/cfC3H Mice<xref ref-type="fn" rid="fn2">2</xref>, <xref ref-type="fn" rid="fn3">3</xref>

Spleen cells from BALB/cCrgl mice responded to murine mammary tumor virus (MuMTV) in cell-mediated immune assays at higher levels than did the spleen cells from the syngeneic BALB/cfC3H mice. Implantation in BALB/cCrgl with a chemically induced mammary tumor and in BALB/cfCrgl mice with spontaneous mammary tumors (SMT) arising in the same strain resulted in sensitization of these animals to the antigens of their tumors. Reactivities peaked 3 weeks after transplantation, whereas no positive reactions could be detected when tumors reached maximum size. A kinetic study with the use of MuMTV antigen(s) showed that the responses of lymphocytes from BALB/cfC3H with SMT followed the same pattern as that obtained with tumor antigens, which indicated that this might be a de novo sensitization. In sharp contrast, a steady type of response to MuMTV was observed with the spleen cells from BALB/cCrgl mice; i.e., the levels of responsiveness to MuMTV did not significantly vary at any stage of tumor development. In vivo studies explored the possible relevance of the in vitro cell-mediated immunity to the host defenses. MuMTV-expressing mammary tumor cells were implanted in BALB/cCrgl and BALB/cfC3H mice. The total incidence of tumors was significantly reduced and a delay occurred in their time of appearance in BALB/cCrgl mice in relation to BALB/cfC3H animals. Thus the in vitro reactivity to MuMTV antigen(s) in BALB/cCrgl mice was found to be coincidental with a degree of protection against the development of MuMTV-expressing mammary tumors.

Antigens and antibodies cross-reactive to the murine mammary tumor virus in human breast cyst fluids.

Human breast cyst fluids were shown to contain low concentrations of IgA (15-78 micrograms/ml) and IgG (33-145 micrograms/ml). The IgA:IgG ratios in individual breast cyst fluids ranged from 1:0.6 to 1:4. These levels are considerably higher than their ratio in serum (1:7). IgA from 33% of the 40 fluids examined, and IgG from 10% of the fluids, reacted with the murine mammary tumor virus (MuMTV). The reactivity was detected by an enzyme-linked immunosorbent assay that measures antibody binding to both the envelope glycoprotein and core protein of the virus. In a second series of experiments. IgA from 28% of 40 breast cyst fluids reacted only with MuMTV while IgA from 30% of the fluids was reactive with both MuMTV and the Rauscher murine leukemia virus. Antigen reactive with antiserum to the 28,000-dalton MuMTV core protein (p28), was also identified in a 165,000-g pellet fraction from breast cyst fluids. In individual fluids, the extent of IgA binding to MuMTV was positively correlated (P less than or equal to 0.01) with the binding of anti-p28 antibody to the pellet of the breast cyst fluid. Fractions with the buoyant density of retroviruses (1.16-1.18 g/ml) or their cores (1.21-1.25 g/ml) were isolated from breast cyst fluids. These fractions contained a DNA polymerase capable of utilizing the reverse transcriptase-specific template, dG12-18 x poly rCm. In addition, they reacted with antiserum to MuMTV p 28 but not with antiserum to the 30,000-dalton Rauscher murine leukemia virus core protein.

Monoclonal antibodies against antigens displayed on a progressively growing mammary tumor.

We have produced lymphocyte hybridomas between mouse myeloma cells and either spleen cells of C3H/f/C57BL/6 mice bearing the Mm5mt/c1 tumor-producing murine mammary tumor virus (MMTV) or spleen cells from Fisher rats inoculated with the same tumor. Two classes of hybridoma-secreted monoclonal antibodies were obtained. In the first class are IVC11, IIIA1, and VE7, each of which precipitated a 52,000-dalton protein from 125I-labeled purified preparations of MMTV and [3H]glucosamine-labeled Mm5mt/c1 cell extracts. A second class of monoclonal antibodies, represented by IC12 and IIIB5, reacted specifically with C3H MMTV-secreting cells in radioimmunoassays (RIA) with whole cells and did not precipitate proteins with labeled virus or metabolically labeled Mm5mt/c cell extracts. IC12 and IIB5 could be distinguished from each other in a RIA in which antigen discs were prepared from a membrane preparation of Mm5mt/c1 cells. In this assay, IC12 antibody was blocked from reacting with discs precoated with goat anti-MMTV, and IIB5 antibody was not.

Cytogenesis of murine mammary tumors in BALB/cfC3H and BALB/cfRIII strains.

Biological and morphological differences in the mammary tumors of BALB/cfC3H and BALB/cfRIII mice are due to differences in the causative viruses. The C3H and RIII variants of the murine mammary tumor virus (MuMTV) might give origin to different mammary tumors by transforming different types of cell, i.e. epithelial or myoepithelial cells. The nature (epithelial or myoepithelial) of the neoplastic cells has been investigated by demonstrating their plasma membrane ATPase activities. We found that in normal murine mammary gland both epithelium and myoepithelium have Mg++ dependent ATPase activity, while the myoepithelium shows in addition an Na+K+ dependent ATPase activity. It is suggested that the results obtained exclude the participation of myoepithelium to the neoplastic growth and we ascribe the differences in mammary tumors of the two strains of mice to differences in the mechanisms of action of the virus variants.

Noncoordinate expression of murine mammary tumor virus gene products

The expression of mouse mammary tumor virus (MMTV) gene products was examined in several different murine mammary tissues which showed no production of complete extracellular MMTV B-particles. Analyses by specific competition radioimmunoassay demonstrated that these mammary tissues contained large amounts of protein immunologically related to, or identical to, the 28,000-dalton major nonglycosylated MMTV core protein (p28), but no immunologically detectable 52,000-dalton MMTV envelope glycoprotein (gp52). Such noncoordinate MMTV genome expression was found in primary and transplanted mammary tumors, and in preneoplastic mammary tissues of BALB/c mice, as well as in normal mammary tissue of Swiss albino mice. These mammary tissues exemplify a naturally occurring animal model system for studying noncoordinate expression of MMTV gene products, and its possible significance for MMTV-mediated oncogenesis.

Reevaluation of the Effect of Mouse Mammary Tumor Virus Infection on the BALB/c Mouse Hyperplastic Outgrowth<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

The BALB/c (C-) mouse hyperplastic outgrowth line (D1) was used to study murine mammary tumor virus (MuMTV) expression in both D1 and tumors derived from D1. D1 was transplanted into virus-infected BALB/cfC3H (C+) and virus-uninfected C- animals. In duplicate studies, tumor incidence was the same in both groups. However, the tumor latency period was longer for D1 transplanted into C+ mice (D1/C+) than for D1 transplanted into C- mice (D1/C-). MuMTV was detected in 85% of D1/C+ outgrowths and in 29% of D1/C+ tumors but was never detected in D1/C- outgrowths or tumors. D1/C- outgrowth and tumors and most of the D1/C+ tissues expressed little or no MuMTV RNA. Some D1/C+ tumors expressed substantial levels of MuMTV RNA. These same tumors also had MuMTV antigen and contained the exogenously acquired C3H-MuMTV provirus in the cellular DNA as shown by DNA fragment patterns following cleavage with Pst I and Eco RI endonucleases. D1/C+ tumors containing no viral RNA were also antigen-negative and lacked the acquired C3H provirus. These studies indicate that D1 has substantially changed in its incidence and in its response to MuMTV. MuMTV infection was not tumorigenic in the traditional sense, a finding that has led to a reevaluation of our current models of virus-host relationships and the biology of precancerous conditions.

Short gastric veins as the major portal of entry for milk-borne murine mammary tumor virus.

The flow pattern of the short gastric veins that drain the fundic portion of the stomach appeared to influence mammary tumorigenesis. In I and C57BL mice, strains that are highly resistant to murine mammary tumor virus (MuMTV)-induced mammary tumorigenesis, the short gastric veins empty directly into the splenic vein outside the spleen, connecting with the portal system. In (C57BL X I)F1 mice, which are highly susceptible to MuMTV-induced mammary tumorigenesis, the short gastric veins empty directly into the superior hilus of the spleen, connecting with the splenic parenchyma. Neonatal splenectomy or cauterization of the short gastric veins of (C57BL X I)F1 mice prior to the introduction of MuMTV by foster-nursing delayed the onset and reduced the final incidence of mammary tumors. When neonatal splenectomy was performed after milk-borne MuMTV introduction, it caused premature appearance of mammary tumors without affecting the final tumor incidence. In A, BALB/c, C3H, DBA/2, GR, and RIII mice, strains that are susceptible to MuMTV-induced mammary tumorigenesis, the short gastric veins also empty into the superior hilus of the spleen. Our findings suggest that susceptibility to mammary taumorigenesis may depend on the direct entry of milk-borne MuMTV into the splenic environment through the short gastric veins that drain the fundus of the stomach.

Gene order of murine mammary tumor virus gag proteins and env proteins

The gene orders of murine mammary tumor virus (MuMTV) gag proteins and env proteins have been determined by pactamycin mapping techniques. A MuMTV producing cell line, Mm5mt, was pulse-labeled with [ 31S]methionine in the presence or absence of 5 × 10 −7 M pactamycin, an inhibitor of initiation of protein synthesis. Both pactamycin and the labeled amino acid were removed after the pulsing period and cells were further incubated in normal growth medium. Virus was harvested from the medium after 24 hr, and the individual protein constituents of the virus were analyzed by SDS-gel electrophoresis. For each protein, the ratio of the radioactivity incorporation in the presence of pactamycin and in its absence was determined. The lower this ratio, the closer would be the protein to the amino terminal of the precursor polyprotein. The following gene orders were derived from these experiments: gag, NH, p10(p28,pp23)p14 COOH, and env, NH 2 gp52gp36 COOH.

Depression of murine macrophage accumulation by low-molecular-weight derived from spontaneous mammary carcinomas.

Extracts prepared from spontaneous mouse mammary adenocarcinomas, as well as plasma and urine from inbred C3H/HeN mice carrying murine mammary tumor virus and bearing such tumors, significantly inhibited the accumulation of macrophages at inflammatory sites in inbred the accumulation of macrophages at inflammatory sites in inbred C3Heb/FeJ mice. Much of the inhibitory activity from tumor cells was associated with products having a molecular weight (¿ 30,000); the inhibitory factor isolated from the plasma and urine of tumor-bearing animals had a molecular weight of 30,000 or less. Liver and spleen tissue, plasma, and urine from non-tumor-bearing animals had a molecular weight of 30,000 or less. Liver and spleen tissue, plasma, and urine from non-tumor-bearing animals had no effect on macrophage accumulation. Tumor cell extracts, plasma, and urine from tumor-bearing mice were shown to be free of infectious lactate dehydrogenase virus, a frequent contaminant of transplanted tumor and a known modifier of macrophage function. These results agreed with earlier reports of inhibitory activity for macrophage accumulation found in the tumors and sera of mice bearing multiple-passaged transplanted tumors and suggested that spontaneously arising neoplasms may subvert immune surveillance by depressing the ability of macrophages to respond to inflammatory stimuli.

Murine mammary tumor virus structural protein interactions: formation of oligomeric complexes with cleavable cross-linking agents.

Murine mammary tumor virus protein interactions in the intact virion structure were studied with the use of the cleavable cross-linking reagents dithiobis(succinimidyl propionate) and methyl 4-mercaptobutyrimidate hydrochloride. Cross-linked oligomeric complexes of murine mammary tumor virus proteins were analyzed by two-dimensional gel electrophoresis. Among the complexes most consistently formed were a heterodimer of the two glycoproteins gp36 and gp52, the homodimer of gp36, and the homotrimer of gp52. A very prominent oligomer formed at higher concentrations of dithiobis(succinimidyl propionate) was a complex of about 230,000 molecular weight, made up of three molecules each of gp36 and gp52. A number of lines of evidence, including electron microscopic analysis, suggest that the 230,000-molecular-weight complex actually represents the murine mammary tumor virus spike structure. Of the murine mammary tumor virus core proteins, p14 forms homooligomers most readily. Upon cross-linking with methyl 4-mercaptobutyrimidate hydrochloride a small amount of what seems to be a heterodimer made up of the N-terminal gag protein p10 and the hydrophobic membrane glycoprotein gp36 can be observed.

Presence of a proteolytic activity in murine mammary tumor virus.

Presence of a proteolytic activity in murine mammary tumor virus.

Immunoferritin Labelling of Murine Leukemia Virus Antigens in thin Sections

We previously described the intracellular localization of murine mammary tumor virus (MuMTV) p28 protein in thin sections (1). In that study, MuMTV containing cells fixed in 3% paraformaldehyde plus 0.05% glutaraldehyde were labelled after thin sectioning using ferritin-antiferritin in an unlabelled antibody technique. We now describe the labelling of murine leukemia virus (MuLV) particles using the unlabelled antibody technique coupled to ferritin-Fab antiferritin. Cultures of R-MuLV in NIH/3T3 cells were grown to 90% confluence (2), fixed with 2% paraformaldehyde plus 0.5% glutaraldehyde in 0.1 M cacodylate at pH 7.2, postfixed with buffered 17 OsO4, dehydrated with a series of etha-nols, and embedded in Epon. Thin sections were collected on nickel grids, incubated in 107 H2O2, rinsed in HEPES buffered saline, and subjected to the immunoferritin labelling procedure. The procedure included preincubation in 27 egg albumin, a four hour incubation in goat antisera against purified gp69/71 of MuLV (3) (primary antibody), incubation in F(ab’)2 fragments of rabbit antisera to goat IgG (secondary antibody), incubation in apoferritin, incubation in ferritin-Fab ferritin, and a brief fixation with 2% glutaraldehyde. The sections were stained with uranyl acetate and examined in a Siemens IA electron microscope at an accelerating voltage of 60 KV.

Virus-Mediated Abrogation of Chicken Lymphocyte Responsiveness to Mitogenic Stimulus

We have found that various sorts of virus particles, including avian leukosis and sarcoma viruses, Sendai virus, Friend leukemia virus and murine mammary tumor virus, are able, upon coincubation with chicken peripheral lymphocytes and either Concanavalin A (Con A) or phytohemagglutinin (PHA), to inhibit the mitogenic responses that normally follow. Such inhibition is not dependent on the use of infectious virus, and can be documented by using particles whose infectivity has been abolished by irradiation with ultraviolet light. Lymphoid cells that had been preincubated with viruses at concentrations approximating 10 particles per cell and for only 2 hours were inhibited by as much as 83% in ability to respond to the lectins employed. These results appear to be mediated, at least in part, by a virus-induced factor with the capacity to abrogate the responsiveness of freshly obtained lymphocytes to mitogenic stimulus. This factor is devoid of interferon activity, as tested in a vesicular-stomatitis-virus plaque reduction assay.

Murine mammary tumor virus deficient in the major glycoprotein: Biochemical and biological studies on virions produced by a lymphoma cell line

A cell line, designated MLA, was established from a spontaneous lymphoma of a DBA/2 mouse. These cells contained large amounts of intracytoplasmic type-A particles, the presumed precursors of mature MuMTV. Electron microscopy revealed that the MLA cells released MuMTV-like virions into the culture fluid, but these virions were devoid of the characteristic envelope “spikes” of MuMTV. Antigenic analysis of virions purified from the culture fluid of MLA cells showed that, by microimmunodiffusion, the glycoproteins of MuMTV, gp49 and gp36, were not detectable, nor was the glycoprotein gp70 of MuLV detectable. Internal structural proteins of MuMTV, p28 and p12, were however, readily detectable in these virions. By the more sensitive radioimmunoassay technique, it was found that the virions produced by MLA cells contained some gp49-related antigen, but the amount present was about 500-fold lower than in other tissue culture-derived MuMTV. MLA cells synthesized a 70,000-dalton precursor of MuMTV glycoproteins. Culture fluids from which the virions were removed by centrifugation did contain gp49, indicating processing of the precursor polypeptide, but this polypeptide was not incorporated into the virions. Molecular hybridization studies revealed that MLA cells contained a large amount of MuMTV-specific RNA; the amount of MuLV-related RNA was about 100-fold lower. Whereas 150–200 MuMTV particles were released into culture fluid per cell per day, few MuLV particles were released. Purified virions from the MLA cells were injected into BALB/c and C57BL weanling females. This resulted in mammary tumor development in BALB/c females, but not in C57BL. The tumors contained MuMTV with the normal complement of envelope spikes and gp49 was detectable in the virions.