Mammary Tumor Virus Antigens Research Articles

HISTOLOGICAL DISTRIBUTION OF MTV ANTIGEN IN MICE DETECTED BY IMMUNO‐PEROXIDASE STAINING

Histological localization of mammary tumor virus (MTV) antigen was investigated using a variety of organs high (DD/Tbr, SHN, SLN, GR) and low (BALB/c) mammary cancer mice strains and immuno-peroxidase staining with MTV antigen. Except for BALB/c strain mice, the mammary gland and mammary tumors were generally positive. Accessory male genital organs including the prostate, seminal vesicle, and coagulating gland also demonstrated a positive reaction, but the testis and female genital organs including uterus and ovaries did not. MTV antigen was also revealed in the serous acini of the salivary gland in both sexes. The site of positive reaction in the accessory male sex organs and salivary gland was located in the apical portion of the secretory epithelial cells and their secretory substance. Localization and intensity of antigenic expression of MTV detected histologically were comparable to results obtained by immunodiffusion test and radioimmunoassay. These evidences support the idea that MTV is transmitted horizontally via seminal fluid or saliva.

マウス汗腺・皮脂腺における乳癌ウイルス抗原発現の免疫組織化学

Mammary tumor virus (MTV) antigens in both sexes of GRS/A, SHN and C3H mice were examined in the sweat and sebaceous glands by immunoperoxidase technique using antiserum against gp52, envelope protein, or p27, core protein. Balb/c mice were used for reciprocal foster nursing with these inbreds to discriminate the expression of endogenous MTV from that of exogenous MTV. Both antigens were first detected around the age of 4 months in the sweat glands of mice with endogenous GR- or SHN- MTV. A linear staining of gp52 was seen along the luminal borders of glandular cells, and the reaction products for gp52 were demonstrated on the apical cell membranes, where no virion could be seen ultrastructurally. A diffuse staining of p27 was found in the cytoplasm of some glandular cells, where MTV particles could not be detected. In the sebaceous gland of the same mice, however, only p27 was first detected at the age of 60 days. A dot-like staining of p27 was found in the perinuclear region of some glandular cells, where an aggregation of intracytoplasmic A particles could be seen under an electron microscope. These positive stainings were unrelated to sex. In such skin appendages of all examined C3H mice and Balb/c mice with GR- or SHN- MTV, no antigen expression could be seen up to the age of 500 days. Therefore, some genes might be able to regulate the expression of endogenous MTV antigens in the skin appendages, while their glandular cells would have no receptor for exogenous MTV, namely the so-called "milk factor".

Tissue and organ distribution of mammary tumor virus antigens in low and high mammary cancer strain mice

Expression of mammary tumor virus (MTV) antigen was measured in a wide variety of organs and tissues of a series of high (GR, SHN, SHNf, DD, SLN, SLNf) and low (DDf, DDD, DDDf, KF, KFf, ddY, C57BL, BALB/c) mammary cancer strain mice. Tests were carried out by microimmunodiffusion (micro-ID) and immunoperoxidase tests on formalin-fixed tissues and radioimmunoassays in extracts for 2 viral proteins, MTVp27 from the viral core and MTVgp52 from the viral envelope. Organs with exocrine function, i.e. the mammary gland, salivary gland, coagulating gland and prostate, were mostly positive. The secretory epithelial cells of these organs showed viral antigen expression. Less positivity was encountered in brain, pancreas, stomach, urinary bladder, epididymis, uterus, thymus, spleen, lymph nodes and kidney, plasma and blood cell pools. Unexpectedly the uterus extracts showed MTV antigen expression, occasionally even by immunodiffusion, especially in mice of various DD stocks, but also in the GR strain. Another striking observation was the detection of MTV antigen expression in salivary glands in C57BL strain mice; most other organs of this strain (including the mammary glands) were negative. The implications of results of this extensive survey for MTV antigen expression are discussed for tumorigenesis by MTV of mammary gland and perhaps other tissues in the mouse.

Suppression of spontaneous mammary tumorigenesis despite Mtv-1 gene expression in hybrid and backcross C3H-AvyfB X C3H/Sm mice.

AbstractMouse mammary tumor virus (MMTV)‐specific RNA and antigen content of normal and neoplastic mouse mammary tissue were measured in strains C3H/Sm and C3H‐AvyfB, in C3H‐AvyfB♀ × C3H/Sm♂ F1 hybrids and in (C3H‐AvyfB × C3H/Sm) × C3H/Sm♂ backcross segregants. Good correlation between MMTV antigen expression in lactating mammary glands and mammary tumor incidence was observed in the parental mouse strains. The high‐mammary‐cancer strain C3H‐AvyfB (90%) had consistently high levels of MMTV antigen in the mammary glands and tumors, whereas mammary tissues from low‐mammary‐cancer subline C3H/Sm (1.6%), whether normal or neoplastic, had little or no detectable MMTV antigen. In contrast, MMTV RNA content of normal C3H/Sm mammary tissue was consistently as high as or higher than corresponding levels in C3H‐AavfB females. These molecular data, along with the relative mammary tumor incidences, suggest that the endogenous MMTV provirus expression gene Mtv‐l present in C3H mice is fully expressed in the C3H‐AvyfB subline but is silent or more probably absent in the C3H/Sm subline. Our earlier genetic crosses between C3H‐AvyfB and C3H/Sm mice were conducted to determine the effect of the Avy gene on mammary tumorigenesis during its segregation in back‐cross (BC) females produced from matings of the F1 hybrid females with C3H/Sm males. Our mammary tumor incidence data presented in the accompanying paper strongly suggested the possibility that C3H/Sm mice possessed a dominant genetic factor(s) which specifically attenuated the tumorigenic effects of the Avy and Mtv‐l genes on the mammary gland without affecting the Avy gene's enhancement of liver tumorigenesis and obesity. Examination of MMTV RNA and antigen content in (C3H‐AvyfB × C3H/Sm) hybrid and BC females showed that the expression of MMTV antigen was completely suppressed in the agouti (AA) BC females. However, yellow F1 (AvyA) and yellow (AvyA) BC females continued to express MMTV antigen in their mammary glands even though mammary tumor incidence was significantly depressed. MMTV RNA levels were similar in all F1 and BC females regardless of the presence or absence of the Avy gene. Mammary tumor incidence was reduced in all the BC females (whose genome is 3/4 derived from C3H/Sm) relative to the F1 females (whose genome is only 1/2 derived from C3H/Sm) suggesting that more than one gene is involved in the suppression of spontaneous mammary tumor development. Cessation of translational expression of endogenous MMTV occurred only in the agouti (AA) BC females, suggesting an association between this phenomenon and the agouti locus of C3H/Sm. Evaluation of the relative MMTV RNA content of nucleic and cyto‐plasmic fractions from C3H/Sm mammary tissues strongly suggests that transport and/or processing of MMTV RNA is defective in these mice. This defect probably accounts for the absence of detectable MMTV antigens in C3H/Sm and agouti (AA) BC mammary tissues and tumors and may contribute to the observed reduction in mammary tumor incidence.

非近交ddマウスにおける乳癌ウイルス (MuMTV) 感染の実態

The expression of mammary tumor virus (MTV) antigen in the milk and various organs of three non-inbred dd mouse stocks (ddO, ddN and ddY) was examined by the immunodiffusion (ID) and micro-immunodiffusion (micro-ID) tests. The rate of MTV antigen expression in the milk was 100% at the first lactation in ddO (6/6) and ddN mice (10/10), and 23% in ddY mice (3/13). Mammary tumor incidence was 13% (mean tumor age: 12.0 months), 32% (9.6 months) and 10% (11.5 months) in ddO, ddN and ddY mice, respectively, In F1 hybrids between MTV-free BALB/c females and dd males, a high level of MTV antigen was detected by the ID test in the milk of (BALB/c X ddO) F1, however, the levels in (BALB/c X ddN) F1 and (BALB/c X ddY) F1 mice were low at the first lactation and elevated with the advance of lactation number. Mammary tumor incidence had a trend to be higher and earlier in these F1 hybrids than in non-inbred dd stocks. The development of mammary tumors and detection of MTV antigen in F1 hybrids indicate the extrachromosomal transmission of MTV by male dd mice. The micro-ID test has shown that the mammary tumors, mammary glands, male genital organs except for the testis and the salivary gland expressed MTV antigen, with a high frequency of suggesting that secondary male genital organs may play an important role in MTV infection in mice.

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.

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

AbstractC3H/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.

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.

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.

Factors interfering with cellular immunological responses to the murine mammary tumor virus in tumor‐bearing mice

T-cell-enriched spleen cell fractions from BALB/c mice bearing virally induced mammary tumors become unresponsive with progressive tumor growth in the lymphocyte blastogenesis test, using purified mammary tumor virus (MTV) as an antigen. reactivity can be restored by mild trypsinization or extensive washing of the cells. When reactive cells from mice immunized with MTV were incubated with the wash fluid of the initially unresponsive cells. MTV-specific blastogenesis was inhibited. Washings of normal cells had no such effect. Leukocytes from tumor-bearing mice also become unresponsive to MTV in the leukocyte adherence inhibition (LAI) assay. Trypsinization or extensive washing does not restore reactivity. However, such treatment of reactive cells from immunized animals abolishes a positive reaction in the LAI assay, using MTV as an antigen. The washings of spleen cells from tumor-bearing mice inhibit the reactivity of leukocytes from immunized animals. Sera from these mice could block the reaction in both cellular immunological tests. The inhibiting factors appeared early after the onset of tumor growth. By means of the Sepharose bead immunofluorescence assay it was established that wash fluid and sera with blocking activity contain MTV antigens as well as anti-MTV antibodies.

Antibody-Induced Modulation and Shedding of Mammary Tumor Virus Antigens on the Surfaces of GR Ascites Leukemia Cells as Compared With Normal Antigens2

The distribution, antibody-induced redistribution, and shedding of murine mammary tumor virus (MuMTV) antigens and the surfaces of GR mouse ascites leukemia (GRSL) cells were studied by the immunoferritin technique and compared with the same activities of thy 1.2 and H-2.8 antigens. MuMTV antigens were redistributed easily and then largely shed from the cell surface; in contrast, H-2.8 antigen moved easily and probably was partially released from the plasms membrane and Thy 1.2 antigen moved slowly and was some what interiorized. The complement-dependent cytotoxicity test was used to study the possibility of antigenic modulation for these cell-surface antigens from the surface of the GRSL cells could be modulated by preincubation with anti-MuMTV serum, in contrast to H-2.8 and Thy 1.2 antigens. The results obtained with the immunoferritin technique and the cytotoxicity test correlated well and sug-ested that the shedding of MuMTV antigens from the cell surfaces may occur in vivo, providing the tumor a way to escape from the immune defense of the host. Thy 1.2 and H-2.8 antigens were present on the envolope of B and C particles, which suggested that these viruses do not select a Thy 1.2 or H-2.8-negative area of the GRSL cell surface as amaturation site.

Complexity of Factors in Sera of Different Mice That Affect MTV-Induced Mammary Tumor Cells

Specific spleen cell activity in microcytotoxicity assay can be altered by pretreatment of target mammary tumor virus (MTV)-induced mammary tumor cells with serum. Serum from both BALB/cfC3H females neonatally infected with MTV and BALB/c females horizontally exposed to MTV antigens will block specific spleen cell activity against isologous mammary tumor cells. On fractionation of sera, blocking factors are localized in the 7s fraction. The 19s fraction contains recruiting factors that are not detectable in the unfractionated serum; these factors are active against isologous tumors and are thus distinct from the tumor-specific recruiting factors previously described in the sera of tumor-bearing females, which are active only against the autologous tumor. Antibodies mediating complement-dependent cell lysis are also detectable after serum fractionation.

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.

Blocking of Spleen Cell Activity against Target Mammary Tumor Cells by Viral Antigens

Spleen cells from BALB/c females exposed to or neonatally infected with mammary tumor virus (MTV) are cytotoxic to MTV-induced mammary tumor cells in microcytotoxocity assay. This activity can be partially or completely blocked by pretreatment of spleen cells with MTV purified from milk. Murine leukemia virus (MuLV) has no effect. T cell responses of virgin and multiparous BALB/cfC3H females are effectively blocked. Non-T cell responses of multiparous BALB/cfC3H females or of virgin BALB/c females are blocked by some but not all of the MTV antigen preparations. MuLV, but not MTV, can block activity of spleen cells from MuLV-sensitized donors against target MuLV-producing tumor cells.

In Vitro Detection of Immune Responses to Mtv-Induced Mammary Tumors: Qualitative Differences in Response Detected by Time Studies

Both BALB/cfC3H females neonatally infected with mammary tumor virus (MTV) and MTV-free BALB/c females possess spleen cells capable of significant activity against target-MTV-induced mammary tumor cells in in vitro microcytotoxicity tests. The responses of the BALB/cfC3H and BALB/c females are different, however, on the basis of time studies. Further, the reactivity characteristic of virgin BALB/cfC3H females is increased in parous females, and part of this reactivity is subsequently lost in multiparous females bearing large tumors. BALB/cfC3H females possess reactive spleen cells as early as 3 weeks of age, indicating that response to MTV antigens develops soon after birth in these neonatally infected mice.

Expression of mammary tumor virus antigen on the membranes of lymphoid cells.

Expression of mammary tumor virus antigen on the membranes of lymphoid cells.

Limited Synthesis of Mammary Tumor Virus Antigens in Lymphoid Tissues and in Certain Leukemias of High Mammary Cancer Strains

Limited Synthesis of Mammary Tumor Virus Antigens in Lymphoid Tissues and in Certain Leukemias of High Mammary Cancer Strains

Inability to Predict Mammary Tumorigenesis in Strain A Mice From Presence of Mammary Tumor Virus or Antigen in the Milk

Strain A females were milked and their milk was tested for mammary tumor virus (MTV) by the hyperplastic alveolar nodule test and for MTV antigen by microimmunodiffusion. An attempt was made to correlate these results with later occurrence of mammary tumors in the females. There was not full agreement between the results of the test for the presence of the MTV and those of the test for MTV antigen, and from these results it was impossible to predict accurately whether a specific female would later develop a tumor. It is concluded that, in this strain, factors other than the MTV can be the determinant in whether the individual female will develop a tumor.

Mammary tumor virus antigen: sensitive immunoassay.

A rapid, sensitive immuno-assay for mammary tumor virus antigen based on inhibition of passive hemagglutination has been developed. The method permits measurement of this antigen in mouse milk from which the fat has been removed.