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.     

Selenium-mediated inhibition of 7,12-dimethylbenz[a]anthracene-induced mouse mammary tumorigenesis

The effect of supplemental selenium on 7,12-dimethyl-benz[a]anthracene(DMBA)-induced mammary tumori-genesis was investigated in severalmouse strains. Selenium, administered as SeO₂ in the drinkingwater, inhibited mammary tumor formation in DMBA-treated (C57BLx DBA/2f)F₁, C3H/StWi and BALB/c female mice. In addition, seleniuminhibited the occurrence of DMBA-induced ductal hyperplasiasin (C57BL x DBA/2f)F₁ and BALB/c mice and mammary tumor virus-inducedalveolar hyperplasias in BALB/cfC3H mice. Selenium did not alterthe growth of established mammary tumors. These results demonstratethat supplemental selenium inhibits both chemical- and viral-inducedmouse mammary tumorigenesis, and secondly, that the developmentof preneoplastic lesions, an early stage in mammary tumorigenesis,is very sensitive to selenium-mediated inhibition.     

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

Mouse mammary tumor virus (MMTV)-specific RNA and antigen content of normal and neoplastic mouse mammary tissue were measured in strains C3H/Sm and C3H-A^vyfB, in C3H-A^vyfB♀ × C3H/Sm♂ F₁ hybrids and in (C3H-A^vyfB × 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-A^vyfB (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-A^avfB 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-A^vyfB subline but is silent or more probably absent in the C3H/Sm subline. Our earlier genetic crosses between C3H-A^vyfB and C3H/Sm mice were conducted to determine the effect of the A^vy 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 A^vy and Mtv-l genes on the mammary gland without affecting the A^vy gene's enhancement of liver tumorigenesis and obesity. Examination of MMTV RNA and antigen content in (C3H-A^vyfB × 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 (A^vyA) and yellow (A^vyA) 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 A^vy 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.     

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.     

Naturally occurring humoral immunity to murine mammary tumor virus (MuMTV) and MuMTV GP52 in mice with low mammary tumor incidence.

Antibodies to murine mammary tumor virus (MuMTV) were found in sera from male and female mice by means of a radiolabelled intact MuMTV precipitation assay. These antibodies were demonstrated both in strains of mice that have a high incidence of mammary tumors and transmit the highly oncogenic MuMTV via the milk (C3H) and in mice that have been foster-nursed to remove the highly oncogenic milk-borne MuMTV (C3Hf) and subsequently have a decreased mammary tumor incidence. Antibodies to MuMTV were readily demonstrated in C3H mice at 6 weeks of age, whereas only marginal antibody activity was detected in C3Hf mice of less than 15 weeks of age. Antibody levels increased with age in both strains, but in C3Hf mice the immune response was also accelerated by pregnancy. Several feral and BALB/c NIV mouse sera with high (¹²⁵I)MMTV precipitating titers also precipitated the major MuMTV envelope glycoprotein (gp52). Maximum precipitation of the (¹²⁵I)gp52 was >50% with a 20% endpoint binding titer of 1:40, whereas the same sera precipitated >80% of (¹²⁵I)MMTV with a titer of >1:2560. These naturally occurring antibodies were specific for gp52 since only MuMTV and purified MuMTV gp52 competed for binding of radiolabelled antigens by limiting dilutions of mouse sera. MuMTV gp52 was found in both C3H and C3Hf mice, predominantly in organs which had secretory functions, such as submaxillary glands and mammary tissue of females and submaxillary, coagulating, and vesicular glands and vas deferens of males. Extracts of other tissues were negative for MuMTV gp52. Neither gp52 nor naturally occurring antibodies for MuMTV were found in BALB/c or C57BL/6 mice.     

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.     

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.     

Separation of high mammary tumor incidence from high hepatoma incidence in backcross mice during segregation of the viable yellow gene

Mammary tumorigenesis in strain C3H-^vyfB female mice (approximately 90% incidence) is not due to exogenous mouse mammary tumor virus (MMTV) but to genetic factors transmitted by either parent. Prominent among these genetic factors is the viable yellow (A^vy) gene which may increase either the virulence of the endogenous MMTV provirus (Mtv-l) by promoting expression or the susceptibility of the mammary tissue to tumorigenesis by endogenous MMTV. Crosses were made between the viable yellow strain, C3H-A^vyfB/He(A^vyA^ay) and C3H/Sm (AA) to give heterozygous (A^vyA) F₁ yellow hybrids from which yellow (A^vy) and agouti (AA) back-cross progeny were produced by mating with C3H/Sm males. A^vyA backcross females were expected to have at least twice as many mammary tumors as the AA back-cross females if the A^vy gene were solely responsible for the high incidence of mammary tumors in C3H-A^vyfB since mammary tumor incidence is 90% in A^vyfB and 40% in (AA)C3HfB. Alternatively, if the high tumor incidence in C3H-A^vyB were due to a more active endogenous MMTV (Mtv-l), then a much smaller difference in the mammary tumor incidence of A^vyA versus AA backcross females would be expected. The more reactive endogenous MMTV would occur equally between yellow and agouti females, since the C3H Mtv-l gene is present on a separate chromosome from the A^vygene. Unexpectedly, the F₁ hybrid females (A^vyA) developed mammary tumors at a relatively low rate (28.9%) although hepatoma incidence remained high (89.5%). Both yellow (A^vyA) and agouti (AA) backcross females showed an even greater reduction in mammary tumor incidence, 7.9% and 3.8% respectively. In contrast, the high rate of spontaneous hepatoma development segregated with the A^vy gene since 86-5% of the yellow (A^vyA) backcross females development hepatomas by 19 months of age, while only 25% of the agouti (AA) backcross females had liver tumors at 24.4 months. The reduction of mammary tumor incidence in (C3H-A^vyfB × C3H/Sm)F₁ hybrids was unexpected since earlier studies had shown that reciprocal F₁, hybrids between strains BALB/c and C3H-A^vyfB continued to show a 90% mammary tumor incidence. The present result is consistent with the possibility that strain C3H/Sm possesses a heritable factor(s) which attenuates the ability of endogenous MMTV to malignantly transform mammary epithelium.     

Autogenous immunity to mouse mammary tumor virus in mouse strains of high and low mammary tumor incidence.

Specific radioimmune precipitation assays were utilized to demonstrate the presence of precipitating antibodies to mouse mammary tumor virus (MMTV) in the high-spontaneous mammary tumor strains of mice: C3H/HeN+, GR/N, BALB/cfC3H, and C57BL/6 X C3H F1 (hereafter called B6C3F1). Antibody titers in C3H/HeN+ mice increased with age, with highest titers observed in tumor-bearing animals. MMTV-precipitating antibodies were not detectable by radioimmune precipitation assay in low-mammary tumor strains (AKR, BALB/c C57BL/6, and C3H/HeN-) but were detectable in MMTV-inoculated BALB/c mice. Appearance of antibodies preceded palpable tumor formation, and antibody titers were directly correlated to virus dose. Natural antibody to MMTV in C3H/HeN+ and B6C3F1 mice coexists with the murine leukemia virus natural antibody as determined by competition radioimmunoassays.     

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.     

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.     

Mode of action of selenium inhibition of 7,12-dimethylbenz[a]anthracene-induced mouse mammary tumorigenesis

Possible mechanisms for the inhibitory effect of selenium on mouse mammary gland tumorigenesis were evaluated in two different mouse models, in 7,12-dimethylbenz[a]anthracene [(DMBA) CAS:57-97-6]treated and hormonally stimulated mammary glands with two dietary levels of Se (0.2 and 2.0 ppm). In (C57BL X DBA/2f)F1 (BD2F1) and BALB/c strains of female mice, Se at 2.0 ppm decreased mammary tumor incidences by 36 and 68%, respectively. Selenium-dependent glutathione peroxidase (GSH-Px) activity in the mammary glands of BD2F1 female mice decreased at 6 months of age and then increased to the highest levels at 9 months of age. Mammary glands from DMBA-treated mice had lower GSH-Px activity than those from control mice. The increase of dietary Se to 2.0 ppm did not overcome this DMBA effect. These results indicate that GSH-Px activity does not correlate with the tumorigenic inhibitory effects of Se. In the hormonally stimulated mammary gland, increasing dietary Se to 2.0 ppm increased GSH-Px activity threefold and decreased mammary-gland-membrane-localized lipid peroxidation by 16%. In vitro peroxidation of hormonally stimulated mammary glands was inversely proportional to the level of GSH present in the incubation mixture. The marginal decrease in lipid peroxidation found in the mammary glands exposed to 2.0 ppm Se could not explain the inhibitory effect of Se on tumorigenesis.     

Genetic diversity of spontaneously developed primary and metastatic mammary tumor cells in mice

Metastatic cells are often considered to be clonal derivatives of one of the primary tumor cell subpopulations. To determine if the cells of spontaneously developed lung nodules in a mammary tumor-bearing mouse represent the major or minor population of the cells in the primary tumor, comparisons were made of the pattern of their mouse mammary tumor virus (MMTV) proviral integrations, and their insertional mutations of the mouse mammary tumor proto-oncogenes, int-1 and int-2. Of the 78 tumor-bearing C3H/He mice sacrificed, seven mice showed metastatic lung nodules, but only four mice had nodule tissues adequate for the present analysis. Examination of the primary tumors and the corresponding lung nodules of these four mice revealed that the number of newly integrated MMTV proviruses and their sites of integration in the genomic DNAs of primary tumors and tumor nodules were variable, and that the int-1 gene was disrupted in three of the primary tumors but not in any of the metastasized tumor tissue. These results support the notion that, at least in some mice, the majority of cells constituting the primary mammary tumors and the corresponding metastases are of different genotypes and raise the possibility that the activation of different int-proto-oncogenes may be involved in the genesis of different cell subpopulations including metastatic cells in the same mouse.     

Activation of H-ras oncogenes in preneoplastic mouse mammary tissues

The mammary hyperplastic outgrowth (HOG) line C4, resulted from serial transplantation of a hyperplastic alveolar nodule which arose in a dimethylbenz(a)anthracene (DMBA) treated mouse. The immortalized C4 outgrowth line, on transplantation into syngeneic mice, develops as preneoplastic, hyperplastic outgrowths and subsequently into malignant carcinomas after a long latent period (greater than 6 months). Treatment of mice carrying C4 HOG transplants with DMBA resulted in a reduced latent period for tumor development (less than 3 months) and an increased tumor incidence. DNA's from C4 HOGs and mammary carcinomas of untreated as well as DMBA-treated mice were analyzed for the presence of oncogenes by the NIH3T3 focus forming assay. Transforming H-ras genes were detected in two of 6 preneoplastic HOGs and 10 of 12 carcinomas from DMBA-treated mice. DNAs from neither the HOGs nor the tumors from untreated mice were positive in this assay. The H-ras locus was then directly examined in the 61st codon by in vitro amplification of each of the tissue DNAs using PCR. The location of the activating mutation was determined by hybridization of amplified DNA to mixed sequence oligonucleotide probes. The specific nature of the mutation was defined by RFLPs using XbaI, TaqI and Sau96I restriction enzymes. Six of the H-ras oncogenes in DMBA-promoted tumors were activated by commonly observed A to T transversions at the 61st codon, while five (including an additional tumor with H-ras oncogene revealed by PCR analysis) contained novel A to G transitions. The H-ras oncogene in one DMBA-treated HOG sample was activated by A to T while the second contained an A to G mutations, representative of both modes of mutational activation involved in this model of mammary tumorigenesis. In summary, DMBA-induced point mutated H-ras oncogenes appear to potentiate the progression of hyperplastic outgrowths (HOG) to mammary carcinomas.     

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.     

Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition

Introduction  

This report describes the isolation and characterization of three new murine mammary epithelial cell lines derived from mammary tumors from MMTV (mouse mammary tumor virus)/activated Neu + TβRII-AS (transforming growth factor [TGF]-β type II receptor antisense RNA) bigenic mice (BRI-JM01 and BRI-JM05 cell lines) and MMTV/activated Neu transgenic mice (BRI-JM04 cell line).     

A congenic line of the DDD mouse strain, DDD/1-Mtv-2/Mtv-2: establishment and mammary tumorigenesis

A single dominant gene on chromosome 18, Mtv-2, controls both the early appearance of mammary tumors and expression of mouse mammary tumor virus (MMTV) in the milk. A congenic DDD mouse strain, DDD/1-Mtv-2/Mtv-2 (DDD-Mtv-2), was developed by introducing this gene from GRS/AJms (GR) into DDD/1 mice by repeating 12 backcrosses and subsequent inbreeding using mammary tumors as a marker for selection. Southern blot analysis of the liver DNA from the resulting congenic mice with EcoRI and MMTV-U3 prove revealed that two DNA fragments corresponding to Mtv-2 were specifically transferred from GR to congenic mice. Detection of MMTV-gp52 antigen in the mammary gland and mammary tumor development in DDDfDDD-Mtv-2 mice demonstrated the production of infectious mature MMTV by Mtv-2 in congenic mice. About 80% of breeding DDD-Mtv-2 females developed mammary tumors in the course of one-year follow-up. The tumor incidence was lower and the tumor age higher than those in GR mice, suggesting less active functioning of the gene on the DDD genetic background. About 70% of these tumors were morphologically classified as pale cell and type P carcinomas peculiar to GR mice. The gene seemed to control the histologic features of mammary tumors. Congenic mice carried an MMTV provirus in an incomplete form on Y chromosome. The DDD-Mtv-2-strain will provide a new model for biological and molecular researches into mouse mammary tumorigenesis.     

A Congenic Line of the DDD Mouse Strain, DDD/1-Mtv-2/Mtv-2: Establishment and Mammary Tumorigenesis

A single dominant gene on chromosome 18, Mtv-2 , controls both the early appearance of mammary tumors and expression of mouse mammary tumor virus (MMTV) in the milk. A congenic DDD mouse strain, DDD/1- Mtv-2 / Mtv-2 (DDD- Mtv-2 ), was developed by introducing this gene from GRS/AJms (GR) into DDD/1 mice by repeating 12 backcrosses and subsequent inbreeding using mammary tumors as a marker for selection. Southern blot analysis of the liver DNA from the resulting congenic mice with Eco RI and MMTV-U3 probe revealed that two DNA fragments corresponding to Mtv-2 were specifically transferred from GR to congenic mice. Detection of MMTV-gp52 antigen in the mammary gland and mammary tumor development in DDDfDDD- Mtv-2 mice demonstrated the production of infectious mature MMTV by Mtv-2 in congenic mice. About 80% of breeding DDD- Mtv-2 females developed mammary tumors in the course of one-year follow-up. The tumor incidence was lower and the tumor age higher than those in GR mice, suggesting less active functioning of the gene on the DDD genetic background. About 70% of these tumors were morphologically classified as pale cell and type P carcinomas peculiar to GR mice. The gene seemed to control the histologic features of mammary tumors. Congenic mice carried an MMTV provirus in an incomplete form on Y chromosome. The DDD- Mtv-2 strain will provide a new model for biological and molecular researches into mouse mammary tumorigenesis.     

The role of high levels of dietary fat in 7,12-dimethyl-benzanthracene-induced mouse mammary tumorigenesis: lack of an effect on lipid peroxidation

The effects of dietary fat on 7,12-dimethylbenzanthracene (DMBA)-inducedmammary tumorigenesis were examined in BALB/c female mice. Inthe first experiment, BALB/c mice were treated with a totaldose of 1,3, or 6 mg DMBA to induce mammary tumors. One weekafter the last dose of DMBA, the mice were placed on a 5% (LF)or 20% (HF) corn oil semi-purified diet. The 20% corn oil dietresulted in a significantly decreased mean tumor latency periodin each of the three groups of mice (p <0.05). However, themammary tumor incidences at 11 months after DMBA treatment werethe following: 6 mg DMBA (LF-14/32, HF-17/32); 3 mg DMBA (LF-13/50,HF-14/50); 1 mg (LF-2/50, HF-6/50). In the second experiment,prolonged hormonal stimulation by pituitary isografts, but nota HF diet, enhanced the incidence of mammary tumors in BALB/cmice given a threshold dose of DMBA (0.5 mg). In a further attemptto examine the mechanism of the enhancing effects of dietaryfat, lipid peroxidation was measured in the microsomal-mitochondrialfractions of the mammary glands by two assays; measurementsof conjugated dienes and TBA reactants (malonyldialdehyde).The high fat diet had no effect on the levels of conjugateddienes in mammary cell membrane preparations and decreased thelevels of TBA reactants (malonyldialdehyde). The results indicatedthat a high fat diet did not lead to enhanced levels of lipidperoxidation in preparations of microsomal mitochondrial membranesfrom mouse mammary glands. The implications of these resultsare discussed with respect to the role of dietary fat as a promoterof mouse mammary tumorigenesis.