Murine Mammary Tumor Virus Research Articles

ML antigen of DBA/2 mouse leukemias: expression of an endogenous murine mammary tumor virus.

Spontaneous, transplantable leukemias of DBA/2 mice express an antigen (ML) which cross-reacts with antigens of murine mammary tumor virus (MuMTV). The MuMTV cross-reactive antigen of the DBA/2 leukemias (ML cells) was found to be a glycoprotein of 78,000 molecular weight containing antigenic determinants of the major MuMTV glycoprotein gp52. No MuMTV particles were produced by the ML cells, although they did contain type A particles--the pronucleocapsids of MuMTV. The ML antigen appeared to be an aberrant form of the intracellular MuMTV env precursor molecular prgp70, which was not processed properly but instead acquired extra carbohydrate groups and was expressed in uncleaved form on the cell surface. Isolation of MuMTV core protein p28 from the leukemic cells and subsequent tryptic peptide mapping analysis showed that the p28 from leukemia cells differed from the p28 of MuMTV isolated from DBA/2 mouse milk. These observations indicate that the MuMTV expressed in DBA/2 leukemic spleen cells is of a different strain than the virus secreted in lactating mammary glands of DBA/2 mice and probably represents the expression of an endogenous DBA/2 provirus.

Cell-Mediated Immunity to Mouse Mammary Tumor Virus Antigens by Patients With Hyperplastic Benign Breast Disease<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

Peripheral blood leukocytes of patients with preoperative breast cancer, benign breast disease, and benign gynecologic disorders and normal healthy females were tested, as blind coded specimens, with murine mammary tumor virus (MuMTV) antigens in the direct and indirect leukocyte migration inhibition (LMI) assays. The incidence of reactivity by patients with breast cancer was low. (From 5 to 35% breast cancer patients reacted, depending on which group of control individuals they were compared to and what antigen was used.) Nonparametric analyses showed no differences between control groups (normal donors and patients with gynecologic disorders) and breast cancer patients with either assay. However, there was a significant difference between benign breast disease patients with hyperplasia and 1) benign breast disease patients without hyperplasia (P less than 0.03) and 2) patients with gynecologic disorders (P less than 0.04) in the direct assay when it was performed blindly with the gp52 antigen. Patients with hyperplasia (benign breast disease as well as breast cancer) had a higher incidence of enhanced migration in the indirect test than breast disease patients without hyperplasia. The enhanced migration to the MuMTV was correlated to enhanced migration to a 3-M KCI extract of the breast cancer cell line MCF-7 in simultaneous tests. Thus the LMI assays with MuMTV antigens do not appear valuable in breast cancer diagnosis, but they may help to identify a small group of benign breast disease patients whose breast pathology is thought to be associated with a high risk for developing breast cancer.

Relationships between morphology and viral etiology in mammary tumors of BALB/cfC3H and BALB/cfRIII mice.

The morphologic pattern of mammary tumors induced by milk-transmitted C3H and RIII murine mammary tumor viruses (MuMTVs) in BALB/c hosts infected by foster nursing were investigated further an compared taking into account several tumor and host variables. Comparison of BALB/cfC3H and BALB/cfRIII mammary tumors under identical tumor and host conditions showed highly significant differences in subgross morphology and histologic structure. Differences in cytology were also observed. Data suggest that mammary tumor morphology in high cancer strain mice is controlled by the causative MuMTV.

Sera from breast cancer patients contain an IgA antibody to a breast cyst fluid component

We have previously demonstrated that aspirated breast cyst fluids from women with benign breast disease contained a particulate component (BCF-PC) that reacted with antiserum to the murine mammary tumor virus (MuMTV). Since women with breast cancer contain antibodies and antigens reactive with MuMTV it was of interest to measure the immune response in breast cancer patients to the BCF-PC. Sera were obtained from breast cancer patients prior to surgery and 5 days postmastectomy, from women with benign breast disease and from healthy women. Analysis for antibody to the BCF-PC was by an enzyme-linked immunosorbent assay. IgA from premastectomy breast cancer patients was significantly more reactive with the BCF-PC than was IgA from women in the other categories ( P < 0.001). In contrast, IgG binding to the BCF-PC did not differ among the various groups. It was further demonstrated that the reactivity of IgA from any particular serum for the BCF-PC remained constant for different BCF-PC isolates. Other fractions of breast cyst fluids were unreactive with IgA from breast cancer patients. Determination of serum IgA levels in these women revealed that the mean IgA level was significantly higher in healthy women than in the other categories ( P < 0.001). Individual IgA levels and levels of IgA antibody to the BCF-PC were found to be not correlated in breast cancer patients. These results indicate that the production of a specific IgA antibody to the BCF-PC increases in sera of patients with breast cancer. Lastly, it was shown that preincubation of patients' sera with disrupted MuMTV, but not intact MuMTV, severely reduced subsequent IgA binding to the BCF-PC. The analysis of sera for IgA antibody to the BCF-PC may be of value for the detection of breast cancer or its recurrence.

Role of natural prostaglandins in the control of murine mammary tumor virus expression.

The regulation of murine mammary tumor virus (MuMTV) production by mammotropic hormones, hormonomimetic substances, and cyclic nucleotides was investigated. The virus produced in control and treated mammary tumor cell cultures was quantitated by measuring the supernatant reverse transcriptase activity in exogenous reaction using poly(rC).oligo(dG) as template-primer. Two days after exposure, the synthetic glucocorticoid, dexamethasone (DXMT), increased spontaneous MuMTV production at optimal concentration (0.1 mumol) up to ten times. Dibutyryl derivative of cyclic AMP had no effect on spontaneous MuMTV production, whereas the drug potentiated suboptimal concentrations of the glucocorticoid. Natural prostaglandins, potent agonists of adenylate cyclase catalyzing intracellular synthesis of cyclic AMP, enhanced both basal (up to five times) and DXMT-stimulated (up to 1.6 times) MuMTV replication. The MuMTV-stimulating activity of prostaglandins decreased in the order of PGA1 greater than PGE1 greater than PGB1 greater than PGF2 alpha. Prostaglandins can be replaced partially by norepinephrine and isoproterenol by enhancing the DXMT-mediated MuMTV stimulation, whereas these drugs remained without effect on spontaneous MuMTV production. Theophylline, an antagonist of cAMP-phosphodiesterase converting cAMP to AMP, enhanced the virus-stimulating activity of DXMT as well as of prostaglandins. The enhancement of MuMTV production by adenylate cyclase agonists do not correlate absolutely with the estimates of intracellular cAMP levels, since the highest amounts of cAMP has been repeatedly observed in cells treated with PGE1 and norepinephrine. The results indicate that besides hormones, other hormone-like substances and cyclic nucleotides may be involved in the complex mechanism of hormone-regulated MuMTV genome expression.

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.

Detection of type-specific antigenic determinants of murine mammary tumor viruses with cell-mediated immune assays.

Exposure of lymphoid cells from normal and mammary tumor-bearing Balb/cCrgl mice to MMTV antigens result in positive responses in blastogenic transformation and migration inhibition reactions. The levels of these responses appear to depend on the murine strain source of the viral preparations. Enhanced reactivities were observed in cultures exposed to purified MMTV from culture fluids of mammary tumor cells of spontaneous origin in the C3H strain when compared to purified MMTV derived from milk of mice from the RIII strain. To ensure the virus relatedness of the differences observed, MMTVs obtained from a common source, i.e., the Crandall feline embryo kidney cell line, were employed in blastogenesis assays. Parallel testing of feline cell-derived C3H, RIII, and GR mammary tumor viruses resulted in different levels of [3H]thymidine incorporation by spleen cells from Balb/cCrgl mice over a range of viral concentrations. These results indicate that it is possible to detect various types of MMTV variants with cell-mediated immune assays. The diverse functional reactivities elicited by these viruses may be related to the differences observed in their bioactivities as determined by incidence and development of mammary tumors and the potential of causing metastases.

Mammary tumor virus DNA contains sequences required for its hormone-regulated transcription

Glucocorticoids regulate the rate of transcription of integrated murine mammary tumor virus (MTV) genes in most clones of MTV-infected rat hepatoma tissue culture (HTC) cells. To determine whether hormonal regulation is mediated from flanking cellular sequences or rather from within the viral DNA, we analyzed the relative rates of trancription of MTV and adjacent HTC sequences in two lines of infected HTC cells, J2.15 and J2.17, each of which contains a single insertion of MTV DNA. In addition, we measured in uninfected HTC cells the transcriptional rates of the corresponding “preinsertion fragments,” which may be viewed as the equivalent sequences bearing deletions of MTV DNA; the two genomic segments into which integration occurred in the two lines are unrelated. Previous work showed that glucocorticoids induce the accumulation of MTV RNA in J2.17, whereas viral transcripts are not detected in J2.15. RNA pulse-labeling experiments indicate that glucocorticoids stimulate the rate of MTV gene transcription in J2.17 but not in J2.15; in contrast, no labeled RNA hybridizing to flanking sequences was detected either in the uninfected or in the infected cells. We conclude that the host site of integration of MTV need not be transcriptionally active or hormonally responsive to permit viral gene expression. Furthermore, these rate measurements indicate that glucocorticoid-stimulated MTV RNA synthesis in J2.17 does not reflect readthrough transcription from a regulated cellular promoter. This notion is independently supported by transcript mapping experiments showing that the 5′ terminus of nuclear MTV RNA is at a site on MTV DNA approximately 1.2 kb downstream from the host-viral junction. Thus our data are consistent with the presence of a hormone-responsive element within the provirus.

Age- and Sex-Related Differences in Antibody Formation And Blastogenic Responsiveness of Splenocytes From RIII Mice Developing Virus-Induced Mammary Adenocarcinoma&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;

Inbred RIII mice, known to be infected neonatally with murine mammary tumor virus so that females develop mammary adenocarcinoma by 12-15 months of age, were examined with regard to antisheep red blood cell antibody responses at the cellular level. Female mice, 3-11 months old, compared to male mice of the same ages had consistent and significant depression of the antibody response of their splenocytes. Furthermore, female mice with adenocarcinoma showed an even greater depression of the antibody response. Spleen sizes were consistently increased in females as compared to those of male mice throughout the first year of life. The blastogenic responsiveness of the splenocytes to the B-cell mitogen Escherichia coli lipopolysaccharide and the T-cell mitogen phytohemagglutinin-P was not significantly different between male and female mice during the same periods, although the responses of the older tumor-bearing female mice to phytohemagglutinin-P were lower than those of non-tumor-bearing female mice. A complex relationship between age, sex, and immune responsiveness was evident in these mammary tumor virus-infected mice, which made it difficult to attribute a specific immune event to emergence of the mammary adenocarcinomas in the female as compared to male mice.

Purified glucocorticoid receptors bind selectively in vitro to a cloned DNA fragment whose transcription is regulated by glucocorticoids in vivo.

Activated glucocorticoid receptor protein, purified to 40-60% homogeneity from rat liver extracts, binds selectively in vitro to a cloned fragment of murine mammary tumor virus (MTV) DNA. The DNA fragment tested contains about half of the sequences present in intact MTV DNA, and its rate of transcription, like that of the intact viral element, is strongly stimulated by glucocorticoids when it is introduced into the genome of a receptor-containing cell. In contrast, the receptor fails to bind selectively to DNA restriction fragments from E. coli plasmids pBR322 and RSF2124 or from bacteriophages lambda and T4. Preliminary experiments to localize regions within MTV DNA responsible for selective binding have revealed thus far one subfragment that fails to bind the receptor and one selectively bound subfragment that maps far downstream from the 5' terminus of the normal RNA transcript. These studies are consistent with the notion that steroid receptors may modulate rates of transcription by recognizing specific DNA sequences within or near the regulated genes.

Induction of Endogenous Mammary Tumor Virus Expression During Hormonal Induction of Mammary Adenoacanthomas and Carcinomas of BALB/c Female Mice&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

Treatment of BALB/c female mice with pituitary isografts, under conditions that established mammary hyperplasia in an anovulatory condition, enhanced mammary tumor development with prior onset of dysplastic foci in lobular parenchyma. Tumor onset began at 8 months (mean onset time, 18 mo); the 25-month incidence was 100%. Adenoacanthomatous tumors appeared first. Adenocarcinomas appeared only after more than 14 months of continuous hormone stimulation. Dysplastic and neoplastic changes occurred while blood levels of the three major mammotropic hormones were physiologic. Murine mammary tumor virus (MuMTV) p28 was detected in all tumors tested, independent of time of tumor appearance or tumor type, although keratinizing cells in adenoacanthomatous tumors did not contribute to MuMTV antigen expression. MuMTV gp52 was detected in only a small fraction of cells in a few tumors. MuMTV RNA contents were above normal in all tumors tested. Neither MuMTV structural antigens nor RNA was induced in normal glands by the same hormone treatment that ultimately resulted in dysplasia and tumor formation and elevated levels of these viral markers in neoplasms. The MuMTV RNA in all hormone-induced tumors was readily distinguishable in base sequence from standard MuMTV RNA but indistinguishable from MuMTV RNA recovered from lactating mammary glands of BALB/c females carrying only endogenous MuMTV proviruses, suggesting that endogenous MuMTV RNA sequences were induced in hormone-induced neoplasms. RNA indistinguishable from MuMTV sequences present in hormone-induced primary tumors was also detected in multiple genomic equivalents in two independently derived hormone-induced premalignant alveolar hyperplasias. MuMTV p28 was detected, but gp52 was not. The same hormone stimulus that generated 100% tumors in normal gland greatly accelerated tumor development in premalignant hyperplasias but did not amplify MuMTV RNA or antigens in either hyperplasias or the tumors derived from them. B-type virions were not detected in these tissues, in either hypophyseal implant-stimulated or virgin hosts. Cell-free virions were not detected in culture. These data suggest that the replication of MuMTV induced in hormone-induced neoplasms is defective. Details of its expression suggest that if involved in events leading to tumors, its involvement is insufficient cause for those tumors.

Independence of H-2 and viral antigens on the cell surface and absence of H-2 antigens on murine leukemia virus and mouse mammary tumor virus particles.

By indirect immunoelectron microscopy we tested for the presence of H-2 antigens on murine mammary tumor virus (MMTV) and murine leukemia virus (MuLV) particles. The association of H-2 antigens and viral antigens on the virus-infected cell surface was investigated with antibody-induced redistribution. Mammary tumor cells and leukemia cell lines with different H-2 genotypes and carrying different MuMTV or MuLV were used. No H-2 antigens could be demonstrated on the envelope of MMTV and MuLV particles, even after the permeabilization of their envelopes with saponin. On the surface of virus-infected cells antibody-induced patching or capping of the viral antigens did not result in copatching or cocapping of the H-2 antigens. In the reciprocal tests no co-redistribution of viral antigens with H-2 antigens was seen. Our experiments failed to show any physical association between H-2 antigens and MMTV or MuLV antigens on the cell surface.

Quantitative of murine mammary tumor virus-related RNA in mammary tissues of low- and high-mammary-tumor-incidence mouse strains.

Lactating mammary glands and hormonally induced mammary tumors of BALB/c mice from three geographically separated breeding colonies were examined by molecular hybridization, using murine mammary tumor virus (MuMTV) cDNA representing the entire viral genome to determine the amount of MuMTV-related RNA expressed in these tissues. The RNA extracted from these tissues by the classical sodium dodecyl sulfate-pronase, phenol-chloroform procedure (method 1) contained barely detectable levels of MuMTV-related sequences. In contrast, both normal lactating mammary glands and hormonally induced mammary tumors of these mice were found to contain approximately one to two copies of the MuMTV genome per cell by using a new procedure in which the RNA was extracted with guanidine derivatives (method 2). No significant differences in the MuMTV-related RNA content of the BALB/c mammary tissues were observed regardless of their colony of origin. Our results suggest that expression of MuMTV RNA does not change in BALB/c mammary glands during transformation to a malignant state and that MuMTV expression does not play a role in tumorigenesis in these mice. In view of the increased recovery of MuMTV-related RNA from BALB/c mice with method 2, we compared the level of MuMTV RNA expression in lactating mammary glands and mammary tumors of other mouse strains, including C57BL/6 and RIII, using both extraction methods. Yields of MuMTV-related RNA from mammary tissues increased by as much as 35- to 40-fold, using method 2 as compared with method 1. Therefore method 2, involving guanidine derivatives, appears to be method of choice for MuMTV-related RNA extraction from the mammary tissues of certain strains of mice, particularly those expressing relatively low levels of MuMTV RNA.

Lymphoproliferative Responses to Mouse Mammary Tumor Virus In Lymphocyte Subsets of Breast Cancer Patients23

Altered immunologic reactions were observed in breast cancer patients as compared to those in normal subjects. Lymphoproliferative responses to murine mammary tumor virus (MuMTV) were significantly enhanced in peripheral blood mononuclear cells from patients with metastatic disease. These reactivities occurred with mammary tumor virus purified from either mouse milk or infected feline kidney cells but not with Rauscher murine leukemia virus. For the assessment of the role of peripheral blood mononuclear leukocyte subpopulations in the responsiveness to MuMTV, the cell preparations were fractionated according to their ability to form spontaneous rosettes with sheep red blood cells (E-rosettes). The effectiveness of the separation was ascertained by means of cell surface markers, i.e., presence of surface immunoglobulins or a T-cell marker. Leu-1 antigen, and mitogen-induced blastogenesis. The responsiveness to the MuMTV antigen(s) was associated with the T-cell subset, identified as the E-rosetting. Leu-1-positive, and surface immunoglobulin-negative population. Although some subjects with the normal population gave positive reactions, the results reveal an apparent association between high levels of responsiveness to MuMTV within the T-lymphocyte subset and breast cancer disease.

Expression of the murine mammary tumor virus in the milk of F1 hybrids of virus negative mouse strains

Milk samples of multiparous females from several mouse strains and their F 1 hybrids were tested for the presence of the envelope gylcoprotein gp 52 of the murine mammary tumour virous. F 1 hybrids resulting from matings of the virus negative strain BALB/c with the virus negative strains AKR, ND2 or RFM were positive for the viral antigen. The progeny of backcrosses to either parent had considerable lower incidences of viral antigen-positive milks. It was concluded that different recessive genes in the BALC/c strain on the one hand and in the AKR, ND2 and RFM strain on the other cause inhibition of endogenous virus expression. In F1 hybrids the two sets of genes complement each other, resulting in virus release.

Immunologic Localization of Murine Leukemia Virus Antigens in Thin Sections

In order to examine the temporal sequence of intracellular events which culminates in the release of infectious virus particles from the cell surface, we have developed immunoferritin techniques to localize viral antigens in thin sections. We previously described the intracellular localization of murine mammary tumor virus p28 protein in thin sections using an unlabelled antibody procedure with ferritin-antiferritin. We now describe the labelling of murine leukemia virus (MuLV) in thin sections using anti-MuLV and Protein A-ferritin.Cultures of F-MuLV in NIH/3T3 cells were grown to 90% confluence, fixed, dehydrated, and embedded in Epon as before. Thin sections were picked up on nickel grids, incubated in 20% H2O2 for 20 min, rinsed in PBS, and subjected to the labelling procedure. The labelling protocol included: preincubation in 5% BSA in PBS, rinse in 1% BSA in PBS, 3 hour incubation in rabbit anti-MuLV, rinse in 1% BSA in PBS, incubation in apoferritin, incubation in Protein A-ferritin (Zymed Labs., Burlingame, CA), rinses in 1% BSA in PBS and PBS, and incubation in 2% glutaraldehyde in PBS.

Murine Mammary Tumor Virus Seroepidemiology in BALB/cfC3H Mice: Correlation with Tumor Development&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN4"&gt;4&lt;/xref&gt;

Serum reactivity to murine mammary tumor virus (MuMTV) was inversely related to mammary tumor risk in 8-to 22-week-old BALB/cfC3H breeding females. Mice at low tumor risk exhibited high-titered serum reactivity to MuMTV (50% end point, greater than or equal to 1:40 by radioimmunoassay) approximately 3-6 months earlier than did the mice at high tumor risk. Maternal MuMTV antibody levels were correlated with the serum anti-MuMTV reactivity of their neonatal offspring (2 wk of age). Serologic antiviral reactivity in infected mice did not change during periods of pregnancy and lactation. All infected animals had detectable serum MuMTV reactivity by 33 weeks of age. The virus-neutralizing capabilities of some of these sera were tested, Sera from some of the young, low-tumor-risk animals that had MuMTV-precipitating antibodies also had virus-neutralizing activity. Conversely, none of the sera from the high-tumor-risk animals had detectable MuMTV-neutralizing antibodies.

Chromatographic evidence that the AAA-coding isoacceptor of lysine tRNA primes DNA synthesis in murine mammary tumor virus

Most of the tRNA encapsulated within the murine mammary tumor virus is tRNA Lys. The reversed-phase chromatographic pattern of tRNA Lys isoacceptors in the viral-“free” tRNA and in the “70 S-associated” tRNA that is released at 65° is similar to the pattern in virus-producing cells. However, the more tightly bound 70 S-associated tRNA Lys is significantly enriched in the AAA-coding isoacceptor. This isoacceptor, but not the AAG-coding one, primes MuMTV 35 S RNA-directed DNA synthesis in vitro.

Purification and translation of murine mammary tumor virus mRNA's

We have studied the functions of the intracellular RNAs of mouse mammary tumor virus (MMTV) by purification and translation in vitro. Two major size classes of MMTV RNA, 35S and 24S RNA, were isolated from MMTV-infected rat (XC) cells and cultured mammary tumor cells by preparative hybridization of whole cell or polyadenylated RNA to cloned MMTV DNA covalently bound to chemically activated paper disks (diazobenzyloxymethyl paper). Genomic-length (35S) RNA was prepared free of 24S RNA by rate zonal sedimentation in sucrose gradients. Experiments using [3H]uridine-labeled cellular RNA indicated that the preparative annealing method was highly specific and capable of effecting a 300-fold enrichment for viral RNA; the recovered RNA appeared to be intact under denaturing conditions and directed synthesis of full-length gag and env polypeptides in vitro. The products of in vitro translation were identified by gel mobility, immunoprecipitation tests with antisera against gag and env products, and partial digestion with Staphylococcus V8 protease. The 35S RNA species directed synthesis of several gag-related polypeptides, including three previously reported in extracts of infected cells; 24S RNA directed synthesis of two polypeptides closely related to env proteins from infected cells. Therefore, 35S RNA includes mRNA's for gag and gag-pol, whereas 24S RNA is the mRNA for env. These results help establish the position of env on the physical map of the MMTV genome and bear upon the coding potential of the genome.

Lack of induction of murine mammary tumor virus expression in cultured mammary glands treated with chemical carcinogens

AbstractChemical carcinogens have previously been found to bring about an epithelial transformation of mouse mammary glands in whole‐organ culture, as evidenced by the escape of lobuloalveoli from the normal hormonal controls of mammary development, and by glandular hyperplasia, dysplasia and metaplasia. In order to assess whether a general induction of endogenous murine mammary tumor virus (MuMTV) genes is associated with the chemical transformation process, we assayed steady‐state levels of MuMTV RNA and protein after treatment of the cultured mammary glands with the carcinogens, 7,12‐dimethylbenz[a]anthracene and N‐2‐fluorenylacetamide. Viral RNA was measured by hybridization with a representative MuMTV‐complementary DNA probe, and viral protein was analyzed by a group‐specific radioimmunoassay for the major MuMTV envelope glycoprotein, gp52. In control glands not treated with carcinogen, there were extremely low levels of MuMTV‐specific RNA in the cultured mammary glands originating from BALB/c mice, and between 10‐ and 50‐fold higher levels in glands from C57BL/6 and C3H mice. Among the untreated glands, only those from C3H mice contained a detectable level of gp52. This order of MuMTV gene expression in the cultured glands of the three strains of mice is consistent with that observed in vivo. Treatment of the cultured mammary glands of these three strains of mice with either of the two chemical carcinogens resulted in all cases in no elevation in the level of either MuMTV RNA or protein. Thus, our studies indicate that the transformation of the cultured mammary glands by the two chemical carcinogens occurs by a process that apparently does not involve a general induction of endogenous MuMTV gene expression.