Murine Mammary Tumor Virus Expression Research Articles

Chromatin-dependent E1A Activity Modulates NF-κB RelA-mediated Repression of Glucocorticoid Receptor-dependent Transcription

The role of chromatin-dependent regulatory mechanisms in the repression of glucocorticoid-dependent transcription from the murine mammary tumor virus (MMTV) promoter by p65 and E1A was investigated by using chromatin and transiently transfected reporters. The p65 RelA subunit of NF-kappaB represses MMTV expression on either transient or integrated reporters. In contrast, the viral oncoprotein E1A represses a transient but not an integrated MMTV. E1A repression is attenuated by chromatin, suggesting p65 but not E1A manipulates chromatin appropriately to inhibit the GR. Coexpression of p65 and E1A additively represses the transient MMTV but restores the transcriptional activation of the chromatin MMTV in response to glucocorticoids. This indicates that E1A has a dominant chromatin-dependent activity that attenuates repression by p65. E1A, p65, and GR bind the MMTV promoter, and chromatin remodeling enhances binding on both repressed and activated promoters. In addition, p65 requires Brg for repression of the integrated MMTV. This suggests that neither p65 repression nor E1A attenuation of repression results from an inhibition of remodeling that prevents transcription factor binding. Furthermore, p300/CBP is also required for both repression and attenuation by p65 and E1A. E1A and p65 mutants that do not bind p300/CBP are inactive, indicative of a requirement for p300/CBP-dependent complex formation for both repression and attenuation with chromatin. These data suggest that both the p65-dependent repression and the E1A-mediated attenuation of repression require the Brg1-dependent chromatin remodeling function and p300/CBP-dependent complex formation at a promoter assembled within chromatin.

Genetic resistance to mammary tumorigenesis in a mouse strain with high murine mammary tumor virus expression

Although II-TES mice release large amounts of murine mammary tumor virus (MMTV) in milk, they are resistant to mammary tumorigenesis. High mammary tumor incidence was observed in ( BALB ca × II-TES)F1 and ( C57BL 6N × II-TES)F1 , whereas no mammary tumors developed in BALB ca × OZ-F)F1 . Mammary tumors developed in 68% of (OZ-F × (OZ-F × II-TES)F1) and 45% of (II-TES × (OZ-F × II-TES)F1). These results suggest that the II-TES mouse carries a recessive gene for mammary tumor resistance which does not inhibit MMTV release, and two independent dominant mammary tumor promoting genes which are inhibited by the resistant gene.

High murine mammary tumor virus expression in milk in a low mammary tumor mouse strain and high incidence of mammary tumor in hybrids produced by crossing with another low mammary tumor mouse strain.

Large amounts of murine mammary tumor virus (MMTV) were expressed in milk from a low mammary tumor mouse strain called II-TES, established by crossbreeding DBA/2 mouse strain with two strains of Japanese pet mouse origin. The reciprocal hybrids of the II-TES strain and OZ-F strain, another low mammary tumor strain of Japanese pet mouse origin, developed early-appearing mammary tumors at a high rate, and large quantities of MMTV were expressed in the milk. These findings suggest that different regulatory genes control MMTV expression and mammary tumorigenesis.

Effect of N-(4-hydroxyphenyl)retinamide on murine mammary tumor cells in culture.

The effects of N-(4-hydroxyphenyl)retinamide (HPR), a synthetic analogue of vitamin A, on cell morphology, cell cycle kinetics, cytoplasmic matrix, and expression of murine mammary tumor virus (MuMTV) in MuMTV-infected murine mammary tumor cells (GR-3A) were determined. Cellular uptake of HPR was rapid and linear, with zero-order kinetics, during the first 30 minutes of incubation. Flow cytometric analysis of cells treated with nontoxic levels of HPR (10 microM) for 48 hours revealed a reduction in percent cells in the DNA synthetic (S) phase of the cell cycle with a concomitant increase in percent cells in the G1 phase of the cell cycle. Dexamethasone-stimulated MuMTV expression was not affected after 48 hours of HPR exposure, whereas the virus expression was significantly reduced in cells treated with HPR for seven days. The reduction in MuMTV expression was preceded by changes in cell morphology (decreased cell-cell contact and reduced cell flattening) and altered F-actin aggregation. Continuous exposure to HPR (10 microM) for 14 days resulted in reduced cell proliferation rates and decreased cell plating efficiency of GR-3A cells. Taken together, these results indicate that HPR is rapidly incorporated into GR-3A cells and that the effects of HPR on cell profileration, cytoskeletal organization, and cell morphology appear to precede the effects of this retinoid on the expression of the etiological agent of murine mammary tumorigenesis, MuMTV.

Low-calorie diet prevents the development of mammary tumors in C3H mice and reduces circulating prolactin level, murine mammary tumor virus expression, and proliferation of mammary alveolar cells.

The effect of carlorie intake on the development of spontaneous mammary tumors in virgin C3H mice was studied. Only about 10% of the mice fed a low-calorie diet [10 kcal/day (1 kcal = 4.184 kJ)] since weaning developed mammary tumors, compared to about 60% of those mice that were reared on high-calorie diets (16 kcal/day or lab chow ad lib). In order to understand the mechanism by which a low-calorie diet decreases the occurrence of mammary tumors in mice, we compared the sex cycle, the amounts of circulating thyroid-stimulating hormone (thyrotropin), growth hormone, and prolactin, the production of type A and B virus particles in the mammary glands, and the morphology of the mammary glands of mice fed low- and high-calorie diets. The amount of serum prolactin and the synthesis of type A and B particles in mammary tissues of mice fed a low-calorie diet was markedly decreased compared to those of age-matched mice fed high-calorie diets. In addition, in young mice fed a low-calorie diet, there were fewer mammary alveolar lesions than in mice fed a high-calorie diet, although the size of the lesions was similar. However, in older mice fed the high-calorie diet, the number and size of these lesions were greater than in the mice raised on the low-calorie diet. The other factors that we studied were not affected by calorie restriction. Our findings suggest that the reduction in serum prolactin level, mammary tumor virus production, and proliferation of mammary alveolar lesions associated with dietary calorie restriction is responsible for lowering the incidence of mammary tumors in mice.

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.

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.

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.

Hormonal regulation of murine mammary tumor virus RNA expression during mammary tumorigenesis in BALB/c mice

The effects of glucocorticoids and prolactin on murine mammary tumor virus (MuMTV) RNA expression in preneoplastic outgrowth lines and mammary tumors in BALB/c mice were investigated. Hyperplastic alveolar nodules (HAN) and a ductal hyperplasia (DH) are induced in virgin BALB/c mice by prolonged hormonal stimulation or treatment with 7,12-dimethylbenz(a)anthracene or both. Mice bearing HAN or DH outgrowth lines and mammary tumors that arose from the outgrowth lines were treated with glucocorticoids or prolactin. MuMTV RNA was quantitated by hybridization with a representative complementary DNA probe specific for MuMTV RNA. Prolactin treatment did not increase MuMTV RNA in the BALB/c HAN or DH outgrowth lines or tumors. MuMTV RNA increased after glucocorticoid treatment in the C3, C4, and C5 HAN outgrowth lines and in tumors that arose from the D1, D2, C4, and C5 HAN and CD8 DH outgrowth lines. No increase in MuMTV RNA with glucocorticoid treatment was observed in the D1 or D2 HAN outgrowth line, in the CD8 DH outgrowth lines, and in tumors that arose from the C3 HAN outgrowth line. The ability of glucocorticoids to stimulate MuMTV expression was specific since the response was dose dependent and specific for glucocorticoid hormones. Glucocorticoid treatment did not increase the level of type C viral RNA in the majority of hormone- or 7,12-dimethylbenz(a)anthracene-induced HAN outgrowth lines or tumors. These observations suggested that glucocorticoids may influence MuMTV expression during mammary tumorigenesis in BALB/c mice.

Autogenous Antibodies Against the Murine Mammary Tumor Virus in Strains of Mice With Low Incidences of Mammary Tumors

The radioimmunoprecipitation assay for the murine mammary tumor virus (MuMTV) was used to detect naturally occurring antibodies against MuMTV in 3 groups of highly inbred mouse strains. 1) Some strains had high incidences of mammary tumors, such as strains GR and C3H. Antibodies against MuMTV were detected in the sera of females of these strains at early ages. 2) Some mouse strains had low incidences of mammary tumors with an intermediate MuMTV expression, such as strains C3Hf, RIIIf, and BALB/c. Some females of these strains developed antibodies against MuMTV. Hormone treatment of these mice resulted in an increase in the proportion of mice carrying antibodies against MuMTV. 3) Some mouse strains were MuMTV-free, such as strains O20, C57BL, and Gr-Mtv2-. No antibodies against MuMTV were detected in the sera of these mice. However, antibodies against MuMTV appeared in the sera of these animals after hormone treatment. The presence of a natural humoral immunity toward MuMTV appeared to be related to the expression of MuMTV in the animals.

Suppressed Murine Mammary Tumor Virus (MuMTV) Expression in RIII Female Mice Treated Neonatally With Goat Antiserum to MuMTV<xref ref-type="fn" rid="FN2">2</xref>

Passive immunization of newborn inbred RIII (R3) mice with the globulin fraction of goat antiserum to murine mammary tumor virus (MuMTV) successfully suppressed MuMTV expression in the milk of some of the treated mice throughout nine successive lactations. No mammary tumors developed in the MuMTV-suppressed mice during the first 9 months, whereas all untreated R3 female breeders expressed MuMTV in the milk of the third lactation, and all developed tumors before 9 months of age (mode and median: 189 days).

Mammary tumors and mammary tumor virus expression in hybrid mice of strains C57BL and GR.

Mammary tumorigenesis in genetic crosses between the high mammary tumor incidence GR and the low incidence C57BL mouse strains is highly correlated with murine mammary tumor virus expression in milk. Although the F1 and first backcross females had a mammary tumor incidence which was consistent with a single dominant gene segregation, the tumor incidence in the critical second backcross segregants disproved the single gene hypothesis. Genetic factors were clearly involved in regulation of virus expression which in turn correlated with both tumor incidence and tumor latency; these complex phenotypes are however best explained as threshold or quasicontinuous characters. As predicted from this model, the age specific incidence of mammary tumors showed a broad peak at 14-19 mo of age with no evidence of an early or late phase. Hematopoietic tumors showed no correlation with virus expression or mammary tumorigenesis suggesting different etiologies for these tumors.

Expression of murine mammary tumor virus-related antigens in human breast carcinoma (MCF-7) cells.

Tested by the indirect immunofluorescence technique, a human breast carcinoma cell in continuous in vitro cultivation (MCF-7) was found to express antigens cross-reactlve with the murine mammary tumor virus (MuMTV), but not with murine leukemia virus or Mason-Pfizer monkey virus. Antiserum dilution, antiserum absorption, and antiserum blocking experiments showed the cross-reactivity observed in MCF-7 cells to be MuMTV-specific. In long-term passage, only 4–6% of the total population of MCF-7 cells reacted specifically with anti-MuMTV sera. The number of specifically reactive cells could be increased up to 9–14% by treating MCF-7 cells with 10-7 or 10-am progesterone. Neither 5-iododeoxyuridine (IUdR), dexamethasone, nor IUdR plus dexamethasone, potent inducers of MuMTV expression in mouse cells, had any effect on MuMTV cross-reactive antligen expression in MCF-7 cells. Late-passage MCF-7 monolayers were phenotypic mixtures of cells. Small cells (S-cells) comprised 80–90% of the cells, and 10–20% were cells two to three times larger (L-cells). L-cells exclusively reacted with MuMTV antisera and responded to progesterone with augmented antigen levels. L-cells were separated from S-cells by cloning. Again, only L-cells In L-cell clones reacted with MuMTV antisera. The number of L-cell clones could be increased from 15% to about 35% by prolonged treatment of cells in the parent monolayer with IUdR; this resulted in a parallel increase in MuMTV-positive L-cells. However, the segregation of L-cells from S-cells and the increase in the number of L-cells by IUdR treatment did not permanently increase the number of MuMTV-positive cells. MuMTV-positive L-cells converted to MuMTV-negatlve L-cells rapidly during cell division such that the number of MuMTV-positive L-cells per clone was inversely related to clone size (I.e., number of cells per clone). To determine the sequence of events in dimunition of antigen synthesis in MCF-7 cell monolayers, earlier MCF-7 passages were examined. Passages 6–10 of one subllne were 50–60% positive in reactivity with anti-MuMTV sera. Approximately 80% of cells in these passages were L-cells. Within four subsequent passages, the percentage of MuMTV-positive cells diminished from 50–60% to 4–6%. During that time, the cultures remained at least 70% L-cells. Two models of growth dynamics were proposed to account for phenotypic Interconversions of MCF-7 cells during in vitro proliferation.

Expression of Mouse Mammary Tumor Viral Polypeptides in Milks and Tissues

A 14,000-dalton polypeptide (p14) from RIII murine mammary tumor virus (MMTV) has been isolated by column chromatography in 6 M GuHCl. Antiserum prepared in rabbits specifically precipitated 125I-labeled p14; in double antibody competition, radioimmunoassays performed with limiting amounts of antibody, both purified p14 and disrupted MMTV, competed specifically with labeled antigen. The expression of this MMTV type B virus antigen could be measured by competition radioimmunoassays in milks, mammary glands, tumors, and tissue culture cells. MMTV expression measured by p14 immunoassay correlated well with the spontaneous incidence of mammary adenocarcinomas in different murine strains but not with type C MuLV p30 antigen expression. Levels of MMTV gp52, the major type-B viral glycoprotein, corresponded to p14 levels, suggesting that their control is comparably regulated. Evidence that this low m.w. polypeptide is present in feral and inbred strains of widely differing geographic origin and in MMTV with apparently different biologic properties suggests surprising conservation of MMTV protein homology.

Mammary tumor virus induction by glucocorticoids. Characterization of specific transcriptional regulation.

Dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), a potent synthetic glucocorticoid, stimulates mouse mammary tumor virus expression 10- to 20-fold in tissue culture cells. This hormone effect was observed at concentrations as low as 1 times 10-10 M and was maximal at 10-7 to 10-8 M. The time course of induction indicated that detectable increases in extracellular viral DNA polymerase were first noted 18 to 24 hours following the addition of dexamethasone, and cells produced the highest polymerase levels at the time monolayers approached confluence. Steroid responsiveness was associated with specific increases in type B murine mammary tumor virus structural polypeptide (gp52(sl) expression and murine mammary tumor virus RNA that quantitatively paralleled the increase in extracellular virus production as measured by electron microscopy and supernatant RNA-dependent DNA polymerase activity. Another virally transformed murine cell line, KA 31, did not contain detectable levels of murine mammary tumor virus gp52(sl) or RNA before or after dexamethasone stimulation; thus induction was noted only in murine cells with pre-existing murine mammary tumor virus expression. No increase in basal levels of type C murine leukemia viral proteins or RNA was detected in dexamethasone-treated mammary cell lines which were producing increased levels of murine mammary tumor virus. Therefore, increases in murine mammary tumor virus gene products are specific for murine mammary tumor virus DNA sequences under these conditions.

Dexamethasone stimulation of murine mammary tumor virus expression: a tissue culture source of virus.

In mouse cell lines derived from mammary adenocarcinomnas, the synthetic steroid dexamnethasone stimulates production of murine mammary tumor virus. Viral RNA and antigens are increased as much as 20-fold, and culture fluid supernatants from steroid-treated cells contain type B particles with reverse transcriptase. These cells provide a possible tissue culture source of this virus and a model system for studying the mechanism of action of corticosteroids and the regulation of transcription of integrated viral DNA.