Mouse Mammary Tumor Virus Gene Research Articles

Lipopolysaccharide and Dexamethasone Induce Mouse Mammary Tumor Proviral Gene Expression and Differentiation in B Lymphocytes through Distinct Regulatory Pathways

Endogenous mouse mammary tumor virus (MMTV) proviral transcripts are up regulated during the normal course of B-lymphocyte differentiation. We report here that the regulatory mechanisms which lead to increased levels of MMTV transcripts in differentiating, lipopolysaccharide (LPS)-stimulated normal B cells and in the inducible B-cell lymphoma line CH12 are at least partially distinct from those controlling increases in immunoglobulin and J-chain gene expression. In studies designed to characterize the stimulatory pathways leading to MMTV expression in CH12 cells, we found that stimulation with either LPS or dexamethasone (Dex), a transcriptional activator of MMTV genes, induced not only MMTV expression but also differentiation to antibody secretion. Only Dex-induced and not LPS-induced MMTV expression and differentiation were inhibited by the glucocorticoid antagonist RU486, demonstrating that Dex and LPS stimulate B cells by distinct molecular pathways. Therefore, in B cells, MMTV expression can be regulated via either the conventional hormone receptor-dependent pathway or a hormone receptor-independent pathway. Furthermore, these results suggest that steroid stimulation of B cells can lead to alterations in the expression of other results suggest that steroid stimulation of B cells can lead to alterations in the expression of other steroid-responsive genes that can become involved in the process of B-cell differentiation.

Influence of mammary cell differentiation on the expression of proteins encoded by endogenous BALB/c mouse mammary tumor virus genes

The interactions between differentiation-associated cellular events in the intact mammary gland or in cultured mammary cells and the post-transcriptional activity of the endogenous mouse mammary tumor virus (MMTV) loci were investigated. The transcriptional activities of the endogenous MMTV proviruses of the BALB/c mouse strain ( Mtv-6, Mtv-8 and Mtv-9) appear to be regulated differentially during pregnancy-induced mammary gland development (J.E. Knepper D. Medina and J.S. Butel J. Virol. 59, 518–521, 1986). Analysis of MMTV-specific proteins at various stages of mammary gland development (virgin, midpregnant, lactating, regressing) established the presence of steady-state levels of a 67,000- M r env precursor-type polypeptide at all physiological stages. However, processing to lower-molecular-weight env-specific proteins, including a predominant 50,000- M r species, was detected only with the transition to the functional mammary gland phenotype. The contributions of cell proliferation, cell-matrix interactions, and modulation of functional activity to the pattern of endogenous MMTV protein expression were investigated using a 3-dimensional collagen type I culture system. Growth and cell-matrix interactions (cell polarization, lumen formation) leading to formation of 3-dimensional duct-like structures were permissive for the synthesis and processing of MMTV-specific proteins; accumulation of high levels of the 50,000- M r env-specific polypeptide was associated with the onset of the fully functional mammary cell phenotype. Expression of MMTV-specific proteins was not due to amplification of a specific cell subpopulation. The potential of the full-length Mtv-8 and Mtv-9 proviruses to be transcribed, as indicated by their methylation status, was not dramatically different between differentiated and undifferentiated mammary cells in culture. This study indicates that MMTV transcriptional activity is reflected at the protein level in mammary tissue of BALB/c mice and that viral protein synthesis and processing may serve as important markers of different physiological stages of mammary epithelial cells. These observations also suggest a general approach to the examination of potential modulatory effects of cellular interactions (cell-cell, cell-matrix or both) known to be important in various differentiated epithelial cell systems for the expression of viral genes.

A glucocorticoid-resistant rat hepatoma cell variant contains functional glucocorticoid receptor.

The mechanism of glucocorticoid resistance was studied in a rat hepatoma cell variant (6.10.2) which contains low levels of glucocorticoid receptor. These cells seem to have lost glucocorticoid-induced transcriptional responses as measured by the induction of expression of stably integrated mouse mammary tumor virus gene and the endogenous tyrosine aminotransferase gene, as well as the transcriptional suppression of glucocorticoid receptor gene expression. However, characterization of the glucocorticoid resistance in 6.10.2 cells revealed that the receptor is indistinguishable from the wild-type receptor with respect to hormone binding and affinity for both nonspecific and specific DNA sequences. The levels of the receptor mRNA and the total immunoreactive protein found in 6.10.2 cells were about 20% of those found in wild-type cells. Further analysis of 6.10.2 cells demonstrated that the receptor was indeed biologically functional. First, treatment of 6.10.2 cells with 8-bromo-cAMP elevated the endogenous glucocorticoid receptor levels 2-fold and restored responsiveness to glucocorticoids. Second, pretreatment of the cells with cycloheximide also led to acquisition of cellular responsiveness to glucocorticoids. We propose that there exists a "threshold" level of glucocorticoid receptor which is required for responsiveness and that under normal culture conditions, the level of glucocorticoid receptor in 6.10.2 cells is below this threshold. However, glucocorticoid responsiveness can be restored by raising the glucocorticoid receptor level above the threshold with 8-bromo-cAMP or, alternatively, by removing the threshold barrier with cycloheximide.

Spontaneous progression of hyperplastic outgrowths of the D1 lineage to mammary tumors: Expression of mouse mammary tumor virus and cellular proto‐oncogenes

Mammary cancer in mice is characterized by progression through defined stages of preneoplasia, with the most common preneoplastic stage being the hyperplastic alveolar nodule (HAN). We determined the relative levels of RNA expression of various cellular proto-oncogenes and endogenous mouse mammary tumor virus genes in outgrowths and tumors of three sublines of the transplantable D1 HAN preneoplastic outgrowth line. The three sublines differed in relative tumor-producing capabilities. Subline D1B produced a high incidence of tumors with short latency periods, whereas sublines D1C and D1D produced low incidences of tumors with long latency periods. No consistent alteration in proto-oncogene expression correlated with relative tumorigenicity, although tumors frequently contained higher levels of one or more proto-oncogene transcripts as compared with preneoplastic tissue. Slightly elevated (2- to 6-fold) levels of different oncogene transcripts were detected in 13 of 17 tumors as compared with outgrowth tissue, including abl (2 tumors), fps (5 tumors), Ha-ras (6 tumors), and Ki-ras (8 tumors). One tumor contained 45 times more Ki-ras-specific RNA than outgrowth tissue because of a comparable amplification of Ki-ras DNA sequences. Elevated levels of Ha-ras occurred more frequently in tumors of a high-incidence subline than in a less-aggressive subline (5/10 vs 1/7), but this difference was not statistically significant. However, consistent changes in MMTV expression accompanied progression from preneoplastic tissues to mammary tumors. All 17 tumors displayed reduced levels of the MMTV-specific long terminal repeat (LTR) transcript (1.6 kb) as compared with HAN tissue; tumors with moderate levels of LTR transcript expressed the 3.8-kb envelope message as well, one not detected in HANs. Expression of the LTR transcript is apparently influenced by factors in addition to the methylation status of endogenous mouse mammary tumor virus genes, which was similar in outgrowths and tumors. As the survey of representative proto-oncogenes failed to identify a uniform change between HAN and tumors, it is likely that other genes are involved in tumor progression in the mammary gland.

Binding of steroid receptors to the hres of mouse mammary tumor virus, chicken and xenopus vitellogenin and rabbit uteroglobin genes: Correlation with induction

Binding to hormone responsive elements (HRE) is an essential step in gene regulation by steroid hormones. Using a combination of in vivo and in vitro studies we have analyzed the interactions of the estrogen receptor with genes from three different systems: the long terminal repeat (LTR) of the mouse mammary tumor virus (MMTV), the chicken and xenopus vitellogenin genes and the rabbit uteroglobin gene. The estrogen receptor binds to all four genes in vitro, but the MMTV LTR does not respond to estrogen in gene transfer experiments. Similarly, the xenopus vitellogenin gene binds the progesterone and glucocorticoid receptors in vitro, but only estrogen induces the xenopus vitellogenin gene in gene transfer. These results suggest that events distal to DNA binding are essential for transcriptional activation, and that the function of the HRE is not simply to position the hormone receptor in the vicinity of the regulated promoter.

Glucocorticoid-resistant lymphoma cell variants that contain functional glucocorticoid receptors.

A mouse T-lymphosarcoma cell line stably infected with mouse mammary tumor virus (MMTV) was used as the parent line for a genetic analysis of two glucocorticoid hormone responses, hormone-induced cytolysis and stimulation of viral gene expression. Variants were selected for survival and elevated expression of MMTV proteins in the presence of the steroid. The MMTV marker provided a sensitive test for glucocorticoid receptor (GR) function in the hormone-resistant variants. This strategy resulted in the isolation of two novel types of hormone-resistant variants. One type of variant with only about 25% of the level of GR found in the parent line was resistant to the cytolytic effects of glucocorticoid but produced increased levels of MMTV gene products in response to the hormone. This variant phenotype demonstrated that the MMTV response requires fewer GR than the cytolytic response. Another variant, which required approximately 100-fold higher concentrations of hormone than the wild-type cells for both responses, apparently contained GR with altered hormone-binding properties.

Glucocorticoid-resistant lymphoma cell variants that contain functional glucocorticoid receptors.

A mouse T-lymphosarcoma cell line stably infected with mouse mammary tumor virus (MMTV) was used as the parent line for a genetic analysis of two glucocorticoid hormone responses, hormone-induced cytolysis and stimulation of viral gene expression. Variants were selected for survival and elevated expression of MMTV proteins in the presence of the steroid. The MMTV marker provided a sensitive test for glucocorticoid receptor (GR) function in the hormone-resistant variants. This strategy resulted in the isolation of two novel types of hormone-resistant variants. One type of variant with only about 25% of the level of GR found in the parent line was resistant to the cytolytic effects of glucocorticoid but produced increased levels of MMTV gene products in response to the hormone. This variant phenotype demonstrated that the MMTV response requires fewer GR than the cytolytic response. Another variant, which required approximately 100-fold higher concentrations of hormone than the wild-type cells for both responses, apparently contained GR with altered hormone-binding properties.

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.

Functional differentiation of virgin mouse mammary epithelium in explant culture is dependent upon extracellular proline.

Depletion of proline from insulin, hydrocortisone, and prolactin-containing medium prior to incubating virgin mouse mammary explants prevents both DNA synthesis and functional differentiation in the mammary epithelial cells; however, DNA synthesis in the mammary stroma and total incorporation of radioactive amino acids into total protein appears to continue without hindrance. Removal of glycine instead of proline had no deleterious effect on either DNA replication in the hormone-stimulated epithelium or in its functional differentiation. Functional differentiation was determined by the induction of casein and alpha-lactalbumin synthesis in the insulin, hydrocortisone, and prolactin (IFPrl)-treated explant cultures. As a control, the induction of mouse mammary tumor virus (MMTV) gene expression, a corticosteroid-regulated function, was also measured. Neither the absence of proline or glycine prevented the glucocorticoid stimulation of MMTV gene expression. In contrast to mammary tissue from virgin mice, explants from nonpregnant primiparous mice responded fully to IFPrl stimulation with respect to DNA, casein, and alpha-lactalbumin synthesis in medium depleted of proline. These data suggest that the uncommitted epithelium of virgin mouse mammary glands requires the presence of exogenous proline in order to respond to lactogenic hormonal signals. We have demonstrated earlier that DNA synthesis is a prerequisite of functional differentiation in virgin mouse mammary explants (Smith and Vonderhaar, 1981, Dev. Biol., 88:167-179; Vonderhaar and Smith, 1982, J. Cell Sci, 53:97-114), although cytological differentiation proceeded unencumbered in explants prevented from synthesizing DNA. Here, without proline, neither cytological nor functional differentiation can be induced; this suggests that proline provides an essential metabolic interlock in the acquisition of lactogenic hormone responsiveness in uncommitted mouse mammary tissue.

Differential expression of endogenous mouse mammary tumor virus genes during development of the BALB/c mammary gland.

Expression of endogenous mouse mammary tumor virus sequences varied over the course of development of the mammary gland during primary pregnancy and lactation in virus-free BALB/c mice. Although RNA from all regions of the genome was detected, both the level and temporal regulation of expression were different for long terminal repeat-, env-, and gag-pol-specific RNAs. Analysis of the methylation status of proviral DNA indicated differential accessibility of the three endogenous units during development. The results demonstrated noncoordinate regulation of mouse mammary tumor virus expression with respect to provirus template utilized and specific transcripts accumulated.

Molecular cloning and long terminal repeat sequences of human endogenous retrovirus genes related to types A and B retrovirus genes.

By using a DNA fragment primarily encoding the reverse transcriptase (pol) region of the Syrian hamster intracisternal A particle (IAP; type A retrovirus) gene as a probe, human endogenous retrovirus genes, tentatively termed HERV-K genes, were cloned from a fetal human liver gene library. Typical HERV-K genes were 9.1 or 9.4 kilobases in length, having long terminal repeats (LTRs) of ca. 970 base pairs. Many structural features commonly observed on the retrovirus LTRs, such as the TATAA box, polyadenylation signal, and terminal inverted repeats, were present on each LTR, and a lysine (K) tRNA having a CUU anticodon was identified as a presumed primer tRNA. The HERV-K LTR, however, had little sequence homology to either the IAP LTR or other typical oncovirus LTRs. By filter hybridization, the number of HERV-K genes was estimated to be ca. 50 copies per haploid human genome. The cloned mouse mammary tumor virus (type B) gene was found to hybridize with both the HERV-K and IAP genes to essentially the same extent.

Mouse mammary tumor virus gene expression regulated in trans by Lps locus

Expression of mouse mammary tumor virus (MMTV) in the lactating mammary glands of uninfected mice varies between strains of mice in a manner largely independent of the proviral content. Previous linkage analysis in the mouse suggested that the Lps locus was associated with steady-state levels of MMTV RNA. The Lps locus mediates the mouse's response to the injection of lipopolysaccharide (LPS) in the responder mouse while mice with the deficient allele are incapable of responding. Injecting LPS-responder mice, C3HfB/HeN, and nonresponder mice, C3Hf/HeJ, with LPS resulted in a threefold increase in the level of MMTV RNA in responder mice but had no effect on nonresponders. The increased level was due to only one of the possible MMTV transcripts: the 1.7-kb transcript containing the open reading frame (orf) of the long terminal repeat (LTR). The level of MMTV-specific transcripts, then, is regulated by the Lps locus, a cellular gene which is not linked to any viral coding sequences and therefore must act in trans.

Alterations in chromatin structure associated with glucocorticoid-induced expression of endogenous mouse mammary tumor virus genes.

Alterations in the chromatin structure of endogenous mouse mammary tumor virus genes accompany glucocorticoid induction of viral RNA synthesis in the C57BL/6 T lymphoma cell line T1M1. These alterations are defined by the appearance of sites of DNase I hypersensitivity within proviral DNA in isolated nuclei, as well as by changes in the moderate nuclease sensitivity of entire proviral transcription units. Induced hypersensitive sites, termed type I, appear with a time course comparable to that required for induction of the rate of viral RNA synthesis and are maintained only in the continuous presence of hormone. Two such sites map to analogous positions in the 5' and 3' long terminal repeats of proviral DNA within, or very near, sequences that have been shown to comprise positions of specific binding of the glucocorticoid receptor in vitro and that are required for hormone-inducible transcription in vivo. A third type I site maps to another position of in vitro receptor binding near the 3' long terminal repeat. Some sites of DNase I hypersensitivity, termed type II, appear not to be markedly hormone dependent; two such sites are present in corresponding positions in each long terminal repeat. Comparison of the moderate DNase I sensitivity of mouse mammary tumor virus proviral DNA suggests that the three different endogenous units in T1M1 cells can be maintained in distinct chromatin conformations that are determined by factors related to the site of provirus insertion. It seems possible that altered chromatin conformations may reflect, or actually encode, important mechanistic features of these hormone-responsive genes.

Alterations in chromatin structure associated with glucocorticoid-induced expression of endogenous mouse mammary tumor virus genes.

Alterations in the chromatin structure of endogenous mouse mammary tumor virus genes accompany glucocorticoid induction of viral RNA synthesis in the C57BL/6 T lymphoma cell line T1M1. These alterations are defined by the appearance of sites of DNase I hypersensitivity within proviral DNA in isolated nuclei, as well as by changes in the moderate nuclease sensitivity of entire proviral transcription units. Induced hypersensitive sites, termed type I, appear with a time course comparable to that required for induction of the rate of viral RNA synthesis and are maintained only in the continuous presence of hormone. Two such sites map to analogous positions in the 5' and 3' long terminal repeats of proviral DNA within, or very near, sequences that have been shown to comprise positions of specific binding of the glucocorticoid receptor in vitro and that are required for hormone-inducible transcription in vivo. A third type I site maps to another position of in vitro receptor binding near the 3' long terminal repeat. Some sites of DNase I hypersensitivity, termed type II, appear not to be markedly hormone dependent; two such sites are present in corresponding positions in each long terminal repeat. Comparison of the moderate DNase I sensitivity of mouse mammary tumor virus proviral DNA suggests that the three different endogenous units in T1M1 cells can be maintained in distinct chromatin conformations that are determined by factors related to the site of provirus insertion. It seems possible that altered chromatin conformations may reflect, or actually encode, important mechanistic features of these hormone-responsive genes.

Dysdifferentiative nature of aging: age-dependent expression of mouse mammary tumor virus and casein genes in brain and liver tissues of the C57BL/6J mouse strain

Many aspects of the aging process could be the result of cells slowly drifting away from their proper state of differentiation. This possibility has been studied by searching for an age-dependent increase in the expression of specific genes in tissues where expression of these genes would not normally be expected. In these studies, cDNA probes of specific genes are used in a DNA⋯RNA hybridization assay to detect possible complementary RNA sequences in tissues of different-aged animals. Using this technique in past experiments, a qualitative increase in the RNA sequence complexity of mouse leukemia virus (MuLV) and a quantitative increase in the amount of alpha- and beta-globin RNA were found with increasing age in the brain and liver of the C57BL/6J mouse strain. We report here a similar age-dependent qualitative increase in the RNA sequence complexity for mouse mammary tumor virus (MMTV) but no quantitative or qualitative age-dependent change in casein RNA sequences for the same tissues and mouse strain.

Asymmetric transcription of mouse mammary tumor virus genes in vivo and in vitro.

Mouse mammary tumor virus (MMTV) DNA fragments were cloned into M13 bacteriophage, and the single-stranded recombinant phage DNAs were used as strand-specific nucleic acid hybridization probes to measure synthesis of plus (genomic) and minus strands of MMTV RNA in cultured cell lines and in cell-free preparations of nuclei. Pulse-labeling studies showed that synthesis of MMTV RNA in three different cell lines was highly asymmetric. In nuclear preparations from a cloned line of MMTV-infected rat hepatoma cells, elongation of nascent MMTV RNA chains and initiation of new MMTV RNA chains with nucleoside (beta-S)triphosphates were also highly asymmetric.

ADP-ribosylation of chromosomal proteins and mouse mammary tumor virus gene expression. Glucocorticoids rapidly decrease endogenous ADP-ribosylation of nonhistone high mobility group 14 and 17 proteins.

The relationship between endogenous ADP-ribosylation of chromosomal proteins and glucocorticoid-regulated mouse mammary tumor virus gene expression was investigated in cultured mouse mammary tumor cells. It was observed that glucocorticoids quickly decreased endogenous (ADP-ribose)n on the nonhistone high mobility group (HMG) 14 and 17 proteins. The half-time for this loss was 8 and 17 min, respectively, for the two proteins. (ADP-ribose)n on HMG 1 and 2 and on histone H1 was less susceptible to hydrolysis during glucocorticoid treatment. The rapid loss of (ADP-ribose)n from HMG 14 and 17 occurred in the same time frame as the induction of mouse mammary tumor virus RNA synthesis by glucocorticoids in these cells (Young, H. A., Shih, T. Y., Scolnick, E. M., and Parks, W. P. (1977) J. Virol. 21, 139-149). 3-Amino-benzamide, a specific inhibitor of (ADP-ribose)n synthetase, increased mouse mammary tumor virus RNA levels with an accompanying decrease in endogenous ADP-ribosylation of HMG 14 and 17. These results show that a decrease in endogenous ADP-ribosylation of HMG 14 and 17 is a consequence of glucocorticoid action and suggest that loss of (ADP-ribose)n from these proteins may be an important event in mouse mammary tumor virus gene expression.

Immunological selection of variant mouse lymphoid cells with altered glucocorticoid responsiveness.

We have devised an immunological procedure to separate cells on the basis of expression of mouse mammary tumor virus (MMTV) gene products. Plastic petri dishes coated with specific antibodies against MMTV proteins bind cells with an efficiency that correlates with the level of MMTV gene expression. Glucocorticoid-sensitive mouse thymoma cell line W7 was infected with MMTV. Clones from the infected population retain the relatively slow cytolytic glucocorticoid response and, in addition, exhibit a rapid induction of MMTV-specific RNA and proteins. By combining our immunological selection with the selection for resistance to hormone-mediated cytolysis, we have isolated variant cells which are resistant to the cytotoxic effect of glucocorticoids but which retain the induction of viral gene products and must therefore have a functional glucocorticoid receptor protein.

Regulation of mouse mammary tumor virus gene expression by glucocorticoid hormones.

Regulation of mouse mammary tumor virus gene expression by glucocorticoid hormones.

Selection of rat hepatoma cells defective in hormone-regulated production of mouse mammary tumor virus RNA.

We have been studying the mechanism of glucocorticoid hormone action by using mouse mammary tumor virus (MMTV)-infected rat hepatoma cells as a model system. J2.17, a clonal cell line that contains one MMTV provirus, induces tyrosine aminotransferase (TyrATase; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), viral RNA, and the cell surface viral glycoprotein gp52 in response to dexamethasone. Using a fluorescence-activated cell sorter and a rabbit antiserum directed against gp52, we selected a cell population that displays a reduced hormone-mediated increase in cell surface gp52. Fourteen clones of this population were assayed for induction of viral gp52 and RNA and of cellular TyrATase. The results of these assays revealed that the clones display a variety of responses to hormone. One clone has retained wild-type responses of both TyrATase and gp52. Six clones exhibit coordinately reduced or abolished responses of both markers. Seven clones show normal induction of TyrATase but reduced or undetectable induction of gp52. These latter clones exhibit reduced production of MMTV RNA and thus may represent a unique class of variants defective in the regulation of MMTV gene expression.