Mouse mammary tumor virus-related sequences in mouse lymphocytes are inducible by 12-O-tetradecanoyl phorbol-13-acetate

cDNA libraries from EL-4 cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) were screened for TPA-inducible sequences by differential hybridization. The most abundant inducible species was a sequence similar to that of mouse mammary tumor virus (MMTV). Induction of the mRNA corresponding to the MMTV-related sequences was already evident 30 min after TPA treatment, whereas the maximum accumulation occurred after 20 h of exposure to TPA. TPA also increased levels of MMTV-related RNA in the normal spleen cells of BALB/c and C57BL/6 mice. The level of RNA expression corresponding to MMTV-related sequences, however, was markedly elevated in EL-4 cells as compared with normal spleen cells. Southern blots of EL-4 cell DNA showed that the MMTV-related sequences were inserted into multiple locations of the EL-4 genome. Sequence analysis revealed that the MMTV-related cDNA clones included a part of the env gene and the right long terminal repeat of MMTV. However, the cDNA sequences were substantially different from published MMTV proviral sequences, most notably because of a contiguous deletion of 491 base pairs in the open reading frame within the U3 region.     

Proteins encoded by the long terminal repeat region of mouse mammary tumor virus: identification by hybrid-selected translation.

The long terminal repeat (LTR) region of mouse mammary tumor virus (MMTV) is known to contain an open reading frame of sufficient length to code for a protein of 36,000 Mr. The coding capacity of the 3' sequences of MMTV genomic RNA has been demonstrated by in vitro translation studies, which have reported the synthesis of four related proteins: p36, p24, p21, and p18. These proteins are overlapping translation products of the same open reading frame, with the smaller ones initiating at internal methionine codons. From the predicted amino acid sequence of the LTR protein, we have selected a region likely to be antigenic, obtained a synthetic peptide of that region, and raised antiserum to the peptide. The antipeptide serum specifically immunoprecipitated all four proteins from in vitro translated genomic 3' MMTV RNA, plus an additional one of 32,000 Mr. Published sequence data of MMRV LTRs show an internal AUG codon at a position which could initiate a protein of 32,000 Mr. The three smaller in vitro translation products (p24, p21, and p18) were consistently synthesized in much greater amounts than the p36 or p32 protein. The relative amount of each in vitro synthesized protein from genomic MMTV RNA could be predicted and was in good agreement with the postulated effect of flanking nucleotides on the efficiency of the respective AUG initiation codon. Polyadenylated RNAs, isolated from various mouse tissues, were selected by hybridization to plasmid DNA containing MMTV LTR sequences immobilized on nitrocellulose. In vitro translation of hybrid-selected mRNAs isolated from BALB/c mouse lactating mammary glands and carcinogen-induced mammary tumors, followed by immunoprecipitation with antipeptide serum, revealed that only one polypeptide was synthesized by the MMTV LTR-specific mRNA, the 36,000 Mr species.     

Collagenase is a major gene product of induced rabbit synovial fibroblasts

We have investigated the effects of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), on rabbit synovial fibroblasts, and found that this agent induced a major switch in gene expression in these cells that was marked by the specific induction of the neutral proteinase, collagenase, and was always accompanied by alterations in cell morphology. Procollagenase synthesis and secretion was first observed 6-12 h after the addition of TPA. The rate of collagenase production (1-5 U, or approximately 0.2-1 micrograms secreted procollagenase protein per 10(5) cells per 24 h) depended on the TPA concentration (1-400 ng/ml) and time of exposure (1-72 h). Procollagenase was the most prominent protein visible by direct silver staining or by autoradiography after SDS PAGE of [35S]methionine-labeled proteins. The two procollagenase bands of Mr 53,000 and 57,000, which migrated as a family of spots on two-dimensional gels and were immunoprecipitated by antibodies to purified rabbit collagenase, accounted for 23% of the newly synthesized, secreted protein in TPA-treated cells. Cell-free translation of mRNA from TPA-treated cells in rabbit reticulocyte lysate produced a single band of immunoprecipitable preprocollagenase (Mr 55,000) as a major product (5% of total) that migrated as a single spot on two-dimensional gels. Secreted procollagenase, preprocollagenase , and active collagenase (purified to homogeneity; specific activity 1.2 X 10(4) U/mg protein) had related peptide maps. Two other major secreted proteins, a neutral metalloproteinase of Mr 51,000 and a polypeptide of Mr 47,000, were also induced by TPA. In contrast to the induction of these four polypeptides, TPA decreased synthesis and secretion of a number of proteins, including collagen and fibronectin. Thus, collagenase is a convenient marker for major alterations in the pattern of protein synthesis and secretion by rabbit synovial fibroblasts treated with TPA.     

Structural analysis of a 1.7-kilobase mouse mammary tumor virus-specific RNA

We have detected a mouse mammary tumor virus (MMTV)-specific 1.7-kilobase (kb) polyadenylated RNA in mammary glands of several mouse strains. In BALB/c mice, it is the only MMTV-specific RNA species present. C3H and GR mammary glands and tumors contain, in addition, 3.8- and 7.8-kb MMTV RNAs. Nuclease S1 analysis was performed to map 1.7-kb polyadenylated RNA. It contains predominantly long terminal repeat (LTR) sequences. The 5' end maps approximately 134 nucleotides upstream from the 3' end of the LTR. Colinearity with complete proviral DNA continues to a site about 153 nucleotides downstream from the left (5') LTR. No sequences from the middle part of proviral DNA were found. Colinearity with proviral DNA is resumed 72 nucleotides upstream from the right (3') LTR. The nucleotide sequence in this area is TTCCAGT, which is a splice acceptor consensus sequence. The anatomy of 1.7-kb RNA indicates that it may serve as a messenger for the 36,700-dalton protein encoded by the LTRs of MMTV.     

Androgen regulation by the long terminal repeat of mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) has long been implicated in mouse mammary carcinogenesis, and it is now well established that the long terminal repeat (LTR) contains regulatory sequences responsible for glucocorticoid-mediated induction of viral RNA. However, we have demonstrated previously that androgens as well as glucocorticoids can regulate MMTV RNA in the S115 mouse mammary tumor cell line. To determine if androgens act directly on the LTR in these cells, plasmids were constructed with the MMTV LTR joined to the coding sequences of genes not normally expressed in the cells. Following transfection of these chimeric genes into S115 cells, we show that the expression of the genes is regulated by both androgens and glucocorticoids. Furthermore, hormonal regulation is also conferred by the LTR on the neighboring guanine phosphoribosyltransferase (gpt) gene. Thus, androgens can act on the LTR of MMTV when the appropriate receptors are present in the cells, and this interaction can influence the expression of additional adjacent genes.     

The chromosomal integration site determines the tissue-specific methylation of mouse mammary tumour virus proviral genes.

Multiple endogenous mouse mammary tumour virus (MMTV) proviral genes are present at different chromosomal locations in inbred mouse strains. Proviral DNA methylation is location and tissue specific. The methylation patterns are stably inherited and appear to be conferred upon the viral DNA by the flanking mouse genomic DNA. In transformed cells, either mammary carcinoma cells, or cells immortalized by SV40 in vitro, the stable pattern of methylation is lost. Although hypomethylation of proviral genes, both in normal and in transformed tissue, accompanies MMTV-specific RNA expression, it is also observed in non-expressing tissues.     

Glucocorticoid hormone interactions with cloned proviral DNA of mouse mammary tumor virus

The molecular details of glucocorticoid hormone regulation of expression of the mouse mammary tumor virus (MMTV) proviral gene have been investigated. Cloned proviral DNA was introduced into cultured cells by a gene transfer procedure. DNA acquired by transfection was shown to be expressed in a hormone regulated fashion. The proviral DNA was fragmented and recombined in vitro with an indicator gene to delimit the hormone response sequence. Inducibility of the indicator gene (thymidine kinase gene from Herpes Simplex Virus, tk) was observed upon recombination with the long terminal repeat (LTR) sequence of MMTV. Further delimitation of the LTR DNA demonstrated that 202 nucleotides located 5' of the RNA initiation site are sufficient to confer glucocorticoid regulation. In vitro interaction of LTR DNA with glucocorticoid hormone receptor complex, showed a preferential affinity to the same sequence which mediated hormonal regulation in transfected cells. Evidence for a direct receptor gene interaction in the process of gene induction was gained by the measurement of the kinetics of induction and the use of a glucocorticoid antagonist (RU 486). The induction of the transfected gene is very rapid, independent of simultaneous protein synthesis and requires a functional glucocorticoid receptor hormone complex.     

Steroid regulation of transfected genes in mouse mammary tumour cells

The regulation of mouse mammary tumour virus (MMTV) RNA by glucocorticoid hormones is well-established and has provided much information on how steroid hormones work. However, we have shown that androgens can also control MMTV RNA accumulation in S115 mouse mammary tumour cells. This novel androgen action could be explained on the basis that the MMTV long terminal repeat (LTR) can respond to several classes of steroid if appropriate receptors are present in the cells. We have used transfection experiments to demonstrate that androgens can act directly on the LTR in S115 cells. Hormonal regulation of transfected chimaeric genes into these cells was effected by androgen and glucocorticoid but not by oestrogen or progesterone, corresponding to the receptor status of the cells. Furthermore, hormonal control was also conferred by the LTR on expression of an independent cotransfected adjacent gene under its own separate promoter, suggesting that effects of an LTR can stretch to neighbouring genes in a type of hormone-enhancer insertion mechanism.     

The endogenous proviral mouse mammary tumor virus genes of the GR mouse are not identical and only one corresponds to the exogenous virus.

The endogenous proviral copies of mouse mammary tumor virus (MMTV) were selected from a gene library of GR mouse DNA. We obtained five different lambda. MMTV recombinant clones. Four of them correspond to the 3' Eco RI fragments of the endogenous proviruses an one comprises an intact MMTV provirus with 2 to 3 kb of flanking mouse genomic DNA. Heteroduplex formation followed by S1 digestion under stringent conditions shows that there is nucleotide sequence heterology among the cloned endogenous proviral copies. Only one endogenous proviral copy, associated with the mtv-2 locus, was found to be totally homologous to the exogenous proviral DNA.     

Dexamethasone increases the number of RNA polymerase II molecules transcribing integrated mouse mammary tumor virus DNA and flanking mouse sequences.

In mouse Ltk- cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the transcribed MMTV DNA as well as on the flanking mouse DNA sequences. We found that the specific increase in the polymerase loading after hormone treatment is proportional to the increase in the amount of stable MMTV mRNA. When the DNA sequences which are responsible for hormone-receptor binding and for the increased MMTV mRNA levels were deleted, no increase in RNA polymerase II loading on MMTV DNA was observed. Nuclear RNA chains which were transcribed in response to hormone treatment were detected not only from the transfected MMTV DNA but also from the mouse DNA sequences adjacent to the 3' end of the provirus.     

Mitogen-activated S6 kinase is stimulated via protein kinase C-dependent and independent pathways in Swiss 3T3 cells

Soluble extracts prepared from quiescent Swiss mouse 3T3 cells that had been briefly exposed to various mitogens exhibited a 2- to 3-fold elevation in phosphorylating activities toward ribosomal protein S6 and a synthetic peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (RRLSSLRA), patterned after a phosphorylation site sequence from S6. Optimal activation of the phosphorylating activity occurred within 15-20 min of exposure of the cells to platelet-derived growth factor (10 ng/ml), epidermal growth factor (100 nM), and insulin (100 nM), and 2-5 min after 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nM) treatment. Fractionation of the cytosolic extracts from mitogen- or TPA-treated cells on Sephacryl S-300, TSK-400, and DEAE-Sephacel columns gave results suggesting that a single stimulated kinase accounted for the enhanced S6 and RRLSSLRA phosphorylating activities. The mitogen-activated kinase had an apparent Mr of about 85,000 as determined with Sephacryl S-300, but eluted with an apparent Mr of 26,000 from a TSK-400 high pressure liquid chromatography column. The S6 kinase was also stimulated in cytosols from insulin-like growth factor 1- (100 nM), vasopressin- (250 nM), prostaglandin F2 alpha- (250 nM), and 10% fetal calf serum-treated cells but not from quiescent cells exposed to beta-transforming growth factor (2 ng/ml). TPA, vasopressin and prostaglandin F2 alpha appeared to stimulate this kinase via a protein kinase C-dependent mechanism, since the responses to these hormones, but not to platelet-derived growth factor, epidermal growth factor, and insulin, were lost in protein kinase C-depleted cells.     

Construction and characterization of a packaging cell line for MMTV-based conditional retroviral vectors

A chimeric provirus in which the 5'LTR of a complete biologically active Mouse Mammary Tumour Virus (MMTV) proviral DNA has been replaced with the Rous Sarcoma Virus LTR has been constructed. Upon transfection into permissive cells, this provirus directs the synthesis of the MMTV gag and env structural proteins, but is impaired in packaging of the RNAs that encode these proteins. Supertransfection of these cells with MMTV based vector constructs results in the production of infectious recombinant virus at a higher efficiency than with previously described helper cell lines. Such a retroviral vector system based on MMTV will allow the study of the effects of conditional expression of inserted genes upon infected cells.     

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing [3H]triamcinolone acetonide [( 3H]TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, [3H]TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. When similar experiments were conducted under conditions of large receptor excess, using 3' [32P]-MMTV LTR DNA, the trace quantity of DNA formed a stable 10-14S complex with DNA-cellulose pretreated cytosols or with untreated cytosols in the presence of excess Escherichia coli competitor DNA. If trace quantities of the 3' [32P]-MMTV LTR DNA were incubated with untreated crude cytosols, much larger complexes were formed, indicating the association of other cytosolic proteins with the MMTV LTR DNA fragment. Activated [3H]TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA, but with lower apparent affinities in the order MMTV LTR DNA fragment much greater than pBR322 fragment containing a single GRE DNA consensus sequence greater than non-GRE-containing pBR322 fragment.(ABSTRACT TRUNCATED AT 250 WORDS)