Glucocorticoid-receptor interaction and induction of murine mammary tumor virus.

The relationship between the cellular uptake of glucocorticoid hormones, the binding of these hormones to specific in vitro receptors, and the induction of mouse mammary tumor viruses in an established mouse mammary tumor cell line was highly correlated. These results suggest that the induction of mouse mammary tumor virus by glucocorticoid hormones is a physiological process acting through a mechanism of high affinity, saturable steroid-receptors. A temperature-sensitive or salt-dependent step following glucocorticoid-receptor interaction was required for nuclear uptake of the steroid. Induction studies with different adrenocorticoids indicate that the synthetic glucocorticoid, dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), is the most potent inducer of mouse mammary tumor viruses and all steroids which caused significant induction were glucocorticoids. Other glucocorticoids appear to stimulate murine mammary tumor virus production by a mechanism similar to that of dexamethasone; for example, corticosterone competes with dexamethasone for binding to the glucocorticoid receptor and blocks the uptake of dexamethasone into cells. Progesterone also blocks the cellular uptake of dexamethasone and can bind to the glucocorticoid receptor at low concentrations (10-7 to 10-8 M) but progesterone does not consistently induce virus at hormone concentrations even as high as 10-4 M. Thus, in this system, binding to a cytoplasmic receptor is necessary but not sufficient for induction by glucocorticoids. Estrogens and androgens interfere with receptor binding and cellular uptake of dexamethasone but only at much higher concentration (10-4 M) than progesterone, and do not induce mammary tumor virus production. Although there was a positive correlation between steroid structure, binding, and biologic induction, other factors clearly affect the physiological manifestations of steroid actions. Mouse cells with comparable cytoplasmic receptor levels and comparable nuclear uptake differed absolutely in their degree of murine mammary tumor virus induction following hormone treatment. Although all mouse cells examined contain comparable levels of murine mammary tumor virus DNA, only cells producing constitutive levels of murine mammary tumor virus RNA could be induced to higher levels by a variety of glucocorticoids.     

Radioimmunolocalization of murine mammary tumor virus proteins in gels

Nycodenz is a new nonionic iodinated gradient medium which readily dissolves in water to give nontoxic, autoclavable solutions. This paper describes the use of diffusion techniques to prepare isotonic Nycodenz gradients with maximum densities up to 1.15 g/ml, which is sufficient to band most types of cells.     

Phosphorylation of murine mammary tumor virus precursor polypeptides.

Phosphorylation of the murine mammary tumor virus (MuMTV) structural proteins was studied in an MuMTV-infected epithelial cell line derived from a BALB/cf C3H mouse mammary tumor. Immunoprecipitation of 32P-labeled cell extracts with monospecific anti-p27 serum revealed that phosphorylation occurred at the stage of the core-protein polyprotein precursor prp75. Two forms of phosphorylated prp75 were found: one migrating with an apparent molecular weight of 80,000, and the other with a molecular weight of 76,000. The 80,000-molecular-weight species was found to be the most heavily phosphorylated. In addition, a relatively stable phosphorylated processing intermediate of 34,000 molecular weight was observed as well. Tryptic peptide mapping analysis of the 32P-labeled viral proteins indicated a precursor product relationship between the intracellular phosphorylated, high-molecular-weight peptides and the mature MuMTV phosphoproteins p23 and p27. Phosphopeptide analysis also suggested that phosphorylation of the viral proteins occurred in discrete steps and that the attached phosphate groups were conserved throughout the processing steps.     

Sialylatin of glycoproteins of murine mammary tumor virus, murine leukemia virus, and Mason-Pfizer monkey virus.

Neuraminidase treatment of mouse mammary tumor virus, Rauscher murine leukemia virus, and Mason-Pfizer monkey virus resulted in loss of their capacity to inhibit hemagglutination of influenza virus. Hemagglutination-inhibition activity of these RNA tumor viruses could be restored by in vitro resialylation catalyzed by sialyl transferase. The major glycoprotein in the intact envelope of desialylated and, to some extent, native virions could be specificallly labeled in vitro with CMP-(14C) sialic acid. These studies further characterize the individual glycoproteins of mouse mammary tumor virus, Rauscher murine leukemia virus, and Mason-Pfizer monkey virus.     

Lysine tRNA is the predominant tRNA in murine mammary tumor virus

The method of aminoacylation and subsequent identification of the esterified amino acids was used to characterize the transfer RNAs in murine mammary tumor virus. Lysine tRNA was the major tRNA in both “free” 4S RNA and “7OS-associated” 4S RNA in virus derived from either tissue culture or mouse milk.     

Structural analysis of the intracellular RNAs of murine mammary tumor virus.

We have characterized murine mammary tumor virus (MuMTV)-specific RNA in several types of cells in which viral DNA is transcribed into RNA: cultured GR mouse mammary tumor cells, S49 lymphoma cells from BALB/c mice, lactating mammary glands from C57BL/6 mice, and mink lung cells infected in vitro with MuMTV. In all cell types studied, there are three distinct species of intracellular viral RNA, with sedimentation coefficients of 35S, 24S, and 13S (or molecular weights of 3.1 X 10(6), 1.5 X 10(6), and 0.37 X 10(6), as determined by rate-zonal sedimentation in sucrose gradients and by electrophoresis in agarose gels under denaturing conditions. These three viral RNA species appear to be present regardless of viral RNA concentration, responsiveness to glucocorticoid hormones, production of extracellular virus, and use of either endogenous or acquired MuMTV proviral DNA as template. The three viral RNAs display characteristics of mRNAs in that they are polyadenylated, associated with polyribosomes, and released from polyribosomes by treatment with EDTA; hence all three species presumably direct the synthesis of virus-coded proteins. The two larger species of viral RNA are probably responsible for synthesis of the structural proteins of the virion, but the function of the 13S RNA is not known. Both of the subgenomic RNAs contain sequences found at the 3' terminus of 35S (or genomic) RNA. However, only the 24S RNA (not the 13S RNA) contains sequences which are located at the 5' terminus of 35S RNA and are apparently transposed during RNA synthesis of maturation, as described for subgenomic mRNA's of other retroviruses.     

Growth inhibition of murine mammary carcinoma by monoclonal IgE antibodies specific for the mammary tumor virus

Summary Two IgE-producing hybridomas were established from spleen cells of Balb/c mice, which had been immunized with mouse mammary tumor virus (MMTV). These IgE monoclonal antibodies (mAbs) reacted specifically with the major envelope glycoprotein (gp36) of MMTV, as established by the immunoblot assay and by passive cutaneous anaphylaxis. The effect of the IgE mAbs (produced by clone A8) on the growth of the MMTV-secreting mammary adenocarcinoma H2712 was investigated in syngeneic C3H/HeJ mice. The mice were inoculated s.c. with either 10⁵ (100 × LD₅₀) or 10⁶ (1000 × LD₅₀) tumor cells and received repeated i.p. injections of 25 µg anti-gp36 IgE mAbs at 4-day intervals for 8 weeks. This treatment prevented the development of subcutaneous tumors in 50% of the animals. Similar protection was observed when the tumor cells (10⁵/animal) were injected i.p. 4 days prior to the beginning of the i.p. treatment consisting of injections of 25 µg mAbs at 4-day intervals for 6 weeks. However, these mAbs did not protect C3H/HeJ mice against the MMTV-negative MA16/c carcinoma cells. Hence, these results support the view that IgE-mediated cytotoxic mechanisms may play an immunologically specific antitumor surveillance role and that laboratory-induced antitumor IgE mAbs have the potential of specific therapeutic agents for in vivo destruction of tumor cells.     

Murine mammary tumor virus pol-related sequences in human DNA: characterization and sequence comparison with the complete murine mammary tumor virus pol gene.

Sequences in the human genome with homology to the murine mammary tumor virus (MMTV) pol gene were isolated from a human phage library. Ten clones with extensive pol homology were shown to define five separate loci. These loci share common sequences immediately adjacent to the pol-like segments and, in addition, contain a related repeat element which bounds this region. This organization is suggestive of a proviral structure. We estimate that the human genome contains 30 to 40 copies of these pol-related sequences. The pol region of one of the cloned segments (HM16) and the complete MMTV pol gene were sequenced and compared. The nucleotide homology between these pol sequences is 52% and is concentrated in the terminal regions. The MMTV pol gene contains a single long open reading frame encoding 899 amino acids and is demarcated from the partially overlapping putative gag gene by termination codons and a shift in translational reading frame. The pol sequence of HM16 is multiply terminated but does contain open reading frames which encode 370, 105, and 112 amino acid residues in separate reading frames. We deduced a composite pol protein sequence for HM16 by aligning it to the MMTV pol gene and then compared these sequences with other retroviral pol protein sequences. Conserved sequences occur in both the amino and carboxyl regions which lie within the polymerase and endonuclease domains of pol, respectively.     

Electrophoretic analysis of the molecular weight of murine mammary tumor virus RNA.

Molecular weight determinations of native and subunit RNAs of murine mammary tumor virus (MuMTV), a type B oncornavirus, were performed by polyacrylamide gel electrophoresis and compared with molecular weights of well-characterized avian cellular RNAs and tobacco mosaic virus RNA. From extrapolations of semilog plots of the molecular weights of the standard RNAs versus relative electrophoretic mobilities and Ferguson plots, the subunit and native RNAs of MuMTV were found to possess molecular weights of 2.93 X 10(6) and 6.45 X 10(6), respectively. These data support the assumption that two subunit molecules comprise the native RNA of MuMTV.     

Synthesis and processing of precursor polypeptides to murine mammary tumor virus structural proteins.

Biosynthesis of murine mammary tumor virus (MuMTV) proteins was studied in the chronically MuMTV-infected epithelial cell line MuMT-73 by using monospecific antisera to the major MuMTV core protein p27 and the major envelope glycoprotein gp47. In pulse-labeling experiments using [35S]methionine, monospecific antisera to p27 precipitated a 75,000-molecular-weight protein as the major intracellular component. Analysis of the same cellular extracts with monospecific antisera to gp47 revealed that the gp47 precursor was a 70,000-dalton protein. After chase periods, there was a loss of label from the precursors and a concomitant increase of labeled extracellular mature viral proteins. The glycoprotein precursor incorporated labeled glucosamine and seemed to be processed more rapidly than the p27 precursor. Considerable amounts of apparently nonvirion-associated gp47 and glycoprotein precursor could be detected in the extracellular culture fluid.     

Mammary tumor virus proviral DNA in normal murine tissue and non-virally induced mammary tumors

The Southern DNA filter transfer technique was used to study the involvement of the endogenous mouse mammary tumor virus (MMTV) in the development of mammary tumors of nonviral etiology. The presence of extra MMTV proviruses in the genomes of these non-virally induced mammary tumors would indicate an integration of the provirus of an activated endogenous MMTV. Acquisition of MMTV proviruses did not seem to be an absolute requirement for the development of hormone or carcinogenically induced mammary tumors in strain BALB/c nor for hormone-induced mammary tumors in mouse strains 020, C57BL, and C3Hf. In some hormone-induced mammary tumors we did observe extra MMTV proviruses in submolar quantities, indicating that reintegration may occasionally occur and that only a part of the tumor cells acquired new MMTV DNA information. Hormone-dependent and -independent primary mammary tumors of the mouse strain GR, which are controlled by the Mtv-2 mammary tumor induction gene, all acquired extra MMTV proviruses. Most of these extra MMTV proviral-DNA-containing fragments appeared present in submolar quantities, suggesting that only part of the tumor cells acquired extra MMTV proviral information. These findings indicate that the initially transformed mammary gland cells of non-virally induced mammary tumors do not necessarily acquire extra MMTV proviral DNA information, in contrast to the MMTV-induced mammary tumors, in which all tumor cells contain extra MMTV DNA information.     

Mouse mammary tumor virus with rearranged long terminal repeats causes murine lymphomas.

Mouse mammary tumor virus (MMTV) is a slowly transforming retrovirus associated primarily with the induction of mammary tumors. It is widely accepted that T-cell lymphomas of various mouse strains are associated with extra proviruses of MMTV. These extra proviruses showed site-specific rearrangements in the U3 region of long terminal repeats (LTRs), consisting of about 400 nucleotide deletions and occasional substitution resulting in unique tandem repeats. However, the question of whether these mutant MMTVs cause lymphomas has not been experimentally resolved. Here we present distinct evidence that they do. We constructed chimeric MMTVs by replacing the LTR of the recently constructed pathogenic MMTV provirus clone with rearranged LTRs of MMTV proviruses obtained from two DBA/2 mouse lymphoma cell lines, MLA and DL-8, and inoculated them into BALB/c mice. These mice developed lymphomas, but no mammary tumors, 4 to 11 months postinoculation, whereas the original pathogenic MMTV clone alone induced mammary tumors. These results showed that the tissue specificity of MMTV tumorigenesis is determined by the LTR structures.     

Purification of immature cores of mouse mammary tumor virus and immunolocalization of protein domains.

The immature capsids of the mouse mammary tumor virus (MMTV), known as intracytoplasmic A particles, have been isolated from murine L1210 leukemia cells. The diameter of the isolated particles was 80 nm as determined by negative staining. Two polypeptides of 77 and 110 kDa were found to be their major polypeptide components, in agreement with the expected sizes of the Gag and Gag-Pro precursor polypeptides of the mature MMTV proteins. Both polypeptides were recognized by antibodies directed toward the matrix (p10) and capsid (p27) proteins of MMTV. Immunogold labeling of p10 on isolated A particles, visualized by negative staining, showed that this protein is located at the surface of the immature capsids, whereas p27 can be detected only in broken or disrupted particles, suggesting that it has an internal location. These observations were confirmed by immunolabeling of both proteins on thin sections of A particle-producing cells. In addition, the viral protease had a more internal position than p27. Since the sequential order of the viral proteins in the Gag precursor is p10-pp21-p27-p14 and that in Gag-Pro is p10-pp21-p27-p30-protease, our results demonstrate the radial organization of the polypeptide precursors forming the intracytoplasmic A particles.     

The purification and characterization of a major glycoprotein of the murine mammary tumor virus.

Affinity chromatography of solubilized murine mammary tumor virus on concanavalin A-Sepharose was clearly affected by different mixtures of detergent present in the elution buffer: A complex consisting of a glycoprotein of 52,000 daltons (gp52), and a glycoprotein of 36,000 daltons (gp36), besides free gp52 were isolated. The gp36 could be purified by gel filtration of the complex in the presence of a high concentration of sodium deoxycholate. The elution of gp36 in the void volume of the Sephadex column and the results obtained with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed strong hydrophobic interactions within the molecule. The glycoprotein was immunochemically characterized by competitive radioimmunoassay and immunoelectrophoresis. No cross-reactivity of gp36 with gp52 or two nonglycosylated viral polypeptides was observed.     

Purification and characterization of the mouse mammary tumor virus protease expressed in Escherichia coli.

The mouse mammary tumor virus (MMTV) protease gene was cloned into pGEX-2T, an Escherichia coli expression vector containing the glutathione S-transferase coding region of Schistosoma japonicum. The chimeric protein was formed by fusion of the glutathione S-transferase with a hexapeptide which contains a thrombin cleavage site, followed by the MMTV protease. Affinity chromatography on a glutathione-Sepharose 4B column was used to isolate the chimeric protein. After thrombin cleavage, the glutathione S-transferase and the protease were separated by gel filtration chromatography on a Sephadex G-75 column. The overall yield of the protease purification procedure was about 1 mg of protease/liter of culture, and the specific activity was 380 pmol/min.micrograms of enzyme. Like other retroviral proteases, the MMTV enzyme was active as a dimer, showed maximum activity at pH between 4 and 6, and could be inhibited by pepstatin A and a phosphinic acid derivative HIV-1 protease inhibitor. Enzymatic characterization of this protease reveals its broad specificity, showing a clear preference for the oligopeptide substrate mimicking the cleavage site at the amino-terminal end of the capsid protein (kcat/Km = 9725.5 M-1.s-1). The chimeric protein was also an active dimer and showed a similar Km (17 microM) for such an oligopeptide, although its kcat was about 10 times smaller. Autocatalytic processing of the MMTV protease was observed after expression of clones containing the natural cleavage site, as it occurs at the amino-terminal end of the viral protease, instead of the thrombin-sensitive sequence.