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Biomedical subjects

R Michalides

Publications and source records attributed to R Michalides.

16 recordsLinked to original sources
PubMed1979

Induction of mouse mammary tumor virus RNA in mammary tumors of BALB/c mice treated with urethane, X-irradiation, and hormones.

The involvement of mouse mammary tumor virus (MTV) in the development of mammary tumors of nonviral etiology in BALB/c mice was studied by measuring the levels of MTV RNA, MTV DNA, and MTV proteins in spontaneously arising and hormonally, chemically, and/or physically induced mammary tumors of BALB/c females. The following results were obtained. (i) Spontaneous mammary tumors contained very low levels of MTV RNA; 4 X 10(-6)% of the the cytoplasmic RNA was MTV RNA. No MTV proteins could be demonstrated by using sensitive radioimmunoassays for MTV proteins p27 and gp52. (ii) Mammary tumors induced by treatments with urethane or X-irradiation alone contained higher levels of MTV RNA; these tumors contained 3- and 19-fold more MTV RNA, respectively, compared with spontaneous mammary tumors. (iii) Mammary tumors induced by combined treatment with urethane and X-irradiation expressed high levels of MTV RNA in the mammary tumors; a 1,724-fold increase in MTV RNA content compared with spontaneous mammary tumors was observed. However, very low levels of MTV proteins gp52 and p27 were detected, suggesting some kind of impairment at the translation of the MTV RNA. MTV RNA was also induced by this treatment in mammary glands and spleens, but not in the livers of tumor-bearing animals. (iv) Balb/c females continuously exposed to prolactin contained high levels of MTV RNA and MTV proteins in stimulated mammary glands and in the hormonally induced mammary tumors. These findings suggest that MTV is not responsible for the maintenance and probably also not for the development of all murine mammary cancers.

Animals↗
PubMed1979

Impaired maturation of mouse mammary tumor virus precursor polypeptides in lymphoid leukemia cells, producing intracytoplasmic A particles and no extracellular B-type virions.

Processing of polypeptides of the mouse mammary tumor virus, a type B retrovirus, was investigated in a transplanted thymic lymphoma cell line of the GR strain (GRSL). This cell line was maintained in vivo in ascites form and in vitro as a suspension culture. GRSL cells produce clusters of intracytoplasmic A particles and are virtually deficient in the production of mature extracellular B-type particles. As control, a mammary tumor cell line of the same mouse strain capable of complete virion synthesis was used. The kinetics of viral polypeptide synthesis were studied by pulse labeling with various isotopes (including (35)S and (32)P), followed by immunoprecipitation of cell lysates with monospecific antisera to the major mouse mammary tumor virus gag and env proteins, p27 and gp52, respectively. Both the primary gag and env precursor polypeptides were synthesized in the GRSL cells, but their conversion into viral proteins was impaired. The major gag precursor, Pr73(gag), was stable over a period of 8 h, and mature viral core polypeptides could not be detected. Also, the highly phosphorylated intermediates in the proteolytic processing of Pr73(gag) in virus-producing cells were absent in GRSL cells. By immunoprecipitation, Pr73(gag) was detected in a GRSL particle fraction with the density of intracytoplasmic A particles. The precursor for envelope proteins, Pr73(env), was turned over without the generation of mature viral envelope components gp52 and gp36. The in vivo-transplanted ascites GRSL cells, however, were shown to express gp52 on the cell surface together with a 73,000-dalton polypeptide, as indicated by cell surface iodination and immunoprecipitation.

Animals↗
PubMed1979

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.

Animals↗
PubMed1978

Identification of the Mtv-2 gene responsible for the early appearance of mammary tumors in the GR mouse by nucleic acid hybridization.

In the mouse strain GR, the Mtv-2 gene controls the expression of large amounts of mammary tumor virus (MTV) antigens in the milk at first lactation. It also controls the early appearance of mammary tumors. We have investigated the number of MTV proviral sequences associated with this Mtv-2 gene by nucleic acid hybridization between MTV [(3)H]cDNA and DNA from GR, B10, and GR-Mtv-2(-) mice. B10 and GR-Mtv-2(-) mice lack Mtv-2 gene expression. The molecular hybridizations revealed that the DNA of GR mice contains 12 copies of MTV proviral sequences, whereas only 4 copies are present in the DNA of B10 and GR-Mtv-2(-) mice. We therefore conclude that the Mtv-2 gene in the GR mouse strain is associated with eight additional MTV proviral sequences. The four Mtv proviral sequences in the GR-Mtv-2(-) DNA might represent another Mtv gene in the GR mouse. Different amounts of MTV RNA are detected in mammary glands at first lactation of B10 and GR-Mtv-2(-) mice, even though both contain four copies of MTV proviral sequences. This indicates a difference between these two mouse strains either in the regulation of expression of these MTV proviral sequences or in the location of these sequences in the murine genome.

Animals↗
PubMed1978

Involvement of mouse mammary tumor virus in spontaneous and hormone-induced mammary tumors in low-mammary-tumor mouse strains.

The involvement of the mouse mammary tumor virus (MTV) in spontaneous and hormone-induced mammary tumors in low-mammary-tumor mouse strains was studied by comparing the amounts of MTV RNA and MTV DNA sequences in mammary tumors and other tissues of mice with an without hormonal treatments. The following results were obtained. (i) Mammary tumors which appeared in C3H mice as a result of an infection with MTV contained more MTV DNA compared with noninfected organs; these mammary tumors also contained more MTV RNA than was present in lactating mammary gland cells. (ii) Hormonal stimulation by administration of excessive amounts of prolactin via hypophyseal isografts in C3Hf and O20 mice resulted in an increased expression of MTV RNA in the mammary glands. This elevated level of MTV RNA expression was, however, not maintained in the hormone-induced mammary tumors. (iii) Spontaneous mammary tumors in BALB/c mice contained similar levels of MTV DNA and MTV RNA sequences as were found in other cells of these animals.

Animals↗
PubMed1977

Differences in mouse mammary tumor viruses. Relationship to early and late occurring mammary tumors.

The murine model has been used extensively to study the various factors involved in the etiology of mammary carcinoma. Inbred mouse strains have been classically categorized into (i) high incidence stains with tumors occurring relatively early in the life of the animal, or (ii) low or moderate incidence strains with tumors occurring later on in life. We have radioactively labeled the RNA genome of the mouse mammary tumor virus (MMTV) from each of several mouse strains. We report here, using the technique of molecular hybridization, that the class of MMTVs responsible for the early occurring mammary tumors in high incidence strains can be clearly distinguished from the MMTVs associated with late occurring mammary tumors in low or moderate incidence strains; we also demonstrate that minor differences in MMTV genomes can also exist within these classes. Our findings show that MMTVs are transmitted via the germ line (as a germinal provirus) in some mouse strains, whereas in other strains, a non-germ line transmission is clearly demonstrated. Biochemical techniques can thus be used to track the mode of transmission of oncogenic viruses. The relationship of these findings to an understanding of the etiology of mammary carcinoma is discussed.

Animals↗
PubMed1977

Biochemical properties of endogenous rat C-type viruses.

The cellular restriction to persistence of inducible C-type viruses of at least two rat strains can be overcome in actively in vitro replicating and/or spontaneously transforming rat embryo cells. Both, SD-RaLV and W-RaLV particles are morphologically and biochemically related to known oncorna viruses: They are of C-type, have an identical buoyant density of 1.15 g/cm3 in sucrose gradients and contain high-molecular-weight RNA. The RaLV-associated DNA polymerase exhibit both exogenous (polynucleotide-templated) and endogenous (viral RNA-templated) reactivity. The enzyme strongly preferred Mn++ over Mg++ ions (Mg++/Mn++ ratio of 0.04 to 0.07), with maximum reactivity at 0.1 mM MnCl2 concentration. The molecular hybridization experiments revealed the endogenous nature of the SD-RaLV and the W-RaLV. From the competition hybridization assay the number of proviral sequences could be calculated to be 84 per haploid cell genome. The role of endogenous rat viruses in the "spontaneous" in vitro transformation is discussed.

Animals↗
PubMed1976

A biochemical approach to the study of the transmission of mouse mammary tumor viruses in mouse strains RIII and C3H.

Mouse mammary tumor virus (MMTV) proviral sequences were detected in the cellular DNA of mammary tumors and livers of RIII and C3H mice by molecular hybridization with radioactively labelled MMTV 60-70S RNA or tritiated MMTV complementary DNA (cDNA). By means of DNA:DNA reassociation kinetics, the DNA of the mammary tumor cells of these two mouse strains were found to contain more MMTV proviral sequences than the DNA of liver cells of these same tumor-bearing mice. Evidence is also presented that the DNA of the liver cells lacks a part (approximately 25%) of the MMTV proviral sequences found in the mammary tumor cells of these mouse strains. The relationship of the extra MMTV proviral sequences found in mammary tumor cells to the early mammary tumor-igenesis seen in these mouse strains is discussed.

Animals↗
PubMed1976

Characterization of mouse mammary tumor viruses from primary tumor cell cultures. II. Biochemical and biophysical studies.

Primary mammary tumor cultures of RIII, GR, DD, BALB/c, and BALB/cfC3H mice were examined for mouse mammary tumor virus (MuMTV) production. Levels of production of 12-32 mug virus protein/day/75-cm2 culture flask could be maintained for 30-50 days with daily virus harvests. The viruses from tumor cell cultures of these mouse strains contained DNA polymerase with a strong preference for Mg++ over Mn++ as the divalent cation, a characteristic of DNA polymerase of MuMTV from mouse milk. These viruses from tumor cell cultures were excellent sources of MuMTV 3H-complementary DNA (complexed to 60-70S RNA) and radioactive 60-70S RNA, sufficiently free of contaminating murine leukemia virus nucleic acids, that can be used in molecular hybridization experiments. The effects of several culture parameters on MuMTV production were also studied.

Cells, Cultured↗
PubMed1976

Characterization of the oncornavirus particles in the plasma of guinea pigs with L2C leukemia.

The inoculation of L2C guinea pig leukemia cells into strain 2 guinea pigs results in the death of the animals within 12 to 15 days. Death is preceded by the simultaneous appearance in the plasma of (i) elevated leukocyte levels, (ii) extracellular virus particles, and (iii) a particle-associated RNA-directed DNA polymerase. This enzyme activity has a cation preference identical to that of the type B bromodeoxyuridine-induced guinea pig virus, i.e., an Mg2+ optimum at 20 mM and no activity using Mn2+. Competitive molecular hybridization studies also revealed that the plasma of leukemic guinea pigs contained approximately 2 X 10(9) genome equivalents per ml of an RNA that is homologous to the RNA of the bromodeoxyuridine-induced guinea pig virus. Morphological observations indicate that most, but not all, of the extracellular particles observed in leukemia plasma are derived from the intracisternal particles seen in the L2C tumor cells. The possibilities that either two viral populations are present or that the in vivo morphogenesis of the type B bromodexoyuridine-inducible guinea pig virus is markedly different from its in vitro morphogenesis are discussed.

Animals↗
PubMed1975

Relationship in nucleic acid sequences between mouse mammary tumor virus variants.

Primary cultures of mouse mammary carcinomas were used as a source of both radioactively labeled and unlabeled 60-70S RNA of mouse mammary tumor virus (MMTV) obtained from various mouse strains. Competition molecular hybridization experiments revealed that, within the limits of the assay, the RNAs of the MMTVs synthesized in culture by the tumors of the mouse strains RIII, GR, A, and C3H, are identical. A comparison of the genomes of the milk-transmitted MMTV(C3H) and the vertically transmitted MMTV(C3Hf) revealed that these two viruses are approximately 75% similar. No nucleic acid sequence homology was observed between MMTV(C3H) 60-70S RNA and the RNAs of murine leukemia virus, Mason-Pfizer virus, or the BrdUrd-induced type-B quinea pig virus.

Animals↗
PubMed1975

Biochemical properties of the bromodeoxyuridine-induced guinea pig virus.

The biophysical and biochemical properties of the virus particles released by guinea pig embryo cells treated with 5-bromo-2'-deoxyuridine (BUdR) have been compared to those of the B-type mouse mammary tumor virus (MMTV) and the C-type Rauscher murine leukemia virus. The high-molecular-weight (60 to 70S) RNA of the BUdR-induced guinea pig virus (GPV) has a molecular weight of 8 X 106 when measred by mixed agarose polyacylamide gel electrophoresis. The virus particles isolated from the tissue culture medium of BUdR-induced guniea pig cells have the following properties in common with MMTV: (i) a buoyant density of 1.18 g/ml in sucrose and 1.21 g/ml in CsCl, and (ii) a DNA polymerase that prefers Mg2+ over Mn2+ in an assay using the synthetic template poly(rC):oligo(dG). No nucleic acid sequence homology between GPV RNA and the viral RNAs of the MMTV, murine leukemia virus, hamster sarcoma virus, or Mason-Pfizer monkey virus could be observed in a competition hybridization assay using the radioactive-labeled GPV 60 to 70S RNA. By this same competition by hybridization assay the frequency of GPV proviral sequences was estimated to be at least 83 per haploid cellular genome of guniea pig cells. No nucleic acid sequences related to be GPV RNA were detected in the DNA of normal tissues of mice, rats, cats, dogs, baboons, or humans by direct RNA-DNA hybridization using radioactive GPV60 to 70S RNA.

Animals↗
PubMed1975

Evidence for an RNA tumor viruses in human milk.

Certain human milks have been shown to contain particles that have the biochemical and biophysical properties that are diagnostic of the known RNA tumor viruses of animals. These properties include 1) a particle density of 1.16-1.19 g/ml 2) a viral reverse transcriptase (RNA-directed DNA polymerase), and 3) a high molecular weight (HMW) 60-70S RNA that contains polyadenylic regions of 200 nucleotides in length. Inner cores, or nucleoids, of these particles have been isolated. They have a density of 1.26-1.27 g/ml and contain the viral reverse transcriptase and 60-70S RNA. Using molecular hybridization, a specific homology was demonstrated between radioactive DNA synthesized from the RNA of the human milk particle and the RNA from human malignant breast tumors. RNA from benign breast tumors and other human tissues were negative in these tests.

Adenocarcinoma↗
PubMed1975

Biochemical characterization of putative subviral particulates from human malignant breast tumors.

Particulates with the properties of cores and/or ribonucleoproteins of RNA tumor viruses have been isolated from Sterox-SL-treated fractions of murine and human mammary adenocarcinomas. These particulates have an RNA-directed DNA polymerase, a 60 to 70 S RNA, and a density of 1.26 g/ml or greater in sucrose equilibrium density gradients. Their uniquely higher densities lead to banding in regions comparatively free of cellular contaminants. These circumstances minimize some of the technical complications of performing the simultaneous detection assay in the presence of cell debris.

Adenocarcinoma↗