Search PubMed⌕ Search

Biomedical subjects

R Michalides

Publications and source records attributed to R Michalides.

At least 37 records · Page 2Linked to original sources
PubMed1986

Genetic marker patterns and endogenous mammary tumor virus genes in inbred mouse strains of Japan.

In order to establish the genetic relatedness of the inbred mouse strains kept in Nara, genetic marker patterns were determined in conjunction with a study on endogenous mammary tumor viral genes in these strains. Isoenzyme patterns combined with patterns of other genetic markers, show that the unrelatedness between various inbred strains of the dd stock is as high or even higher as between strains of known different origin and geneology. Based on endogenous viral gene patterns the dd stock derived mice can be subdivided into three group, DDD, DDN, DDO, KF and DD/Tbr. The DD/Tbr and its foster-nursed substrain (DD/Tbrf) have the lowest number of endogenous viral genes, i.e. two, while the other strains carry 4-6 such genes. The SLN and SHN strains, derived from a Swiss stock, have a similar pattern of viral genes different that of all other strains studied, also strains of Swiss origin from other sources, such as the NFS and the GR.

Animals↗
PubMed1985

Characterization and chromosomal distribution of endogenous mouse mammary tumor viruses of European mouse strains STS/A and GR/A.

The endogenous mouse mammary tumor virus (MMTV) proviral copies in two genetically dissimilar mouse strains, STS/A, a European mouse strain, and BALB/c, were characterized. STS/A carries the same four MMTV proviral copies as GR.Mtv-2-; these strains share also most of the isoenzyme markers and are therefore highly related. Cellular DNA of GR.Mtv-2- contains a partial MMTV provirus that is not present in STS/A. GR.Mtv-2- is derived from GR; they differ in the locus Mtv-2 that contains one MMTV provirus. Expression of this Mtv-2 endogenous MMTV provirus is directly linked to mammary tumorigenesis in GR. MMTV proviral loci were studied using restriction enzyme analysis and the Southern transfer procedure using liver DNAs from recombinant inbred strains between BALB/c and STS/A. All segregating MMTV-specific EcoRI fragments were identified to MMTV proviral loci and most of these were localized by studying the cosegregation of the Mtv units and known chromosomal markers. Since STS/A, GR.Mtv-2-, and GR are highly related, the five complete endogenous MMTV proviruses of GR were located on the following chromosomes: Mtv-2 on chromosome 18, Mtv-3 on 11, Mtv-19 on 1, Mtv-20 on 4, whereas Mtv-8 has tentatively been located on chromosome 18 by Callahan et al. (R. Callahan, D. Gallahan, and Ch. Kozak (1984), J. Virol. 49, 1005-1008). GR and GR.Mtv-2 furthermore contain two incomplete MMTV proviral elements, one of which is also present in STS/A.

Animals↗
PubMed1985

Rearrangements in the long terminal repeat of extra mouse mammary tumor proviruses in T-cell leukemias of mouse strain GR result in a novel enhancer-like structure.

Male GR mice develop T-cell leukemia at low frequency late in life. These leukemia cells invariably contain large amounts of mouse mammary tumor virus (MMTV) RNA and MMTV proteins and have extra MMTV proviruses integrated in their DNA. We show here that the extra MMTV proviruses are all derived from the endogenous MMTV provirus associated with the Mtv-2 locus and that the T-cell leukemias are clonal with respect to the acquired MMTV proviruses. The extra MMTV proviruses in six transplantable T-cell leukemia lines studied had rearranged, shortened long terminal repeats (LTRs); each T-cell leukemia, however, had a different LTR rearrangement within its extra MMTV provirus. The alteration within the extra LTRs of T-cell leukemia line 42 involved deletion of 453 nucleotides and generation of a tandem repeat region consisting of regions flanking the deletion. This alteration generated a sequence similar to the adenovirus enhancer core sequence. The viral RNAs in the T-cell leukemias contained corresponding alterations in their U3 regions. These results demonstrate that expression of MMTV in T-cell leukemias of GR mice may be the consequence of the generation of a novel enhancer, which could also stimulate expression of any adjacent cellular oncogene.

Animals↗
PubMed1983

A new locus (Mtv-4) for endogenous mammary tumor virus expression and early mammary tumor development in the SHN mouse strain.

The SHN mouse strain, which has a high incidence of mammary cancer, developed by inbreeding and selection from Swiss stock mice by Dr. H. Nagasawa and co-workers (Meiji University, Tokyo, Japan), harbored an endogenous mammary tumor virus (MTV) responsible for a high frequency of mammary tumors early in life. The locus was called "Mtv-4" and was only comparable with Mtv-2 of the GR mouse strain in its inducing capacity of mammary cancer. Molecular hybridization with 32P-labeled MTV complementary DNA showed that the characteristic Mtv-2 bands of the GR strain were absent in the SHN strain.

Animals↗
PubMed1982

Acquisition of proviral DNA of mouse mammary tumor virus in thymic leukemia cells from GR mice.

Male mice of strain GR develop T-cell leukemia at a low frequency late in life. These leukemia cells contain large amounts of mouse mammary tumor virus (MMTV) RNA and MMTV proteins in a precursor form (Nusse et al., J. Virol. 32:251-258, 1979). We used restriction enzyme analysis and molecular hybridization to identify MMTV proviruses in the DNA of these leukemia cells. GR leukemia cells contained additional integrated MMTV proviruses at various sites in the genome. This amplification of MMTV proviruses in GR leukemia cells is not restricted to one particular endogenous MMTV provirus of strain GR. The number and location of the extra MMTV proviruses present in transplants of GR leukemia cells did not change upon serial transplantation of the leukemia cells. Acquisition of MMTV proviruses was also found in a similar leukemia, L1210 of the DBA/2 mouse strain, but not in three other leukemias, SL2 of DBA/2, BW5147 of AKR, and a spontaneous thymoma of BALB/c. The two main classes of MMTV RNA, 35S and 24S, were present in the cytoplasmic RNA of GR leukemia cells, indicating that the aberrant processing of MMTV precursor proteins is not due to anomolously sized RNAs. We could not detect extra RNAs in GR leukemia cells which would represent read-through transcripts of cellular genes adjacent to the extra MMTV proviruses, initiated by a promoter signal in the right MMTV long terminal repeat sequence. These data suggest that acquisition of MMTV proviruses may coincide with the onset of leukemogenesis in GR male mice.

Animals↗
PubMed1982

Mammary tumor virus DNA as a marker for genotypic variance within hormone-responsive GR mouse mammary tumors.

Mammary tumors of GR mice acquire extra mammary tumor virus (MMTV) DNA information within their DNA during tumor growth and development. These extra MMTV genes have been used by us as genotypic markers to investigate the heterogeneity of GR mammary tumors and their loss of hormone dependence during serial transplantation. Our studies reveal that the various subpopulations of cells within individual GR mammary tumors are characterized by differences in number and location of acquired extra MMTV DNA fragments. Losses of certain of these extra MMTV DNA fragments occur when mammary tumors become hormone independent, indicating a loss of hormone-dependent cells. The study of MMTV DNA markers also reveals that low levels of autonomous cells are already present in some hormone-dependent mammary tumors at an early stage of their development. The genotypic analysis strongly indicates that mammary tumor progression is not due to phenotypic adaptation but to clonal selection of the more aggressive sublines.

Animals↗
PubMed1981

Mammary tumor induction loci in GR and DBAf mice contain one provirus of the mouse mammary tumor virus.

The mammary tumor induction genes Mtv-1 in mouse strain DBAf and Mtv-2 in strain GR control the complete expression of the endogenous mouse mammary tumor virus (MMTV). We have used a combination of genetic, biochemical and molecular biological methods to identify and correlate specific copies of the endogenous MMTV proviral genes with the biological properties of the tumor induction genes Mtv-1 and Mtv-2. These Mtv induction genes contain specific MMTV proviral information, as was concluded from restriction enzyme analysis and molecular hybridization of DNAs of congenic mouse strains and of progenitors of backcross populations. The congenic strains differed from the parental strains GR and 020 only in the Mtv-2 gene, one lacking the Mtv-2 gene (GR/Mtv-2-) and one having obtained this gene (020/Mtv-2+). The gain or loss coincided with two Eco RI cellular DNA fragments containing MMTV DNA information. Since Eco RI cuts the exogenous proviral variant of MMTV DNA once, we assume that these two cellular DNA fragments contain one MMTV provirus. The same cellular DNA fragments containing MMTV DNA information segregated together with MMTV expression in the offspring population of the backcross. In a similar backcross analysis of the induction gene Mtv-1 it was also demonstrated that the Mtv-1 gene comprises one MMTV provirus. These data indicate that Mtv induction genes contain specific but different MMTV proviral genes and that nly a limited number of the MMTV proviruses present in the cellular DNA is associated with the control of proviral expression.

Animals↗
PubMed1981

Structure, stability, methylation, expression and glucocorticoid induction of endogenous and transfected proviral genes of mouse mammary tumor virus in mouse fibroblasts.

A sequence of mouse genomic DNA containing an endogenous mouse mammary tumor virus (MMTV) provirus, which was isolated by molecular cloning in lambda vector phage, has been reintroduced into cultured mouse L cells, using the thymidine-kinase cotransfection technique. Individual cell clones that acquired the transfected MMTV proviral DNA have been isolated. The transfected DNA remains stable in these cells and does not become methylated. It is transcribed into MMTV RNA, And the transcription is stimulated by glucocorticoid hormones.

Animals↗
PubMed1981

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.

Animals↗
PubMed1980

Quantification of mouse mammary tumor virus structural proteins in hormone-induced mammary tumors of low mammary tumor mouse strains.

The expression of the mouse mammary tumor virus (MMTV) in hormone-induced mammary tumors was investigated by means of a radioimmunoassay for two major MMTV proteins, gp52 and p27. MMTV proteins were isolated on lectin affinity- and ion-exchange chromatography columns. The purified viral proteins were electrophoretically homogeneous and retained immunoreactivity after labelling with 125iodine. Standard competition assays showed that group-specific antigenic determinants were reacting. Mammary tumors were induced in three strains of mice with a low natural incidence of mammary tumors, C57BL, O20 and C3Hf, by a combined hormone treatment, consisting of hypophyseal isografts and administration of progesterone and estrone. Mammary tumors and mammary glands of hormone-treated animals were extracted and used for competition radioimmunoassays. In general, the tumorigenic hormone treatment resulted in enhanced amounts of MMTV proteins in the mammary glands, compared to the amounts found in lactating mammary glands of untreated animals. The levels of MMTV proteins in the mammary tumors were lower than in the mammary glands.

Animals↗
PubMed1980

Analysis of secondary modifications of mouse mammary tumor virus proteins by two-dimensional gel electrophoresis.

The structural proteins of mouse mammary tumor virus (MMTV) were analyzed by two-dimensional electrophoresis on isoelectric focusing and sodium dodecyl sulfate gels. Many of the viral proteins displayed heterogeneity in charge due to variable contents of carbohydrates (in particular, sialic acid) and phosphate residues. Neuraminidase treatment of the virions influenced the isoelectric pattern of the envelope glycoproteins. The glycoproteins of an MMTV variant which was attenuated by replication in feline kidney cells had different isoelectric points. This suggested that the acquisition of an altered carbohydrate configuration had changed the host range of the virus. The major MMTV structural core protein, p27, consisted of two species, which had identical iodinated tryptic peptide compositions but differed in phosphate contents. Another MMTV phosphoprotein, p21, was separated into four different phosphorylated species. Phosphorylation of p21 could be performed in vitro by the MMTV virion-associated protein kinase. This enzyme also has a high affinity for MMTV p30 as a substrate. Possible functions of this enzyme are discussed.

Electrophoresis, Polyacrylamide Gel↗
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↗