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N H Sarkar

Publications and source records attributed to N H Sarkar.

At least 37 records · Page 2Linked to original sources
PubMed1985

Molecular cloning, characterization, and genetic mapping of an endogenous murine mammary tumor virus proviral unit I of C3H/He mice.

We have cloned and characterized a novel endogenous murine mammary tumor virus proviral unit of the C3H/He strain of mice. The cloned proviral unit is 16 kilobase pairs (kbp) in size and is composed of a 5.6-kbp 5' EcoRI segment of an endogenous provirus with 10.4-kbp flanking cellular sequences. A comparison of the restriction map of the cloned proviral DNA with that of an endogenous provirus of the GR strain of mice has revealed minor differences in restriction sites on the two proviruses. The restriction enzyme SstI, which does not cleave the 5' EcoRI fragment of GR DNA, cleaves the C3H/He proviral sequences once; MspI has an additional site in the C3H/He proviral sequences. By using a subcloned fragment containing unique cellular sequences as a hybridization probe, we (i) mapped the C3H/He proviral unit to chromosome 14 by using mouse-hamster somatic cell hybrids, and (ii) demonstrated that this proviral unit is also present in the genome of DBA/2 mice. From these results we conclude that the C3H/He strain of mice acquired this proviral unit from DBA stock by genetic transmission. Our data also indicate that the murine mammary tumor virus sequences present in the gag-specific proviral unit of C3H/He mice extend at least 2.45 kbp downstream of the EcoRI site in the genomic DNA. Since the structural organization and chromosomal location of this proviral unit are distinct from those of previously reported proviral units represented by similar-sized (16.7-kbp) EcoRI fragments, we tentatively propose to designate this proviral unit Mtv-7a.

Animals↗
PubMed1984

Expression of an 86-kilodalton glycoprotein in an idiopathic mammary adenocarcinoma of a BALB/c mouse.

An epithelial cell line (BALB/c-ST) was established from a mammary adenocarcinoma that developed spontaneously in a 20-month-old BALB/c mouse. Like uninfected normal tissues, the cell line was found to contain three endogeneous murine mammary tumor virus (MuMTV) proviruses, but MuMTV particles and antigens were not detected. The cultured cells, after being inoculated subcutaneously, produced tumors in syngeneic mice, and sera from a high percentage of the tumor-bearing mice specifically immunoprecipitated an 86-kilodalton antigen from the extracts of BALB/c-ST cells. This antigen was found to be glycosylated, but whether or not it was exposed to the cell surface could not be demonstrated by cell-surface iodination and immunoprecipitation studies. The 86-kilodalton antigen, a glycoprotein designated gp86, was not detected in immunoprecipitates from extracts of normal mammary cells or from the extracts of GR, C3H, and BALB/cfC3H mammary tumor cells. After being infected in vitro with RIII-derived MuMTV but not with C3H-derived MuMTV, the BALB/c-ST cells appeared to undergo a phenotypic change in that they did not produce tumors in syngeneic mice, and the expression of gp86 was inhibited. Our results indicate that the expression of gp86 in the mammary cells of BALB/c mice is a consequence of neoplastic transformation and that MuMTV infection modulates its expression.

Adenocarcinoma↗
PubMed1984

Fatty acid-induced modifications of mouse mammary alveolar lesions in organ culture.

A newly developed adult RIII mouse mammary gland organ culture system was used to examine the effects of fatty acids and a prostaglandin synthase inhibitor on the survival and/or growth of lactogenic hormone-independent mammary alveolar lesions (MAL). The number of MAL per gland that persisted in the absence of lactogenic hormones was increased in cultures treated with arachidonic acid (24.0 +/- 3.4), and was decreased after treatment with stearic acid (4.0 +/- 3.4) or indomethacin (5.0 +/- 0.6). Arachidonic acid also induced higher incorporation of [3H]thymidine into the cellular DNA of MAL containing glands. A comparison of [3H]arachidonic acid uptake into intact (MAL containing) glands and parenchyma-free fat pads revealed a selectively higher incorporation of the labeled fatty acid in the intact glands. Arachidonic acid exposure produced higher cumulative amounts (4.02 ng) of prostaglandin E2 (PGE2) which was significantly inhibited (1.66 ng) by indomethacin (p less than 0.001). In contrast, exposure to stearic acid did not lower PGE2 levels below that of controls. A concomitant increase in MAL number and PGE2 production by arachidonic acid and a decrease in both caused by indomethacin suggest that arachidonic acid conversion to PGE2 may have a facilitative role in the survival of MAL.

Animals↗
PubMed1983

Expression and disposition of the murine mammary tumor virus (MuMTV) envelope gene products by murine mammary tumor cells.

Three murine mammary tumor virus (MuMTV)-producing epithelial cell lines derived from murine mammary tumors were examined in order to identify the MuMTV-specific cell surface antigens and their distribution on the cell surface, to study the kinetics of the MuMTV envelope precursor processing, virus assembly, and release, and to characterize the soluble MuMTV antigens that are shed into culture medium. Cell surface labeling experiments showed that only the mature MuMTV envelope glycoproteins gp52 and gp36 were exposed on the cell surface, and that gp52 was more abundant than gp36. In cells producing large quantities of MuMTV, expression of gp52 on the cell surface was shown by immunoelectron microscopy to be localized predominantly on the surface of budding virions and not on smooth areas of the cell surface where virus was not budding. The cell surface associated gp36 was found not to be incorporated into budding virions. A few cells in all three cell lines were found to produce only a few or no MuMTV particles and in these cells, unlike in the high virus-producing cells, considerable quantities of gp52 were expressed on the surface membrane. All three cell lines were found to shed large amounts of the MuMTV env precursor polyprotein as well as the mature non-virion-associated glycoprotein, gp52, into the culture medium. The envelope precursor protein (P75env) that was shed into the culture medium was found to differ from the predominant form of the cellular env precursor (Pr70env) in that (1) P75env migrated with an apparent higher molecular weight than Pr70env in SDS gels; (2) Pr70env contained only the core oligosaccharide, whereas P75env contained fucose in addition to the core sugars; (3) two-dimensional gel electrophoretic analysis showed that Pr70env could be resolved into three to four components migrating in the basic region of the isoelectric focussing gel (pH 7-8), whereas P75env was resolved into 9-13 components migrating in a more acidic region of the gel (pH 5-7). The molecular structure of the exfoliated gp52 was found to be similar to that of the gp52 that was incorporated into the virions although the virion-associated gp52 was not the source of the gp52 in the medium. Our quantitative pulse-chase studies suggest that of the two populations of MuMTV env precursors that are present in MuMTV-producing cells, only Pr70env is processed intracellularly to give rise to the mature MuMTV envelope proteins gp52 and gp36.

Animals↗
PubMed1983

Murine mammary tumor virus related antigen in human male mammary carcinoma.

An antigen immunologically related to mouse mammary tumor virus (MuMTV) and the major envelope glycoprotein, gp52 of MuMTV, was identified in tissue sections of human male and female mammary carcinomas using the peroxidase-antiperoxidase technique. The specificity of the reaction was established by absorption studies. Positive reactions with the gp52 antiserum were seen in mouse and human mammary carcinomas, but not in normal mammary tissues, mammary tissues with benign diseases and in other primary malignant neoplasms. Almost all (32/36, 89%) male mammary carcinomas were positive for the gp52 related antigen. A lesser proportion of tumors among female patients (14/50, 28%) were positive. The gp52 positive tumors were significantly larger than the gp52 negative tumors in female patients (P less than 0.05). Gp52 positive tumors were also larger than gp52 negative tumors in male patients, but the difference was not statistically significant. Gp52 reactivity was also detected in metastatic mammary carcinoma in axillary lymph nodes of male and female patients. The presence of gp52 related antigen was not apparently related to tumor grade or lymphocytic infiltrate in the primary tumor. The data do not permit a firm conclusion regarding nodal status in men; no correlation of gp52 activity and nodal status in women was evident. These results indicate that mammary carcinomas in men as well as in women have an antigen related immunologically to MuMTV gp52. Other than tumor size, the antigen seems to be unrelated to major prognostic factors. The significance of the antigen with respect to etiologic features and prognosis in breast cancer remains to be determined.

Adenocarcinoma↗
PubMed1983

Strain-specific determinants on the major phosphoprotein of murine mammary tumor viruses.

The major phosphoprotein (p23) of murine mammary tumor virus (MuMTV) was purified for C3H virus, using a combination of alkyl agarose chromatography and gel filtration. Monospecific antiserum was raised against glutaraldehyde crosslinked polymers of purified p23, and the antiserum was then used with iodinated p23 to develop a competition radioimmunoassay. Phosphoprotein p23 was shown to contain both group- and type-specific antigenic determinants in assays using detergent-disrupted virions of MuMTV strains C3H, RIII, GR, and DBA/2 as competing antigens. Tryptic and chymotryptic peptide mapping analysis of p23 polypeptides isolated from MuMTV strains C3H, RIII, GR and DBA/2 showed that the p23s of each virus strain could be clearly distinguished from each other.

Antigens, Viral↗
PubMed1983

Effect of malignant transformation upon the cellular retinoid binding proteins in cultured murine mammary cells.

Cellular retinol (CRBP) and retinoic acid binding proteins (CRABP) were measured in normal (C57Bl) and Murine Mammary Tumor Virus (MuMTV)-induced murine mammary tumor cells (C57BlfRIII) grown in monolayer culture. High speed supernatant fractions (cytosols) from transformed cells contained elevated levels of CRBP and CRABP (0.276 and 1.410 pmol/mg protein, respectively) compared to cytosol from non-transformed murine mammary cells which contained only low levels of CRBP (0.099 pmol/mg protein). Our findings suggest that malignant transformation of mammary epithelial cells, induced by MuMTV infection, results in changes in the expression of cellular retinoid binding proteins and possibly in the sensitivity of these cells to various retinoids.

Animals↗
PubMed1983

Evidence for a prokaryotic promoter in the murine mammary tumor virus long terminal repeat.

The long terminal repeat (LTR) of C3H murine mammary tumor virus (MuMTV) is approx. 1.3 kb long. HaeIII digestion of a cloned PstI fragment containing the left-end LTR generated four fragments of sizes 0.56, 0.41, 0.34 and 0.14 kb, one of which (0.41 kb) had a promoter activity in Escherichia coli. This was demonstrated by replacing the bacterial promoter for the neomycin-resistance (NmR) gene in the plasmid pKC56 with the HaeIII fragments. Only the 0.41-kb fragment that contains sequences from the U3 region of the LTR was found to contain a promoter, as shown by the expression of the drug-resistance phenotype in the recombinant plasmid. The strength of this promoter was comparable to or greater than that found with the parental NmR gene promoter. S1 nuclease mapping of the NmR gene transcript indicated that the initiation of this transcript occurred within the 0.41-kb LTR fragment from a site approx. 10 bp upstream from the 3' end. A comparison of the known DNA sequences in the MuMTV LTR with those found in bacterial promoters revealed that a 'Pribnow box', the initiation signal for the prokaryotic promoters, is present in the 0.41-kb LTR fragment upstream from the initiation site. Furthermore, in a recombinant plasmid that contained the complete LTR the same promoter sequences appeared to be involved in the initiation of RNA transcription. The 0.34-kb LTR fragment, which contains sequences derived from the U3 and U5 regions of the LTR, did not possess promoter activity in E. coli. However, it was found to induce deletions of adjacent plasmid DNA sequences. The deletions were specifically initiated from the downstream end of the LTR-fragment insert. The presence of a prokaryotic promoter in the MuMTV LTR, together with the observation that certain LTR sequences can induce deletions, analogous to those caused by transposable elements, in recombinant plasmids suggest that the MuMTV LTR may have evolved from such elements.

Base Sequence↗
PubMed1983

Integration of new endogenous mouse mammary tumor virus proviral DNA at common sites in the DNA of mammary tumors of C3Hf mice and hypomethylation of the endogenous mouse mammary tumor virus proviral DNA in C3Hf mammary tumors and spleens.

To understand the molecular mechanisms by which the endogenous murine mammary tumor virus (MuMTV) proviruses are expressed and produce late-occurring mammary tumors in C3Hf mice, we analyzed, by the use of restriction enzymes and the Southern transfer procedure, genomic DNA from normal organs of mammary tumor-bearing and tumor-free mice and from 12 late-occurring C3Hf mammary tumors. We found, by using the restriction enzymes EcoRI and HindIII, that in addition to the preexisting endogenous MuMTV proviruses, new MuMTV-specific proviral DNA was integrated into new sites in the host genome in all 12 of the tumors that we examined. PstI digests of C3Hf tumor DNA revealed that the new proviral DNA found in C3Hf tumors was of endogenous origin. Moreover, the respective sizes of at least one of the new DNA fragments generated by EcoRI or HindIII digestion were the same in at least 50% of the C3Hf tumors analyzed, suggesting that the integration site of this new proviral DNA could be at the same location in the host genome of these tumors. Our results may imply that mammary tumorigenesis in C3Hf mice results from activation of cellular oncogenes by an MuMTV proviral DNA promoter. Specific hypomethylation of MuMTV proviral DNA was detected in the mammary tumors and spleens of C3Hf tumor-bearing mice. Our results indicated that most, if not all, of the hypomethylated MuMTV proviral DNA sequences were derived from the endogenous MuMTV provirus located at the MTV-1 locus, a locus responsible for the production of MuMTV antigens and increased incidence of mammary carcinoma in C3Hf mice. In spleens of non-tumor-bearing mice of ages 3, 6, 9, and 12 months, there was progressive hypomethylation of proviral DNA with increasing age, suggesting a possible correlation between demethylation of MuMTV proviral DNA in the spleens of C3Hf mice and the expression of endogenous MuMTV.

Aging↗
PubMed1982

Prostaglandin regulation of murine mammary tumor virus production: a basis for some of the glucocorticoid and prolactin actions on mammary tumor cell cultures.

Hormonal regulation of mouse mammary tumor virus (MuMTV) production has been studied in cell cultures derived from mammary carcinomas of GR mice. The purpose of this study was to define the role of prolactin in dexamethasone-induced MuMTV production and to evaluate the mechanism(s) of action of these hormones. Results of our investigations in vitro establish a central role for prostaglandins, in particular PGF2 alpha. Prolactin treatment up to 1000 ng/ml increased the MuMTV production only slightly when added alone but a maximal stimulatory response was expressed when prolactin was added simultaneously with dexamethasone 2 micrograms/ml. This prolactin-related enhancement of MuMTV production was probably mediated by PGs since it was inhibited by indomethacin, an inhibitor of prostaglandin synthesis. When higher concentrations of indomethacin were used in dexamethasone-treated cultures, MuMTV production, although inhibited, was restored in part by addition of exogeneous PGF2 alpha. PGF2 alpha added alone stimulated the MuMTV production and, in combination with dexamethasone, showed a synergistic effect on stimulation of MuMTV production. Two compounds, PGF2 alpha and TXA2 (measured as TXB2), were produced in significant concentrations by these cultures. These findings suggest that PGF2 alpha exerts a regulating role on production of MuMTV in GR cells and indicate that the actions of both dexamethasone and prolactin are mediated at least in part by prostaglandins.

Animals↗
PubMed1982

Low-calorie diet prevents the development of mammary tumors in C3H mice and reduces circulating prolactin level, murine mammary tumor virus expression, and proliferation of mammary alveolar cells.

The effect of carlorie intake on the development of spontaneous mammary tumors in virgin C3H mice was studied. Only about 10% of the mice fed a low-calorie diet [10 kcal/day (1 kcal = 4.184 kJ)] since weaning developed mammary tumors, compared to about 60% of those mice that were reared on high-calorie diets (16 kcal/day or lab chow ad lib). In order to understand the mechanism by which a low-calorie diet decreases the occurrence of mammary tumors in mice, we compared the sex cycle, the amounts of circulating thyroid-stimulating hormone (thyrotropin), growth hormone, and prolactin, the production of type A and B virus particles in the mammary glands, and the morphology of the mammary glands of mice fed low- and high-calorie diets. The amount of serum prolactin and the synthesis of type A and B particles in mammary tissues of mice fed a low-calorie diet was markedly decreased compared to those of age-matched mice fed high-calorie diets. In addition, in young mice fed a low-calorie diet, there were fewer mammary alveolar lesions than in mice fed a high-calorie diet, although the size of the lesions was similar. However, in older mice fed the high-calorie diet, the number and size of these lesions were greater than in the mice raised on the low-calorie diet. The other factors that we studied were not affected by calorie restriction. Our findings suggest that the reduction in serum prolactin level, mammary tumor virus production, and proliferation of mammary alveolar lesions associated with dietary calorie restriction is responsible for lowering the incidence of mammary tumors in mice.

Animals↗
PubMed1982

Restriction endonuclease mapping of the proviral DNA of the exogenous RIII murine mammary tumor virus.

Cellular DNA containing integrated murine mammary tumor virus (MuMTV) was isolated from FeI/C6 feline kidney cells and CCL64 mink lung cells infected with milkborne RIII MuMTV. By using restriction enzyme HpaI, intact RIII MuMTV provirus (length, 8.7 kilobases [kb]) was excised from the cellular DNA. Subsequent restriction endonuclease analysis of this HpaI fragment with KpnI, HindIII, EcoRI, BamHI, BglII, PstI, SstI, SalI, and XhoI enabled us to construct a map of the RIII virus genome. A comparison of this map with the maps of the GR and C3H MuMTV's revealed that there are greater sequence differences between the RIII virus and the GR and C3H MuMTV proviruses than there are between the GR and C3H proviruses. The following are features of the restriction map unique to the RIII provirus: the presence of three BamHI and two EcoRI cleavage sites, a HpaI cleavage site in the terminal 3'-5' repeat unit of the provirus, and the absence of an XhoI cleavage site. Another distinguishing feature of the RIII provirus is that the sizes of some of the restriction fragments produced by cleavage of the RIII provirus with PstI are different from the sizes of the fragments obtained by PstI cleavage of the GR and C3H proviruses. Like the GR proviral DNA, the RIII proviral DNA has three SstI (SacI) cleavage sites, whereas the C3H provirus has only two SstI sites. HpaI digestion of MuMTV-infected mink lung cell DNA revealed only one class of provirus (an 8.7-kb fragment); however, we observed several minor classes of RIII proviral DNA in addition to the major class of provirus DNA in infected cat kidney cells. PstI digestion of the HpaI 8.7-kb fragments from both feline and mink cells generated a 3.7-kb DNA fragment identical in size to a PstI-generated fragment that has been found in GR and C3H milkborne virus-infected cells. Although a fragment similar in size to the milkborne 3.7-kb PstI fragment has been found as an endogenous component in many C3H and GR mouse tissues, we did not observe such an endogenous fragment in the RIII mouse strain. Therefore, the 3.7-kb fragment may be useful as a marker for the milkborne RIII MuMTV provirus in RIII mice.

Animals↗
PubMed1982

ML antigen of DBA/2 mouse leukemias: expression of an endogenous murine mammary tumor virus.

Spontaneous, transplantable leukemias of DBA/2 mice express an antigen (ML) which cross-reacts with antigens of murine mammary tumor virus (MuMTV). The MuMTV cross-reactive antigen of the DBA/2 leukemias (ML cells) was found to be a glycoprotein of 78,000 molecular weight containing antigenic determinants of the major MuMTV glycoprotein gp52. No MuMTV particles were produced by the ML cells, although they did contain type A particles--the pronucleocapsids of MuMTV. The ML antigen appeared to be an aberrant form of the intracellular MuMTV env precursor molecular prgp70, which was not processed properly but instead acquired extra carbohydrate groups and was expressed in uncleaved form on the cell surface. Isolation of MuMTV core protein p28 from the leukemic cells and subsequent tryptic peptide mapping analysis showed that the p28 from leukemia cells differed from the p28 of MuMTV isolated from DBA/2 mouse milk. These observations indicate that the MuMTV expressed in DBA/2 leukemic spleen cells is of a different strain than the virus secreted in lactating mammary glands of DBA/2 mice and probably represents the expression of an endogenous DBA/2 provirus.

Animals↗
PubMed1981

Antibodies reactive with murine mammary tumor virus in sera of patients with breast cancer: geographic and family studies.

Sera of patients with breast cancer, of healthy women from the United States, East India, East Africa, and China, and of healthy women of American and Parsi families in which breast cancer occurred in several family members were assayed for levels of antibody reactive with the murine mammary tumor virus (MuMTV) by an enzyme-linked immunosorbent assay. Increased levels of antibody to MuMTV (absorbance greater than or equal to 0.4) were found in sera of 18.6% of American patients with breast cancer and of 2.8% of healthy American women and in 38% of patients from India and 61.9% from East Africa (healthy, 26.9%). In contrast, antibody reactive with MuMTV was found in less than 5 of women with breast cancer from mainland China (healthy Chinese, 5.0%). Differences in serum MuMTV antibody levels between breast cancer patients in the four groups were found to be significant (P less than 0.0001). Studies of two families from the United States and of one Parsi family from India with genetic propensity to breast cancer showed that high levels of antibody to MuMTV were found in 33%, 71%, and 23% of healthy family members, respectively. The antibody to MuMTV was readily absorbed with purified MuMTV and gp52. In contrast, fetal calf serum, murine type c retroviruses, or erythrocytes from various species failed to absorb the antibody.

Animals↗