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Biomedical subjects

M R Banerjee

Publications and source records attributed to M R Banerjee.

At least 37 records · Page 2Linked to original sources
PubMed1981

Cloning of mouse beta-casein gene sequences.

Casein messenger RNAs (mRNAcsn) were purified from lactating mammary glands of BALB/c mice and used as a starting material for cloning of casein gene sequences. Double-stranded casein cDNA (ds-cDNAcsn) was prepared and blunt-end ligated to HindIII-specific DNA linker molecules. After digestion with HindIII, the dsDNAcsn was inserted into the HindIII site of plasmid pBR322, using T4 DNA ligase. Escherichia coli strain RH202 was transformed with the hybrid plasmids, and transformants were selected for resistance to ampicillin. Electrophoresis of HindIII-digested hybrid plasmid DNAs, followed by Southern transfer and hybridization to [32P]cDNAcsn, revealed that one of the hybrid-plasmid-containing colonies, designated pCas51, contained a 400-bp insert which hybridized to the [32P]cDNAcsn. Purification of the individual casein mRNAs (mRNAcsn alpha, beta, and gamma) and solution hybridization of nick-translated insert DNA to each of these revealed that pCas51 contained sequences complementary primarily to mRNAcsn beta.

Animals↗
PubMed1981

Sequential expression of preneoplastic and neoplastic characteristics of mouse mammary epithelial cells transformed in organ culture.

Epithelial cells transformed by 7,12-dimethylbenz[a]anthracene (DMBA) in cultures of whole mammary organs of inbred BALB/cAnN female mice were characterized. Nodule-like alveolar lesions (NLAL) induced by DMBA in the mammary gland in vitro after transplantation into gland-free mammary fat pads of syngeneic hosts produced 22 of 44 hyperplastic alveolar outgrowths, and 7 produced mammary carcinomas in the first generation. Enzymatically dissociated mammary cells from glands containing NLAL but no normal alveoli produced 8 hyperplastic outgrowths; 1 of these outgrowths was tumorigenic. Dissociated cells from DMBA-treated glands containing the lobuloalveolar structures produced 26 of 48 hyperplastic alveolar outgrowths, and 8 of these were tumorigenic. Twelve lines of these mammary hyperplasias produced hyperplastic alveolar outgrowths and mammary tumors in a sequential manner up to 8-20 transplant generations. Thus the sequential stages of mammary cells transformed in organ culture are similar to the sequential stages of preneoplasia to neoplasia associated with murine mammary carcinogenesis in vivo.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1980

Absolute requirement of glucocorticoid for expression of the casein gene in the presence of prolactin.

Second thoracic mammary glands of immature BALB/c female mice were stimulated to pregnancy-like lobuloalveolar (LA) development aftr 6 days of incubation in a corticosteroid-free step I culture medium containing insulin, prolactin, estradiol, progesterone, and growth hormone. A low basal level (0.0009%) of casein mRNA (mRNAcsn) sequences was detectable in the LA glands by a specific cDNA probe. Subsequent incubation of the LA glands for 3 days in medium containing insulin and prolactin or insulin and cortisol failed to elicit mRNAcsn above the basal level, indicating that neither prolactin nor cortisol alone can support casein gene expression. However, an increase in mRNAcsn levels was observed when the 3-day incubation with insulin and cortisol or insulin and prolactin was followed by 3 days of culture in presence of insulin, prolactin, and cortisol. When a 3-day incubation with insulin and prolactin was followed by 3 days in insulin and cortisol medium, mRNAcsn levels in the gland remained similar to the basal level. However, a 20-fold increase in the mRNAcsn levels ensued when the LA glands were sequentially incubated for 3 days in insulin and cortisol and then for another 3 days in insulin and prolactin medium. After a preincubation in insulin and cortisol medium, the LA glands retained residual cortisol during subsequent incubation in insulin and prolactin medium, and the mRNAcsn levels in these glands were related to the level of residual cortisol present. When mRNAcsn and the residual cortisol level reached a minimum, addition of fresh cortisol to the medium caused a 20-fold increase in the mRNAcsn levels. This indicates that cortisol is a limiting factor in insulin and prolactin medium and its presence is absolutely required for casein gene expression.

Animals↗
PubMed1980

Identification of somatomedin-like polypeptides produced by mammary tumors of BALB/c mice.

A transplantable mammary tumor derived from an outgrowth of nodule-like alveolar lesions induced by 7,12-dimethylbenz[a]anthracene in cultures of whole mammary gland was shown to produce a family of somatomedin-like polypeptides when cultured in vitro. Minced mammary tumor tissue as well as monolayer cultures of tumor cells produced similar polypeptides when incubated in serum- and hormone-free medium. The polypeptides released into the medium ranged in molecular weight from 20,000 to 7,000 as determined by Sephadex G-50 chromatography under acidic conditions and analytical gel electrophoresis in acetic acid:urea. Because of their relative abundance, the M.W. 7000 polypeptides were partially purified and characterized. This preparation markedly stimulated DNA synthesis in chicken embryo fibroblast cultures and competed for the binding of 125I-labeled multiplication-stimulating activity to these cells. The binding of 125I-labeled epidermal growth factor to the surface of mouse embryo fibroblasts, in contrast, was not affected.

Animals↗
PubMed1979

Glucocorticoid modulation of prolactin receptors on mammary cells of lactating mice.

Prolactin receptors were monitored by measuring 125I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (Kd) of 0.99 . 10(-9) M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydrocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (Kd) of the receptors.

Adrenal Glands↗
PubMed1979

Neoplastic transformation of epithelial cells in whole mammary gland in vitro.

Biological characteristics of nodule-like alveolar lesions (NLAL) induced by 7,12-dimethylbenz[a]anthracene (DMBA) in organ culture of whole mammary gland (BALB/c female mice) were assessed after transplantation into gland-free mammary fat pads of syngeneic virgin mice. (i) Tissue-fragment explants from NLAL areas of the gland produced abnormal lobuloalveolar (LA) outgrowths in 3 of 10 fat pads. (ii) Transplantation of dissociated cells of NLAL-derived LA outgrowths into 36 fat pads showed 100% LA outgrowths and 3 (8%) of these 36 outgrowths produced mammary carcinomas. (iii) The explants of dissociated cells from whole mammary glands treated with DMBA in culture produced full or partial LA structures in 2 of 56 outgrowths. (iv) The explants of dissociated cells prepared from outgrowths derived from outgrowths derived from explants as in iii produced 9 LA outgrowths in 16 instances; mammary tumor incidence in these outgrowths was 3 of 16 (18%). (v) The explants of tissue fragments from LA outgrowths as in iv produced LA outgrowths in 20 of 20 fat pads; mammary carcinomas appeared in 16 of 20 (80%) of these outgrowths. No NLAL was detectable in control glands treated with dimethyl sulfoxide (solvent for DMBA); explants of the control glands consistently produced ductal outgrowths and no tumor. This accomplishment of chemical carcinogen-induced neoplastic transformation of epithelial cells in vitro provides a model for studying carcinogenesis in an entire isolated organ.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1979

Glucocorticoid modulation of casein gene transcription in mouse mammary gland.

The influence of cortisol and prolactin on casein gene expression in the mammary gland of lactating BALB/c mice was measured by using a specific cDNA probe to 15S casein mRNA (cDNAcsn). Casein mRNA (mRNAcsn) level in the mammary gland was decreased by 85% 5 days after adrenal ablation, but then was increased 4.4-fold 12 hr after a single injection of hydrocortisone-21-acetate. An 80% decrease in serum prolactin level, induced by the prolactin inhibitor 2-bromo-alpha-ergocryptin (CB-154), did not alter the level of mRNAcsn in the gland. Specific transcription of the casein gene in nuclei isolated from lactating mammary glands was measured by cDNAcsn hybridization to the in vitro synthesized Hg-CTP-containing RNA (Hg-RNA), which was purified by SH-agarose chromatography. The level of the mRNAcsn in Hg-RNA synthesized in the isolated nuclei was 0.09% and this was decreased 85% by alpha-amanitin, indicating that the mRNAcsn sequences in the Hg-RNA were the products of RNA polymerase II-directed DNA-dependent RNA synthesis. Transcription of the mRNAcsn in isolated nuclei was decreased by 70% 5 days after adrenalectomy and a single injection of the glucocorticoid then increased the transcription level 2-fold at 6 hr. Essentially no alteration of the level of transcription was detectable in mammary nuclei isolated from lactating mice with 80% decreased serum prolactin level, induced by CB-154 treatment. The results thus demonstrate a glucocorticoid involvement on the modulation of casein gene expression at the transcriptional level of control.

Adrenalectomy↗
PubMed1979

The influence of chromatin structure on the distribution of DNA repair synthesis studied by nuclease digestion.

The influence of chromatin structure on the distribution of DNA repair synthesis was studied by enzymatic digestion of "repair labeled" nuclei of mouse mammary cells: "repair labeled" nuclei were isolated from pregnancy mammary tissue fragments, treated in vitro with methylmethanesulfonate (MMS) or methylnitrosourea (MNU), and pulse-labeled with 3H-thymidine in the presence of hydroxyurea in the culture medium. Micrococcal nuclease digestion of "repair labeled" nuclei indicates that at early hours after treatment with the alkylating agents 70-80% of the total repair synthesis is located in the linker portion of the nucleosome. However, 6-12 hours after treatment DNA repair synthesis is more evenly distributed throughout the core and linker portion of the nucleosome. "Repair labeled" mammary cell nuclei were also digested with DNase I under conditions selective for transcriptionally active chromatin. A two-fold higher level of repair synthesis was found in the transcriptionally active chromatin of "repair labeled" nuclei isolated from MMS or MNU treated mammary fragments, pulse-labeled at different times after treatment. The results indicate that structural constitution of the chromatin may influence the distribution of DNA repair synthesis both at the nucleosome level, and at higher levels of chromatin organization. This may be due to 1) nonrandom base alkylation in chromatin or 2) areas in chromatin with increased accessibility for the repair enzymes to the alkylated bases.

Animals↗
PubMed1978

Prolactin receptor on dissociated mammary epithelial cells at different stages of development.

Properties of prolactin receptors were measured by monitoring [125I]prolactin binding to specific receptor sites on collagenase-dissociated mammary epithelial cells of virgin, pregnant and lactating mice. On a Scatchard plot the data generated a straight line and the estimated dissociation constant (Kd) and number of receptor sites on lactating cells were 0.9 x 10(-9) and 1540 per cell. The [125I]prolactin binding was inhibited in presence of unlabeled prolactin and other lactogenic polypeptide hormones, but not by nonlactogenic polypeptide hormones. The [125I]prolactin binding was sensitive to pronase and trypsin but not to DNAase, RNAase and hyaluronidase. Scatchard plot analysis further showed that while the number of receptors on mammary cells was variable at different stages of endocrine regulated developmental changes of the gland, Kd of the hormone--receptor complex generally remained similar. The high level of prolactin receptors on mammary cells of virgins was reduced during pregnancy and the lactating mammary cells showed a highly elevated level of prolactin receptors. The results demonstrate that specific prolactin receptors can be measured on collagenase dissociated mammary epithelial cells and this method permits a direct assessment of the number of receptors on a per cell basis rather than indirect estimates, based on average DNA or protein content of the tissue, composed of heterogeneous cell types.

Animals↗
PubMed1978

RNA synthesis in isolated nuclei of lactating mammary cells in presence of unmodified and mercury-labeled CTP.

Isolated nuclei of lactating mouse mammary gland were capable of supporting DNA-dependent RNA synthesis in vitro in presence of unmodified and mercurated CTP (Hg-CTP) at high ionic condition at 25 degrees C. In presence of unmodified CTP, [3H]UMP incorporation into RNA increased linearly upto 180 min. The kinetic pattern of the reaction and the rate of RNA synthesis were essentially similar when CTP was replaced by Hg-CTP. Both in unmodified and Hg-CTP containing reactions, 70-80% of RNA synthesis was inhibited by alpha-amanitin. Presence of poly(A) in a small portion of the in vitro synthesized messenger-like RNA was detectable by oligo(dT) cellulose chromatography. Both poly(A)+ and poly(A)- RNAs sedimented with a clear peak around 15S region in a formamide-sucrose denaturing gradient. The Hg-RNA after separation from endogenous nuclear RNA by SH-agarose affinity column chromatography also sedimented around 15S region in a formamide-sucrose gradient. The Hg-RNA synthesized in the isolated mammary cell nuclei in vitro should now permit monitoring hormonal regulation of specific gene (casein) transcription in the mammary cells by molecular hybridization of the Hg-RNA with cDNA to casein mRNA.

Amanitins↗
PubMed1978

DNA repair in normal and preneoplastic mammary tissues.

The purpose of this investigation was to obtain a comparative measure of DNA repair synthesis in hormone-dependent normal mammary tissue and hormone-independent D1 hyperplastic alveolar nodule (HAN) outgrowth. Treatment of mammary fragments in culture with 5 mM hydroxyurea inhibits 98% of the semiconservative DNA synthesis. Treatment of mammary fragments with methyl methanesulfonate in the presence of hydroxyurea results in a 4- to 7-fold higher incorporation of [3H]deoxythymidine into the mammary cell DNA than does treatment with hydroxyurea alone. This hydroxyurea-resistant alkylating agent-induced [3H]deoxythymidine incorporation was studied by CsCl density gradient centrifugation and has been found to represent DNA repair replication. Similar levels of repair replication were found in both normal and preneoplastic D1-HAN outgrowth. Autoradiographic analysis of mammary fragments and D1-HAN outgrowth treated with methyl methanesulfonate plus hydroxyurea revealed that 30 to 50% of the epithelial cell nuclei were lightly labeled. No detectable repair synthesis was found in fat or stromal cells. The average number of grains per labeled nuclei was the same for both explants. These results suggest that a reduced DNA repair capacity is not associated with the increased sensitivity of D1-HAN outgrowth to the tumorigenic effect of chemical carcinogens.

Animals↗
PubMed1977

Hormone-inducible casein messenger RNA in a serum-free organ culture of whole mammary gland.

The whole second thoracic mammary gland of estradiol-17beta + progesterone primed 3- to 4-week-old BALB/c female mice was induced to pregnancy-like lobulo-alveolar morphogenesis after 6-day cultivation in a serum-free culture medium containing a "growth promoting" hormone mixture, insulin + prolactin + growth hormone (somatotropin) + estradiol + progesterone. No radioimmunologically detectable casein was present in these glands. Subsequent cultivation for another 6 days in a "lactogenic" medium with the hormones insulin + prolactin + cortisol produced abundant milk-like secretory material in the alveolar lumen. RNA of the mammary gland after estradiol + progesterone priming or cultivation in the "growth-promoting" medium failed to show a measurable amount of casein mRNA activity when assayed in a cell-free protein synthesis system derived from Ehrlich ascites ribosomes, rabbit reticulocyte factors, and tRNA. However, the glands sequentially cultivated in the "growth-promoting" and the "lactogenic" media showed a high level of casein mRNA activity in the heterologous cell-free protein synthesis system. Sodium dodecyl sulfate/polyacrylamide gel electrophoretic characteristics of the immunoprecipitable (by antibody to mouse milk casein) polypeptides directed by the mammary RNA induced in organ culture medium containing the lactogenic hormones were similar to the characteristics of the polypeptides directed by mammary polysomes of lactating mice. These results demonstrate hormonal induction of a specific mRNA in a sequential two-step culture of an entire organ in a serum-free chemically defined medium.

Animals↗