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1.
Cancer Res ; 53(3): 668-74, 1993 Feb 01.
Article in English | MEDLINE | ID: mdl-8425201

ABSTRACT

The TM preneoplastic mammary outgrowth lines were established in vivo from mammary epithelial cell lines and have been characterized with respect to their tumorigenic, morphological, and functional properties. The TM outgrowth lines were then established as in vitro cell lines. A comparison of the growth factor dependencies of the TM preneoplastic lines and their progenitor cell lines grown in monolayer cell culture indicated that the TM preneoplastic cell lines exhibited a decreased dependence on epidermal growth factor for growth in vitro. The exception to this result was the TM3 cell line which still exhibited a marked dependence on epidermal growth factor for growth. An examination of several genes for mRNA levels indicated that the expression of c-neu, c-H-ras, c-myc, and retinoblastoma was not elevated in those TM preneoplasias which exhibited a decreased dependence on epidermal growth factor. Additionally, there was no evidence that c-H-ras was activated in the preneoplastic outgrowths or tumors. In contrast, mouse mammary tumor virus long terminal repeat mRNA was increased in preneoplasias and tumors, whereas gelsolin mRNA was decreased in tumors but not in preneoplasias. The down-regulation of gelsolin mRNA is consistent with recent reports in human breast cancers. These results together with those reported in another paper (D. Medina et al., Cancer Res., 53: 663-667, 1993) indicate that the TM3 outgrowth line is a minimally deviated preneoplasia which does not share many of the molecular alterations exhibited in tumorigenic TM preneoplastic outgrowth lines. These data, along with other recent data, are interpreted in a hypothesis which views the three essential characteristics of the mammary preneoplastic state as independent and dissociable genetic alterations. Importantly, each of the three characteristics is independently isolated in one or more of the in vivo outgrowth populations. These outgrowth lines should allow identification of the nature and function of the molecular alterations associated with each stage of mammary preneoplasia.


Subject(s)
Growth Substances/pharmacology , Mammary Glands, Animal/pathology , Mammary Glands, Animal/physiology , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Precancerous Conditions/genetics , Precancerous Conditions/pathology , Proto-Oncogenes/genetics , Animals , Base Sequence , Cell Division/drug effects , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Epithelium/pathology , Epithelium/physiology , Female , Genes, myc/genetics , Genes, ras/genetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Tumor Cells, Cultured
2.
Cancer Res ; 52(7): 1924-32, 1992 Apr 01.
Article in English | MEDLINE | ID: mdl-1372532

ABSTRACT

A series of mouse mammary epithelial cell lines has been established by a protocol that gives highly reproducible results. The mammary epithelial cell lines, designated as FSK lines, were judged to be epithelial based on positive immunostaining for keratin-intermediate filaments, negative immunostaining for vimentin-intermediate filaments, hormonal induction of casein, and the ability to exhibit ductal and alveolar mammary morphogenesis in vivo. The FSK cell lines are dependent on epidermal growth factor and insulin in a low serum (1%) medium. Conditioned medium from spindle cell cultures replaced the requirement for serum and increased the growth of FSK3 and FSK4 4-5 times in collagen gels and 12-14 times in monolayer culture, respectively. Following injection into the mammary fat pad at passages 2-11, the FSK cell lines generated stable transplantable hyperplastic alveolar outgrowth lines. The in vivo outgrowth lines were judged as preneoplastic based on their stable alveolar morphology in vivo and an increased susceptibility for tumorigenesis. The FSK cell lines and their derivative in vivo outgrowth lines provide a new and potentially productive system to examine critical molecular alterations involved in the development of mammary preneoplasias. Furthermore, the reproducibility of the in vitro culture system provides the assurance that stable cell lines of mouse mammary epithelial cells can be generated easily and at will.


Subject(s)
Cell Line , Mammary Glands, Animal/cytology , Mammary Neoplasms, Experimental/pathology , Precancerous Conditions/pathology , Adenocarcinoma, Bronchiolo-Alveolar/pathology , Animals , Carcinoma, Intraductal, Noninfiltrating/pathology , Cell Division/drug effects , Culture Techniques , Epidermal Growth Factor/pharmacology , Epithelial Cells , Female , Fluorescent Antibody Technique , Keratins/analysis , Mammary Glands, Animal/pathology , Mice , Mice, Inbred BALB C , Neoplasm Transplantation
3.
Carcinogenesis ; 10(3): 541-6, 1989 Mar.
Article in English | MEDLINE | ID: mdl-2924398

ABSTRACT

Labeling studies with 75selenium (75Se) have suggested the existence of selenium-binding proteins in addition to glutathione peroxidase (GSH-Px) in rodent tissues. Three selenium-binding proteins of apparent mol. wt 56, 14 and 12K on SDS-PAGE were isolated from mouse liver using Sephadex G-150 and DEAE-Sephadex chromatography. The proteins were electroeluted from SDS-PAGE gels and injected into rabbits to elicit antibodies. Western immunoblot experiments indicated that the 56K protein was distinct from the 14 and 12K proteins. The latter two proteins appeared to be immunologically related, perhaps as differentially processed variants. The 56 and 14/12K proteins appeared to be distinct from GSH-Px and the 57K plasma selenium-binding proteins. These results indicate that the mouse liver contains at least two selenium-binding proteins distinct from GSH-Px. The existence of the antibodies should permit experiments which help to examine the role of these proteins in the biological function of selenium in mammals.


Subject(s)
Carrier Proteins/analysis , Glutathione Peroxidase/analysis , Liver/analysis , Selenium/metabolism , Animals , Carrier Proteins/immunology , Carrier Proteins/physiology , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Female , Mice , Mice, Inbred BALB C , Molecular Weight
4.
Cancer Lett ; 43(3): 227-36, 1988 Dec 15.
Article in English | MEDLINE | ID: mdl-3203341

ABSTRACT

Two highly selenite sensitive cell lines with different growth rates were used to evaluate the effect of cell growth phase on selenite retention, selenite distribution, selenite inhibition of DNA synthesis and presence of selenoproteins. Autoradiography of log and confluent phase MOD cells revealed a uniform retention of selenite in log phase cells and a marked lack of uniformity of selenite retention in confluent phase cells. A higher total percentage of selenite was retained and covalently incorporated into proteins by confluent phase cells. Levels of the 58K selenoprotein, but not the 26K and 23K selenoproteins, were higher in confluent versus log phase cells. The results suggest that the 58K selenoprotein accumulated in cell populations where DNA synthesis was inhibited in contrast to cells actively undergoing cell proliferation. In addition, the 58K selenoprotein was the only major selenoprotein present in both log and confluent phase cells during and before inhibition of DNA synthesis. The implications of these results are discussed in terms of potential combination chemoprevention protocols in animal tumor experiments.


Subject(s)
Mammary Neoplasms, Experimental/pathology , Proteins/analysis , Selenium/pharmacology , Animals , Autoradiography , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line/drug effects , DNA, Neoplasm/biosynthesis , Electrophoresis, Polyacrylamide Gel , Mammary Neoplasms, Experimental/metabolism , Mice , Selenious Acid , Selenoproteins
5.
Anticancer Res ; 8(1): 65-71, 1988.
Article in English | MEDLINE | ID: mdl-3358640

ABSTRACT

The effect of selenite on mitochondrial morphology, selenoprotein synthesis, membrane potential, phosphorylating respiration, P/O ratio and respiratory control index were evaluated relative to selenite inhibition of DNA synthesis in murine mammary epithelial cell lines. No inhibition of mitochondrial function was noted. Mitochondrial inclusions and most mitochondrial specific selenoproteins were not observed until after DNA synthesis was inhibited. Pyruvate, methionine, hydroxybutyrate, dexamethasone or carnitine had no effect on the inclusions. The results provide evidence for the hypothesis emphasizing the importance of cytosolic selenoproteins modulating the growth inhibitory effects of selenite.


Subject(s)
Mammary Glands, Animal/metabolism , Mitochondria/metabolism , Proteins , Selenium/pharmacology , Animals , Carnitine/metabolism , Cell Line , DNA/biosynthesis , Dexamethasone/pharmacology , Hydroxybutyrates/metabolism , Intracellular Membranes/drug effects , Membrane Potentials/drug effects , Methionine/metabolism , Mice , Mitochondria/drug effects , Molecular Weight , Oxidative Phosphorylation/drug effects , Oxygen Consumption/drug effects , Protein Biosynthesis , Pyruvates/metabolism , Selenious Acid , Selenoproteins
6.
Anticancer Res ; 8(1): 51-63, 1988.
Article in English | MEDLINE | ID: mdl-2451895

ABSTRACT

The subcellular and macromolecular distribution of 75Se-selenite was determined in murine mammary epithelial cell lines which demonstrated marked differences in their growth response to 5 microM selenite. MOD cells responded sooner to the inhibitory effects of selenite than COMMA-D cells. The MOD cells also incorporated a slightly higher percentage of 75Se-selenite into proteins and attained a higher ratio of selenoprotein to selenonucleic acids than did COMMA-D cells. Most selenium and selenoproteins were located in the cytoplasm as revealed by autoradiography and subcellular fractionations. These data suggest that a cytoplasmic selenoprotein may be an intermediate for selenite's nontoxic growth inhibitory effects.


Subject(s)
Mammary Glands, Animal/cytology , Selenium/metabolism , Animals , Autoradiography , Cell Compartmentation , Cell Division , Cell Line , Chromatin/metabolism , DNA/metabolism , Mammary Glands, Animal/metabolism , Mice , Proteins/metabolism , RNA/metabolism , Selenoproteins , Subcellular Fractions/metabolism
7.
Exp Cell Res ; 172(1): 192-203, 1987 Sep.
Article in English | MEDLINE | ID: mdl-3653254

ABSTRACT

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D line. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of beta-casein mRNA in the presence or absence of prolactin. The heterogeneous COMMA-D line, but none of the clonal lines, was induced by the presence of prolactin to produce significantly increased levels of beta-casein MRNA. The inducibility of beta-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. Individual matrix components of laminin, fibronectin, heparan sulfate, heparan, or hyaluronic acid were not effective as substrata for the induction of beta-casein mRNA. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.


Subject(s)
Caseins/genetics , Genes , Mammary Glands, Animal/metabolism , Transcription, Genetic , Animals , Antigens/analysis , Cell Line , Epithelial Cells , Epithelium/drug effects , Epithelium/metabolism , Female , Immune Sera , Mammary Glands, Animal/cytology , Mice , Prolactin/pharmacology , RNA, Messenger/analysis , RNA, Messenger/genetics
8.
Cancer Res ; 47(17): 4686-93, 1987 Sep 01.
Article in English | MEDLINE | ID: mdl-3621162

ABSTRACT

The levels of milk-specific mRNAs (alpha-casein, whey acidic protein) and proteins (casein, alpha-lactalbumin) were examined in the DIM series of mammary preneoplastic outgrowth lines and tumors. The constitutive production of casein protein was variable among the preneoplastic DIM outgrowth lines maintained in virgin mice. Outgrowth line DIM-2 consistently expressed a very low level of casein mRNA and protein but no detectable alpha-lactalbumin protein. Outgrowth line DIM-4 expressed a high level of casein protein and no alpha-lactalbumin; however, by transplant generation 9, the levels of casein mRNA and protein were significantly decreased but remained greater than those found in DIM-2. Outgrowth line DIM-3 expressed high levels of casein protein during all transplant generations examined and sporadically exhibited detectable amounts of alpha-lactalbumin. The level of beta-casein mRNA in DIM-3 was 7 times greater than seen in DIM-4 outgrowths, but still only 2% of that measured in the normal mammary gland from lactating mice. The majority of tumors derived from the DIM outgrowth lines expressed very low levels of beta-casein mRNA and total casein protein, although occasionally tumors were observed with very high levels of casein expression. Immunoblot analysis of cellular extracts indicated that alpha, beta-, and gamma-caseins were expressed in the three outgrowth lines to varying degrees. Whey acidic protein mRNA attained barely detectable levels in the best of cases. Only outgrowth line DIM-3 responded to a normal lactogenic stimulus (lactating mouse) with significantly increased levels of milk-specific products. As determined by avidin-biotin peroxidase staining, the percentage of alveoli expressing beta-casein and alpha-lactalbumin proteins in lactating mice increased from 20 and 0%, respectively, in virgin mice to 85 and 40%, respectively, in lactating mice. Similarly, the levels of mRNA for beta-casein and whey acidic protein increased 8- and 5.5-fold, respectively. These results indicate that the cell populations selected by serial transplantation of preneoplastic mammary tissues fall into at least three categories with respect to expression of mammary-specific differentiation products: uninducible, inducible, constitutive. The DIM-3 outgrowth line appears to represent a highly inducible cell population. As concluded in earlier investigations, there was no correlation between secretory activity, morphology of the outgrowths, and tumor-producing capabilities in virgin mice. The effect of a normal lactogenic stimulus on the tumor potential of the DIM-3 line is currently under investigation.


Subject(s)
Mammary Neoplasms, Experimental/analysis , Milk Proteins/analysis , Precancerous Conditions/analysis , Adenocarcinoma/analysis , Animals , Caseins/analysis , Cell Differentiation , Female , Histocytochemistry , Lactalbumin/analysis , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , Milk Proteins/genetics , Precancerous Conditions/pathology , RNA, Messenger/analysis
9.
J Natl Cancer Inst ; 76(6): 1143-56, 1986 Jun.
Article in English | MEDLINE | ID: mdl-2423737

ABSTRACT

Immunocytochemical and in vivo transplantation methods were used to study the characteristics of a series of newly developed mammary epithelial cell lines. These mouse mammary cell lines were derived from mid-pregnant primiparous BALB/c female mice and were routinely grown in Dulbecco's modified Eagle medium supplemented with 1% fetal bovine serum, insulin, transferrin, epidermal growth factor, and selenite. Of the 6 cell lines, 1 cell line, COMMA-D, produced normal and preneoplastic mammary outgrowths when it was transplanted into mammary fat pads of syngeneic mice. One cell line, MOD, produced only mammary adenocarcinomas. The other 4 cell lines, COMMA-F, COMMA-T, MOMA-1, and MOMA-2, produced neither normal nor neoplastic outgrowths. Immunocytochemical staining with polyclonal antibodies to keratin and vimentin intermediate filament proteins revealed that 5 of the 6 cell lines were epithelial. The sixth cell line, MOMA-2, was apparently of fibroblastic origin. The COMMA-D cell line was unique compared to the other cell lines with respect to several characteristics. The cell line was morphologically heterogeneous as determined by phase-contrast microscopy, cytologically heterogeneous as determined by immunocytochemical staining, and heterogeneous with respect to DNA content. Finally, the full morphogenic potential of COMMA-D included not only normal mammary ductal and preneoplastic mammary alveolar outgrowths but also adenocarcinomas and fibrosarcomas. The expression of this morphogenic potential upon transplantation in vivo was drastically diminished after passage 14. The significance of the cellular heterogeneity with respect to expression of mammary-specific morphogenesis is not understood at this time; however, conceivably, the observed heterogeneity reflects an essential requirement for morphogenesis in vivo. The transplantation and immunocytochemical characteristics provide the descriptive basis for further studies on these cell lines to determine the cell lineages involved in morphogenesis and preneoplastic transformation in vivo.


Subject(s)
Mammary Glands, Animal/cytology , Animals , Cell Differentiation , Cell Line , DNA/analysis , Epithelial Cells , Female , Fluorescent Antibody Technique , Guinea Pigs , Histocytochemistry , Immune Sera/immunology , Keratins/analysis , Keratins/immunology , Mammary Glands, Animal/transplantation , Mammary Neoplasms, Experimental/etiology , Mice , Morphogenesis , Precancerous Conditions/etiology , Time Factors , Vimentin/analysis , Vimentin/immunology
10.
Biol Trace Elem Res ; 8(1): 19-35, 1985 Aug.
Article in English | MEDLINE | ID: mdl-24259225

ABSTRACT

The steady state levels of growth inhibitory doses of inorganic selenium were examined in five different mammary epithelial cell lines: MOD, COMMA-D, COMMA-F, COMMA-T, and YN-4. The retention of selenium was monitored using a radioactive isotope,(75)Se. Growth inhibition correlated with high levels of selenium in the cell. Generally, the retention of intracellular selenium was not dependent upon cell density, cell number, net growth rate, or tumorigenicity of the mammary cell lines. One cell line, COMMA-D, exhibited an unique response wherein the amount of selenium retained was low and the growth inhibitory effects of selenium were negligible when the cells were exposed to selenium at low density. However, at high cell densities, the COMMA-D cells responded like the other four cell lines. The growth inhibitory effect of selenium was reversible; upon removal of selenium from the medium, cells start synthesizing DNA within 24h. The retention of selenium was influenced by constituents in the growth medium. In particular, cysteine, but not methionine, purines, or pyrimidines altered selenium retention and counteracted the growth inhibitory effects of selenium. These results indicated that the mammary cell lines, particulary COMMA-D and MOD are good model systems to examine the uptake, retention, localization, and function of inorganic selenium under conditions where it acts as a growth inhibitory agent.

11.
Cancer Res ; 44(10): 4361-5, 1984 Oct.
Article in English | MEDLINE | ID: mdl-6467198

ABSTRACT

Previous results have documented that Na2SeO3 has a biphasic effect on the growth of mammary cells in vitro. In the experiments reported herein, the effects of selenium on several parameters of cell proliferation in the YN-4 mouse mammary cell line were investigated. The biphasic effect of selenium on cell growth was confirmed; i.e., 5 X 10(-8) M selenium stimulated cell growth, whereas 5 X 10(-6) M delayed cell growth and 5 X 10(-5) M was cytotoxic. The inhibition of cell growth by 5 X 10(-6) M selenium was reversible when this dose was removed from the growth medium. The increased cell growth at 5 X 10(-8) M selenium was reflected by an increased cell number, increased uptake of [3H]thymidine into DNA, increased DNA labeling index, and an increased rate of DNA synthesis. The decreased cell growth at 5 X 10(-6) M selenium was reflected by a decrease in all of these parameters of cell growth kinetics. The differential effects of selenium were manifested by 48 hr after addition of selenium to the cell culture medium. The results indicate that one of the mechanisms of selenium-mediated inhibition of carcinogenesis may be due to an inhibition of cell proliferation of responsive cells.


Subject(s)
DNA Replication/drug effects , Mammary Glands, Animal/cytology , Selenium Compounds , Selenium/pharmacology , Animals , Cell Division/drug effects , Cell Line , Cell Survival/drug effects , Epithelial Cells , Female , Kinetics , Mice , Selenic Acid , Selenium/toxicity
12.
Proc Natl Acad Sci U S A ; 81(12): 3756-60, 1984 Jun.
Article in English | MEDLINE | ID: mdl-6587390

ABSTRACT

An epithelial cell line, designated COMMA-1D, was derived from mammary tissue of BALB/c mice in the middle of pregnancy. This line, in continuous cell culture for 12 months, exhibits several characteristics distinctive of normal mammary epithelial cells, including induction of casein synthesis in vitro and normal duct morphogenesis in the cleared mammary fat pads of syngeneic mice. The cells also form domes in high density culture and are positive for keratin intermediate filaments by indirect immunofluorescence. COMMA-1D cells have a near diploid number of chromosomes and do not grow in suspension culture or produce tumors in syngeneic hosts. This cell line should prove useful for studies examining the regulation of normal cellular differentiation of mammary cells as well as transformation of epithelial cells to the preneoplastic and neoplastic phenotypes.


Subject(s)
Mammary Glands, Animal/cytology , Animals , Caseins/biosynthesis , Cell Differentiation , Cell Division , Cell Line , Epithelial Cells , Mice , Morphogenesis
13.
Cancer Res ; 43(5): 2100-5, 1983 May.
Article in English | MEDLINE | ID: mdl-6187443

ABSTRACT

The effects of selenium on three mammary epithelial cell lines (YN-4, WAZ-2t, and CL-S1) grown in vitro were examined by immunocytochemical and transmission electron microscopy technique. The primary effect of selenium at the ultrastructural level was the appearance of electron-dense inclusions within the mitochondrial matrix. The mitochondrial inclusions were seen in all three cell lines, although most readily induced in YN-4 cells, the cell line which is most sensitive to selenium-mediated growth inhibition. Selenium at 5 x 10(-8) and 5 x 10(-6) M did not alter cytoplasmic microtubules or intermediate filament networks, as determined by immunocytochemical staining. Immunocytochemical staining for cytoplasmic filaments and microtubules, and transmission electron microscopy observations, supported the contention that cells from all three cell lines were epithelial in origin, since they contained abundant desmosomes and were uniformly positive for keratin intermediate filaments. Whereas line YN-4 was negative for vimentin intermediate filaments, a minority (5 to 24%) of the cells in lines CL-S1 and WAZ-2t stained positively. In addition, the tumorigenicity of these three cell lines was assessed by in vitro growth assays and in vivo transplantation assays. Cell lines YN-4 and WAZ-2t, but not line CL-S1, were tumorigenic in syngeneic mice. All tumors were mammary adenocarcinomas. Cytochalasin B-induced multinucleation assay and growth as multicellular spheroides correlated positively with in vivo tumorigenicity, whereas saturation density and growth in low Ca2+ medium were not correlated with tumorigenicity. It is speculated that one of the early effects of selenium-mediated growth inhibition may be a modulation of mitochondrial function.


Subject(s)
Mammary Glands, Animal/drug effects , Selenium/pharmacology , Animals , Cell Line , Cytoskeleton/ultrastructure , Epithelium/drug effects , Epithelium/metabolism , Epithelium/ultrastructure , Fluorescent Antibody Technique , Inclusion Bodies/ultrastructure , Keratins/metabolism , Mammary Glands, Animal/metabolism , Mammary Glands, Animal/ultrastructure , Mice , Microscopy, Electron , Microtubules/ultrastructure , Mitochondria/ultrastructure
14.
Cancer Lett ; 15(3): 301-10, 1982.
Article in English | MEDLINE | ID: mdl-7116332

ABSTRACT

The uptake and subcellular localization of selenium-75 was examined in 3 established mammary cell lines in vitro. Previous studies had demonstrated that cell line YN-4 exhibited a biphasic growth response to selenium, whereas cell lines CL-S1 and WAZ-2t exhibited only an inhibition of growth. All 3 cell lines incorporated selenium-75 readily over a 48-h incubation period and the concentration of selenium-dependent glutathione peroxidase was correlated with the level of selenium-75 uptake. However, neither of these 2 parameters could be related to the unique biphasic growth response exhibited by cell line YN-4. The intracellular localization of selenium-75 was similar in lines CL-S1 and YN-4 with the label incorporated primarily in the cytosol and secondarily in the mitochondria. Although the unique growth response of line YN-4 could not be attributed to the incorporation parameters examined herein, the stable incorporation of selenium-75 at high levels should make these lines useful for further studies on the subcellular localization of selenium into organelles and macromolecules.


Subject(s)
Mammary Neoplasms, Experimental/metabolism , Radioisotopes , Selenium/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Cytosol/metabolism , Glutathione Peroxidase/metabolism , Mice , Microsomes/metabolism , Mitochondria/metabolism
15.
Cancer Lett ; 13(4): 333-44, 1981 Sep.
Article in English | MEDLINE | ID: mdl-7306960

ABSTRACT

The effect of selenium was examined on the growth potential of normal, preneoplastic and neoplastic mammary cells grown in primary monolayer cell cultures and on 3 established mammary cell lines. Selenium, present as Na2SeO3 in serum-free DMEM, inhibited all mammary cell cultures at 1 x 10(-5) M. Selenium, at 5 x 10(-8) M, stimulated the growth of primary cell cultures of normal mammary cells and C4 preneoplastic cells and the established cell line YN-4, but not the growth of D2 preneoplastic cells and tumors in primary cell cultures and of established cell lines CL-S1 and WAZ-2t. The differential responses of cells from preneoplastic outgrowth lines C4 and D2 and of D2 primary tumors in vitro correlated with the sensitivity of these same cell populations to selenium-mediated inhibition of growth and tumorigenesis in vivo. The differential responses among the 3 cell lines will allow further comparative studies on the cell biological basis of selenium function.


Subject(s)
Mammary Glands, Animal/cytology , Mammary Neoplasms, Experimental/pathology , Precancerous Conditions/pathology , Selenium/pharmacology , Animals , Cell Division/drug effects , Cell Line , DNA/biosynthesis , DNA, Neoplasm/biosynthesis , Dose-Response Relationship, Drug , Female , Mammary Glands, Animal/metabolism , Mammary Neoplasms, Experimental/metabolism , Mice , Mice, Inbred BALB C , Precancerous Conditions/metabolism
16.
Cancer Res ; 40(11): 3982-7, 1980 Nov.
Article in English | MEDLINE | ID: mdl-6162540

ABSTRACT

The conditions for growing preneoplastic mammary epithelial cells in primary cell cultures with serum-free factor-defined medium were established in a series of experiments. The cells, grown in Dulbecco's modified Eagle's medium with 0.6 mM Ca2+, epidermal growth factor, transferrin, insulin, and Na2SeO3, were fibroblast free, exhibited saturation densities similar to those of cells grown in serum-containing medium, and actively incorporated [3H]thymidine into DNA, as demonstrated by biochemical and autoradiography assays. The total growth fraction over a seven-day experiment was 77%. Fetuin was sufficient as a serum substitute for attachment of cells to the dish, although it was apparently not necessary for continued growth of the cells. The possible benefits of growing preneoplastic mammary epithelial cells in serum-free factor-defined medium are discussed.


Subject(s)
Mammary Neoplasms, Experimental/pathology , Precancerous Conditions/pathology , Animals , Calcium/pharmacology , Cell Division/drug effects , Cells, Cultured , Culture Media , Epithelium/pathology , Female , Mice , Neoplasm Transplantation , alpha-Fetoproteins/pharmacology
17.
Cancer Res ; 40(2): 329-33, 1980 Feb.
Article in English | MEDLINE | ID: mdl-7356516

ABSTRACT

Epithelial cell cultures of normal mammary gland, preneoplastic hyperplastic nodule outgrowth lines, primary tumors, and transplanted tumors, all derived from BALB/c mice, were examined for their response to cytochalasin B to determine if cells of primary mammary tumors multinucleated and if preneoplastic hyperplastic alveolar nodule cells responded differently than cells of primary tumors. Established tumorigenic and nontumorigenic cell lines were also examined as positive and negative controls. The standard assay conditions were optimized at 1 microgram CB per ml for 48 hr. The results, expressed as the mean percentage of cells exhibiting three or more nuclei per cell were: normal mammary cells, 5%; preneoplastic mammary cells, 4%; primary mammary tumor cells, 36%; transplanted mammary tumors, 70%; tumorigenic established cell lines, 80%; and nontumorigenic established cell lines, 5%. The frequency of tumor cells exhibiting multinucleation increased with serial transplantation in vivo and with serial passage in vitro. The results demonstrate that neoplastic cells within a primary tumor exhibit uncontrolled nuclear division and that uncontrolled nuclear division is a distinguishing characteristic between preneoplastic and neoplastic mammary cells.


Subject(s)
Cell Nucleus/drug effects , Cytochalasin B/pharmacology , Mammary Neoplasms, Experimental/diagnosis , Precancerous Conditions/diagnosis , Animals , Cell Line , Cell Nucleus/ultrastructure , Female , In Vitro Techniques , Mammary Neoplasms, Experimental/ultrastructure , Mice , Mice, Inbred BALB C , Precancerous Conditions/ultrastructure
18.
J Clin Microbiol ; 8(2): 171-6, 1978 Aug.
Article in English | MEDLINE | ID: mdl-100506

ABSTRACT

Radioimmunoprecipitation (RIP) assays were developed to selectively quantitate class-specific antibodies to purified hemagglutinins (HA) of type A influenza virus in nasal secretions. Rabbit anti-human secretory piece of immunoglobulin A (IgA) and rabbit anti-human IgG were used as second antibodies. A third antibody, goat anti-rabbit IgG, was incorporated into the system to separate immune complexes formed between iodinated HA, nasal wash test specimen, and second antibody. The utilization of this reagent avoided the need for large quantities of IgA and IgG antibody-negative carrier secretions. Nasal was specimens obtained from 14 adults immunized with an inactivated type A influenza virus vaccine were evaluated by RIP and viral neutralization assays. Significant homologous postvaccination secretory IgA and IgG antibody levels were demonstrable in 13 (93%) of individuals by RIP, whereas only 5 (36%) exhibited rises by viral neutralization tests. Moreover, the geometric mean IgA and IgG antibody levels were at least 20- and 37-fold greater than the neutralizing antibody titer. The pattern of heterologous immunoglobulin-specific antibody responses tended to be similar to those observed with the homologous HA subunit.


Subject(s)
Antibodies, Viral/analysis , Hemagglutinins, Viral/immunology , Influenza A virus/immunology , Mucus/immunology , Nasal Mucosa/metabolism , Radioimmunoassay/methods , Adult , Humans , Immunodiffusion , Immunoglobulin A/analysis , Immunoglobulin G/analysis , Influenza Vaccines , Neutralization Tests , Vaccines, Attenuated
19.
J Physiol ; 247(3): 657-86, 1975 Jun.
Article in English | MEDLINE | ID: mdl-238034

ABSTRACT

1. The influence of internal and external Na concentrations on Ca movements have been measured in pinch-off presynaptic nerve terminals (synaptosomes). Ca uptake is enhanced when external Na (Nao) is replaced by Li, choline or dextrose, in Na-loaded synaptosomes. Depletion of internal Na (Nai) abolishes the stimulatory effect of external Na removal. 2. Ca uptake from Na-depleted media is proportional to [Na]i -2, and averages about 1-5 mumole Ca/g synaptosome protein per minute when [Na]i is approximately 137 mM. This may correspond to a Ca influx of about 0-1 p-mole/cm-2 sec. 3. External Na is a competitive inhibitor of the Nai-dependent Ca uptake. The interrelationship between [Na]o, [Ca]o and Ca uptake indicate that two external Na ions may compete with one Ca at each uptake site. 4. The distribution of particles with Nai-dependent Ca uptake activity parallels the distribution of synaptosomes in the preparative sucrose gradient. Thus, this Ca uptake activity is probably a property of the pinched-off nerve terminals per se, and not of the mitochondria which may contaminate the synaptosome fraction. 5. The Nai-dependent Ca uptake mechanism requires an intact surface membrane, since synaptosomes subjected to osmotic lysis lose the ability to accumulate Ca by this route. 6. Ca efflux into Ca-free media is largely dependent upon the presence of external Na. The curve relating Ca efflux to [Na]o is sigmoid, and suggests that more than one external Na ion (perhaps 2 or 3) is needed to activate the efflux of each Ca ion. 7. The net Ca gain exhibited by Na-loaded synaptosomes incubated in Na-depleted media can be accounted for by the increased Ca uptake and decreased Ca loss observed under these conditions. 8. Treatment of synaptosomes with cyanide or 2,4-dinitrophenol decreases Ca uptake and enhances Ca efflux into Na-containing media. This results in a net loss of Ca from the terminals, even in the presence of external Ca. 9. In contrast to the Ca efflux from synaptosomes, the Ca efflux from brain mitochondria is not dependent upon external Na, and is reduced by succinate, a substrate which is known to fuel mitochondrial respiration. 10. The temperature coefficient (Q10) of the Nai-dependent Ca uptake is about 3. 11. The Nai-dependent Ca uptake is reduced at low pH. The relationship between this Ca uptake and pH approximates a titration curve with a pKa of about 5-6. 12. The data indicate that Ca transport in rat brain presynaptic terminals may involve a carrier-mediated Na-Ca exchange mechanism, and that some of the energy required for Ca extrusion may come from the Na electrochemical gradient across the surface membranes.


Subject(s)
Calcium/metabolism , Sodium/pharmacology , Synaptosomes/metabolism , Animals , Biological Transport, Active/drug effects , Brain/metabolism , Choline/pharmacology , Cyanides/pharmacology , Dinitrophenols/pharmacology , Glucose/pharmacology , Hydrogen-Ion Concentration , In Vitro Techniques , Kinetics , Lithium/pharmacology , Mitochondria/metabolism , Rats , Stimulation, Chemical , Synaptosomes/drug effects , Temperature
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