Noncoordinate expression of murine mammary tumor virus gene products.
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Animals↗
Biomedical subjects
D Medina
Publications and source records attributed to D Medina.
Selenium-mediated inhibition of mouse mammary tumorigenesis.
The effects of selenium of mouse mammary tumorigenesis were examined in 2 systems. BALB/cfC3H (MuMTV-S positive) given 2 and 6 ppm selenium in the drinking water and maintained as breeding mice, had significantly lower incidences of mammary tumors (48% and 12%, respectively) than untreated control mice (82%). Selenium at 4 ppm inhibited the tumor-producing capabilities of only 1 of 4 preneoplastic outgrowth lines maintained in BALB/c (MuMTV-S negative) mice. Selenium at 2, 4 and 6 ppm had no effect on the growth rate of primary mammary tumors transplanted subcutaneously in BALB/c mice. Selenium inhibited mammary tumorigenesis without interference of normal reproductive function and weight gain.
Animals↗
Distinction of normal, preneoplastic, and neoplastic mouse mammary primary cell cultures by water nuclear magnetic resonance relaxation times.
Normal, preneoplastic, and neoplastic primary cultures of mouse mammary epithelial cells were distinguishable on the basis of water proton nuclear magnetic resonance (NMR) relaxation times--i.e., spin-lattice relaxation time (T1) and spin-spin relaxation time (T2). T1 values were 916 +/- 24 msec for normal cells, 1,029 +/- 24 msec for preneoplastic cells, and 1,155 +/- 42 msec for neoplastic cells. This method of distinction between normal and neoplastic cells (P less than 0.001) and normal and preneoplastic cells (P less than 0.005) supported previous findings in whole tissues. NMR relaxation times resulted in better distinction between these cell populations than any other technique except direct histology. The T1 and T2 values of water protons in cells grown in primary culture were higher than those of established mouse mammary cancer cell lines. The differences in T1 and T2 did not correlate with cellular hydration. The data suggested a basic difference in water-macromolecular surface interactions among normal, preneoplastic, and neoplastic cells.
Animals↗
Characterization of progesterone receptors, estrogen receptors, and estrogen (type II)-binding sites in the hormone-independent variant of the MXT-3590 mouse mammary tumor.
A hormone-independent mouse mammary tumor line was derived by transplantation of a hormone-dependent line (MXT-3590) into ovariectomized-adrenalectomized animals and subsequent transplantation of surviving tumors. Although the tumor line is hormone independent, it contains a full complement of estrogen and progesterone receptors which are similar to those found in hormone-dependent tumors. This similarity includes the criteria of nuclear translocation of the estrogen receptor and subsequent stimulation of progesterone receptor synthesis. In addition to the estrogen receptor, a secondary estrogen-binding site (type II) is also present; this binding site was previously described in the hormone-dependent companion tumor line. Thus, while this variant tumor line contains the biosynthetic pathways that control estrogen and progesterone receptor synthesis, its growth does not depend on these steroids. Therefore, it may provide a good model for the study of this class of hormone-independent tumors.
Animals↗
Growth of preneoplastic mammary epithelial cells in serum-free medium.
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.
Animals↗
Mammary tumorigenesis in 7,12-dimethybenzanthracene-treated C57BL x DBA/2f F1 mice.
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9,10-Dimethyl-1,2-benzanthracene↗
Comparative responses of normal and malignant mouse mammary cells to modulation of surface properties.
We previously demonstrated that epithelial cells growing in primary cultures derived from normal and neoplastic mouse mammary tissues are indistinguishable on the basis of morphology, surface topography, agglutinability by concanavalin A, or of number, cytoplasmic location, or organization of microtubules and actin-containing microfilaments. In the present study, the modulation and interrelationships of these features were investigated using the drugs colchicine and cytochalasin B and the enzymes trypsin, collagenase, and hyaluronidase. Untreated cells of both types were only weakly agglutinable by concanavalin A. Pretreatment of the cells with colchicine, cytochalasin B, or hyaluronidase increased cytoagglutination by the lectin dramatically, whereas neither trypsin nor collagenase significantly altered reactivity of the cells. Binding studies utilizing fluorescein-tagged concanavalin A indicated that the enhanced agglutinability did not correlate with any particular distribution of lectin receptors on the cells. At the doses used, colchicine and cytocholasin B induced disruption of microtubules and microfilament networks, respectively, but no effects on either type of cytoskeletal element were observed after hyaluronidase, trypsin, or collagenase. The increased agglutinability was not associated with any specific surface conformation since scanning electron microscopy revealed that untreated cells and cells exposed to colchicine, hyaluronidase, and collagenase were flat and possessed variable numbers of small microvilli. In contrast, following incubation with cytochalasin B or trypsin, the cells underwent retraction and rounding, and developed complex surface structures such as blebs, ruffles, and filopodia. Thus, no single factor appeared responsible for determining the agglutinable or nonagglutinable state of the mammary cells. Morover, no differences were detected in the surface responses of the normal and malignant cells under any of the conditions used.
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Distinction between preneopastic and neoplastic mammary cell populations in vitro by cytochalasin B-induced multinucleation.
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.
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Mouse mammary tumor virus genome expression in chemical carcinogen-induced mammary tumors in low- and high-tumor-incidence mouse strains.
Involvement of mouse mammary tumor virus (MMTV) in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis was investigated in low- (BALB/c) and high- (BALB/cfC3H) mammary-tumor-incidence mouse strains. Both strains contain endogenous MMTV integrated into the cellular genome. Additionally, BALB/cfC3H mice are infected with exogenous MMTV-S which is responsible for a higher incidence of mammary tumors in breeding females. Administration of DMBA to virgin mice of both strains resulted in a moderate frequency of mammary tumors within 40 wk after treatment. No differences were found in DMBA-induced tumor incidences at 18 wk (6% and 7%) or at 38 wk (29% and 36%) after treatment of BALB/c and BALB/cfC3H mice, respectively. Expression of MMTV in these tumors was examined by assaying for the presence of MMTV RNA by hybridization using MMTV-specific cDNA and by immunohistochemical staining utilizing antibodies against MMTV 52,000-dalton glycoprotein, gp52, and 28,000-dalton internal protein, p28. Of 16 BALB/c tumors assayed, 11 did not contain detectable levels of MMTV RNA and the remaining 5 tumors contained only low levels (0.0005-0.0010%) of viral RNA. Importantly, MMTV RNA was not detected in 5 of 27 BALB/cfC3H tumors. The other BALB/cfC3H tumors contained quantities of MMTV RNA ranging from 0.0006 to 0.4170%. Most BALB/cfC3H tumors with detectable levels of MMTV RNA also synthesized viral proteins gp52 and p28. Thus, expression of the complete MMTV genome is not requisite for maintenance of the tumor phenotype in DMBA-induced mammary tumors in either BALB/c or BALB/cfC3H virgin mice under 1 year of age.
9,10-Dimethyl-1,2-benzanthracene↗
Manifestation of hyperplastic alveolar nodules and mammary tumors in "viable yellow" and non-yellow mice.
The time course of appearance of hyperplastic alveolar nodule(s) (HAN) and mammary tumor(s) (MT) was determined in untreated virgin "viable yellow" (Avy/A) and non-yellow (A/a) (C3H/HeNIcrWf X VY/Wf)F1 female mice. The first HAN was detected in a yellow female at age 16 weeks, the first period in which mice were killed. HAN were not found in the non-yellow mice until they were 19 weeks old. The incidence of HAN increased to 92% among yellow females and to 75% among non-yellow females by 36 weeks of age. MT were first observed at age 22 weeks in yellow mice and at 28 weeks in non-yellow mice. The incidence of MT at 36 weeks was 75% among yellow mice and 22% among non-yellow mice. Type B adenocarcinoma was the predominant class of MT found in the yellow mice. The time courses of appearance and incidence of HAN and MT in the non-yellow F1 mice were similar to those observed in inbred C3H female mice. MT first appeared in each population when the incidence of HAN bearers had reached 40--45% regardless of age, body weight, or number of HAN per HAN bearer. Apparently, the phenotypic effects of the Avy gene primarily stimulated the multiplication of nodule-transformed cells to form HAN and thus indirectly enhanced MT formation.
Adenocarcinoma↗
Murine mammary tumor virus expression during mammary tumorigenesis in BALB/c mice.
Steady-state levels of murine mammary tumor virus (MuMTV) RNA were quantitated during mammary tumorigenesis in BALB/c mice by molecular hybridization with a representative MuMTV complementary DNA (cDNA) probe. Hyperplastic alveolar nodule (HAN) lines are preneoplastic mammary lesions that were induced in BALB/c mice by hormones alone or in combination with 7,12-dimethylbenz(a)anthracene and give rise to mammary tumors. The hormone-induced HAN lines D1 and D2 contained detectable amounts of hybridizable MuMTV sequences. MuMTV RNA sequences were also observed in five of the six transplanted BALB/c mammary tumors that were examined. Similar levels of hybridizable MuMTV RNA were observed between the D1 or D2 HAN line and mammary tumors derived from each HAN line. The D2 HAN line as well as D2, C4, and CD8 mammary tumors accumulated RNA that was apparently homologous to most of the MuMTV genome. Thermal denaturation of hybrids indicated extensive sequence homology between the MuMTV cDNA and hybridizable RNA in the BALB/c HAN lines and mammary tumors. A low level of type C viral RNA was observed in the BALB/c HAN lines and most mammary tumors by molecular hybridization with a cDNA to Moloney murine leukemia virus. These data demonstrate that MuMTV sequences are frequently expressed in hormone-induced BALB/c HAN lines and mammary tumors derived from HAN lines or ductal hyperplasias induced in BALB/c mice by hormones and/or a chemical carcinogen. The transition from the preneoplastic to the neoplastic state in BALB/c mice does not appear to be due to a change in the steady-state levels of MuMTV RNA since the hormone-induced HAN lines and mammary tumors had similar levels of hybridizable MuMTV RNA.
9,10-Dimethyl-1,2-benzanthracene↗
Hormonal regulation of murine mammary tumor virus RNA expression during mammary tumorigenesis in BALB/c mice.
The effects of glucocorticoids and prolactin on murine mammary tumor virus (MuMTV) RNA expression in preneoplastic outgrowth lines and mammary tumors in BALB/c mice were investigated. Hyperplastic alveolar nodules (HAN) and a ductal hyperplasia (DH) are induced in virgin BALB/c mice by prolonged hormonal stimulation or treatment with 7,12-dimethylbenz(a)anthracene or both. Mice bearing HAN or DH outgrowth lines and mammary tumors that arose from the outgrowth lines were treated with glucocorticoids or prolactin. MuMTV RNA was quantitated by hybridization with a representative complementary DNA probe specific for MuMTV RNA. Prolactin treatment did not increase MuMTV RNA in the BALB/c HAN or DH outgrowth lines or tumors. MuMTV RNA increased after glucocorticoid treatment in the C3, C4, and C5 HAN outgrowth lines and in tumors that arose from the D1, D2, C4, and C5 HAN and CD8 DH outgrowth lines. No increase in MuMTV RNA with glucocorticoid treatment was observed in the D1 or D2 HAN outgrowth line, in the CD8 DH outgrowth lines, and in tumors that arose from the C3 HAN outgrowth line. The ability of glucocorticoids to stimulate MuMTV expression was specific since the response was dose dependent and specific for glucocorticoid hormones. Glucocorticoid treatment did not increase the level of type C viral RNA in the majority of hormone- or 7,12-dimethylbenz(a)anthracene-induced HAN outgrowth lines or tumors. These observations suggested that glucocorticoids may influence MuMTV expression during mammary tumorigenesis in BALB/c mice.
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Serial transplantation of chemical carcinogen-induced mouse mammary ductal dysplasias.
BALB/c and (C57BL times DBAf)F1 mice were treated with 7,12-dimethylbenz[a]anthracene and urethan, respectively, to induce mammary dysplasias. Nine transplantable outgrowth lines of dysplastic tissue were established by transplantation of the primary lesions into the cleared mammary fat pads of syngeneic mice; 8 of the 9 lesions were ductal in origin. The growth and tumorigenic potentials of these 9 lines were followed over 6-9 transplant generations. Most outgrowth lines exhibited a rapid decline in growth potential and/or a progression to papillomatosis and subsequent carcinoma by transplant generation 7. The ductal outgrowth lines and mammary tumors established from urethan-induced dysplasias in hybrid mice were ovary-dependent for tumorigenesis and tumor growth. The transplantation experiments confirmed the hypothesis that ductal dysplasias have high tumorigenic potentials and can be classified as "high-risk" lesions, similar to murine mammary tumor virus-induced hyperplastic alveolar nodules.
9,10-Dimethyl-1,2-benzanthracene↗
Microtubules and actin-containing filaments of normal, preneoplastic, and neoplastic mouse mammary epithelial cells.
The distribution and organization of microtubules (MT's) and actin-containing microfilaments (MF's) were examined in epithelial cells of primary cultures established from normal, preneoplastic, and neoplastic mouse mammary tissues and in cells of three clonal culture lines derived from murine mammary adenocarcinomas. No consistent differences in the cytoskeletal components were found among cell populations of the primary cultures as revealed either by indirect immunofluorescence using antibodies to tubulin and actin or by electron microscopy. Overall, the majority of cells in the three types of primary cultures possessed elaborate complexes of MT's and actin filaments after fluorescent staining with the appropriate antibodies, and abundant MT's and MF's were found in the cells at the ultrastructural level. Similar patterns of MT's and MF's were observed in cells of two of the clonal mammary tumor lines. Cells of the third line, however, exhibited intricate networks of MT's but had a reduction in actin cables detectable by the immunofluorescence procedure. Moreover, MF's were difficult to locate by electron microscopy. The results suggest that the lesion(s) in growth control in the neoplastic mammary cells may not involve any gross alterations in MT's or MF's.
Actins↗
Opposing effects of cryostat sections of preneoplastic and neoplastic mouse mammary lesions on in vitro migration of peritoneal exudate cells.
Cryostat sections of normal mouse tissues and of preneoplastic HAN and neoplastic mammary tumors were used as "antigens" in MMI tests. Nonspecific inhibition of normal and sensitized PEC migration was induced by HAN and some normal tissues, including normal mammary gland. This inhibition did not require the presence of lymphocytes, was not species specific, and could be blocked by sera from HAN-bearing mice. Cryostat sections of mammary tumors did not inhibit, indeed occasionally enhanced PEC migration. Further, the presence of tumor cryostats and eluates interfered with inhibition induced by HAN cryostats and by PPD with PEC from donors sensitized to that antigen. Histologic examination of HAN and of mammary tumor tissue revealed inflammatory cells to be distributed diffusely in the former and localized peripherally around the latter type of lesion.
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In situ lymphoid cells of mouse mammary tumors. IV. Comparison of functional activity of lymphoid cells separated from mammary tumors to that of spleen and lymph node cells of tumor-sensitized mice.
Lymphoid cells isolated form several types of mouse mammary tumors are capable of stimulating tumor cell growth or survival in MCT assays. Lymph node and spleen cells of mice bearing such a tumor are specifically cytotoxic to the tumor cells. Surgical removal of the tumor is followed in 4 to 7 days by the appearance of stimulatory capacity in spleens and lymph nodes. By day 10, cytotoxic cells specific for the sensitizing tumor are again detected. These reach a peak on day 13. By day 17 no reactivity is detectable. The functional distribution of tumor-reactive lymphoid cells is different between tumor masses and peripheral lymphoid organs.
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Characterization and estrogen stimulation of cytoplasmic progesterone receptor in the ovarian-dependent MXT-3590 mammary tumor line.
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Animals↗
Enhancement of the tumorigenicity of preneoplastic mammary nodule lines by enzymatic dissociation.
Preneoplastic mammary nodule lines D1, D2, and C4 were enzymatically dissociated with collagenase, hyaluronidase, and pronase or with only collagenase and hyaluronidase to produce high yields of viable single cells; 10(5) cells were injected into the cleared mammary fat pads of syngeneic BALB/cCrgl mice. In 11 experiments involving three different preneoplastic nodule lines with different tumor potentials, all dissociated nodule cell lines showed a marked increase in tumorigenicity as compared to the same tissues transplanted as 1-mm3 pieces. The results could not be explained on the basis of differences between the amounts of cells transplanted in the two procedures. In a second series of experiments, normal mammary cells from virgin, pregnant, or lactating mice were mixed in different ratios with 10(5) nodule cells and injected into the mammary fat pads. The presence of normal cells reversed the marked increase in the tumorigenicity of enzymatically dissociated nodule cells to a level equal to or less than the tumorigenicity of control transplants (1-mm3 pieces). The growth of 10(5) mammary tumor cells was not inhibited when tumor cells were mixed with 3 x 10(5) normal cells and transplanted into the mammary fat pads. These results showed that enzymatic dissociation can lead to an increase in tumor potential of preneoplastic mammary nodule lines, and they supported the hypothesis that nodule cells, but not neoplastic cells, are sensitive to the growth-inhibitory effects of normal mammary cells.
Adenocarcinoma↗