ABSTRACT
We have developed a murine mammary tumor cell line, MC4-L4, and after 15 passages, a spindle-shaped population became evident. The cuboidal cells, MC4-L4E, cloned by limit dilution, proved to be epithelial tumor cells. When inoculated in syngeneic mice, they gave rise to invasive metastatic carcinomas expressing estrogen and progesterone receptors. These tumors regressed after anti-progestin treatment and stopped growing after 17-beta-estradiol administration. In vitro, they were insensitive to medroxyprogesterone acetate (MPA), 17-beta-estradiol, and EGF and were inhibited by TGFbeta1. They expressed mutated p53 and estrogen receptors alpha; progesterone receptors were undetectable. Cells were polyploid and shared the same four common marker chromosomes present in the parental tumor in addition to an exclusive marker. Spindle-shaped cells, MC4-L4F, were selected by differential attachment and detachment and proved to be non-epithelial non-tumorigenic cells. They were cytokeratin negative, showed mesenchymal features by electron microscopy, differentiated to adipocytes when treated with an adipogenic cocktail, were stimulated by TGFbeta1 and EGF, showed a wild-type p53, and did not exhibit the marker chromosomes of the parental tumor. Although they expressed estrogen receptors alpha, they were insensitive to 17-beta-estradiol in proliferation assays. Co-cultures of both cell types had a synergic effect on progesterone receptors expression and on cell proliferation, being the epithelial cells, the most responsive ones, and 17-beta-estradiol increased cell proliferation only in co-cultures. Cytogenetic studies and data on p53 mutations rule out the possibility of an epithelial mesenchymal transition. Their unique characteristics make them an excellent model to be used in studies of epithelial-stromal interactions in the context of hormone responsiveness in hormone related tumors.
Subject(s)
Breast Neoplasms , Cell Line, Tumor , Mammary Glands, Animal/cytology , Stromal Cells , Animals , Antineoplastic Agents, Hormonal/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Culture Techniques/methods , Cell Differentiation , Cell Line, Tumor/cytology , Cell Line, Tumor/physiology , Cell Shape , Coculture Techniques , Epidermal Growth Factor/metabolism , Estradiol/metabolism , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Genetic Markers , Hormone Antagonists/metabolism , Humans , Keratins/metabolism , Medroxyprogesterone Acetate/metabolism , Mice , Mice, Inbred BALB C , Mifepristone/metabolism , Mutation , Neoplasm Transplantation , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Stromal Cells/cytology , Stromal Cells/metabolism , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1 , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
We report the establishment of two mouse mammary cancer cell lines, MC7-2A and MC7-2B obtained from a mouse mammary carcinoma induced by medroxyprogesterone acetate (MPA) and maintained by syngeneic transplantation in BALB/c mice. They are epithelial (express cytokeratins) and express both estrogen receptors alpha (ERalpha) and progesterone receptors (PRs) isoforms A and B (western blots). In vitro, MPA inhibited 3H-thymidine uptake, starting from concentrations as low as 10(-13) M in MC7-2A and 10(10) M in MC7-2B; the antiprogestin RU 486 exerted a stimulatory effect at 10(-14) M in both cell lines; 17-beta-estradiol (E2) also exerted a stimulatory effect starting at 10(-10) M in MC7-2A and at 10(-13) M in MC7-2B. When transplanted in syngeneic mice, both cell lines originated adenocarcinomas that gave rise to lung metastases within 3 months. In in vivo studies, in MC7-2A, the antiprogestin inhibited completely tumor growth, E2 induced a slight although significant ( p < 0.05) stimulatory effect and MPA stimulated tumor growth while MC7-2B cells were unresponsive to all treatments. ER and PR were also expressed in tumors as assessed by immunohistochemistry. Two marker chromosomes were identified by FISH as translocations between chromosomes 4 and 7, and between chromosomes X and 2; the third marker chromosome remains unidentified. All these markers were also present in the parental tumor. A new marker, a centric fusion of chromosomes 2, was acquired in both cell lines. Considering that there are very few murine breast carcinoma responsive cell lines, these cells represent new tools in which the regulatory effect of hormones can be studied.
Subject(s)
Adenocarcinoma/pathology , Genetic Markers , Mammary Neoplasms, Animal/pathology , Neoplasm Metastasis/physiopathology , Receptors, Estrogen/biosynthesis , Receptors, Progesterone/biosynthesis , Tumor Cells, Cultured , Animals , Antineoplastic Agents, Hormonal/pharmacology , Blotting, Western , Estradiol/pharmacology , Female , Gene Expression Regulation, Neoplastic , Immunohistochemistry , In Situ Hybridization, Fluorescence , Lung Neoplasms/secondary , Lung Neoplasms/veterinary , Medroxyprogesterone Acetate/pharmacology , Mice , Mice, Inbred BALB C , Receptors, Estrogen/analysis , Receptors, Progesterone/analysisABSTRACT
Metastatic mammary carcinoma tumor lines 59-2-HI and C7-2-HI originated in female BALB/c mice treated with medroxyprogesterone acetate and are maintained by syngeneic transplantation. Both lines express estrogen (ER) and progesterone receptors (PR) and regress completely after estradiol (E(2)) or antiprogestin treatment. The BET tumor line, of similar origin and biological features, regresses only after E(2) treatment. To investigate possible differences between E(2)- and antiprogestin-mediated effects we evaluated the morphological features, mitosis and apoptosis, and the differential expression of cell-cycle inhibitors associated with tumor regression. Treatments started when tumors reached 50-100 mm(2). After 24-96 h, tumors were excised and processed for morphological and immunohistochemical studies. Regression was associated with a significant and early decrease in the number of mitosis and with higher percentages of apoptotic cells. These phenomena were accompanied by an increase in p21 and p27 expression in the E(2) and antiprogestin-responsive lines treated with E(2), RU 38.486 or ZK 98.299 (P < 0.05). In BET tumors treated with E(2), p21 expression remained within basal levels and only p27 increased (P < 0.05). p53 was low in control 59-2-HI and C7-2-HI tumors and increased after treatment (P < 0.05) whereas BET untreated tumors already expressed high levels of p53 and MDM2. Although the immunohistochemical findings were compatible with alterations of p53, SSCP evaluation failed to disclose the presence of mutations, suggesting that the defective expression of p21 is related to an impaired p53 pathway. UV irradiation failed to increase p21 expression in BET, but was able to induce p53 and p21 in 59-2-HI and C7-2-HI tumors. The absence of an increased expression of p21 in E(2)-regressing BET lesions suggests that this protein is not necessary for estrogen-induced regression, but may be essential for antiprogestin action. Our results also suggest that p53/MDM2 alterations may be one of the mechanisms responsible for selected hormone resistance in breast carcinomas.
Subject(s)
Cell Cycle Proteins/metabolism , Estrogens/pharmacology , Mammary Neoplasms, Experimental/metabolism , Progestins/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Apoptosis , Cyclin-Dependent Kinase Inhibitor p27 , Female , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , MitosisABSTRACT
La mayoría de los cánceres de mama humanos son carcinomas ductales que expresan receptores hormonales, transitan por distintos estadios de hormono-dependencia y dan metástasis a distancia. No existen modelos experimentales cuyos tumores reúnan todas estas características. El modelo de inducción de carcinomas mamarios inducidos por acetato de medroxiprogesterona en el ratón, desarrollado hace años en nuestro laboratorio, es muy adecuado ya que la mayoría de los tumores inducidos son ductales, desarrollan metástasis en ganglios axilares y en pulmón, expresan altos niveles de receptores de estrógenos y progesterona cumple un rol proliferativo, mientras que en los tumores humanos, si bien cada vez hay más evidencias que sugieren que esta hormona cumple un rol importante, se les ha adjudicado a los estrógenos el rol protagónico. El objetivo de nuestras líneas de trabajo en general está orientada a jerarquizar el rol de los progestágenos en cáncer de mama. Para que un modelo experimental resulte interesante, debe reproducir en forma fidedigna la enfermedad humana y tener características intrínsecas tales como reproducibilidad, bajo costo, posibilidad de estudios in vitro, y uso de animales estándar. Nuestro modelo permite la posibilidad de diseñar estudios de carcinogénesis hormonal para evaluar cofactores que afecten el desarrollo de los tumores... (TRUNCADO)
Subject(s)
Antineoplastic Agents, Hormonal , Mice, Inbred BALB C , Carcinoma, Ductal, Breast/chemically induced , Carcinoma, Ductal, Breast/pathology , Karyotyping , Tumor Cells, Cultured , Cell Differentiation/physiology , Cell Division , Cell Division/physiology , Immunohistochemistry , Cell Line , Disease Models, Animal , Neoplasms, Hormone-Dependent/metabolism , Mammary Neoplasms, Experimental/etiology , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/chemically induced , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mammary Neoplasms, Experimental/drug therapy , Progesterone/physiology , Progesterone/toxicity , Progestins/physiology , /metabolism , Receptors, Estrogen , Receptors, ProgesteroneABSTRACT
La mayoría de los cánceres de mama humanos son carcinomas ductales que expresan receptores hormonales, transitan por distintos estadios de hormono-dependencia y dan metástasis a distancia. No existen modelos experimentales cuyos tumores reúnan todas estas características. El modelo de inducción de carcinomas mamarios inducidos por acetato de medroxiprogesterona en el ratón, desarrollado hace años en nuestro laboratorio, es muy adecuado ya que la mayoría de los tumores inducidos son ductales, desarrollan metástasis en ganglios axilares y en pulmón, expresan altos niveles de receptores de estrógenos y progesterona cumple un rol proliferativo, mientras que en los tumores humanos, si bien cada vez hay más evidencias que sugieren que esta hormona cumple un rol importante, se les ha adjudicado a los estrógenos el rol protagónico. El objetivo de nuestras líneas de trabajo en general está orientada a jerarquizar el rol de los progestágenos en cáncer de mama. Para que un modelo experimental resulte interesante, debe reproducir en forma fidedigna la enfermedad humana y tener características intrínsecas tales como reproducibilidad, bajo costo, posibilidad de estudios in vitro, y uso de animales estándar. Nuestro modelo permite la posibilidad de diseñar estudios de carcinogénesis hormonal para evaluar cofactores que afecten el desarrollo de los tumores... (TRUNCADO)(AU)