Establishment and characteristics of two new human mammary carcinoma lines in nude mice with special reference to the estradiol receptor status and the importance of stroma forin vivo andin vitro growth (original) (raw)
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Growth of human breast carcinomas in nude mice and subsequent establishment in tissue culture
1980
slicing (9, 14), treatment with trypsin (13), and collagenase (1). A different approach was taken by Cailleau et a!. (3) who developed a number of cell lines from pleural effusions. Using this same technique of culturing cells from pleural effusions, Soule et a!. (19) developed a human breast carcinoma cell line that has been reported to have an estradiol receptor (2) and to produce a-lactalbumin (18). In this paper, we report on the establishment of 4 transplant able human breast tumors in the nude mouse and the devel opment of 3 cell strains from 2 of these transplantable tumors. MATERIALS AND METHODS Nude Mouse Colony. A colony of nude mice on the BALB/ c background was developed from breeding stock purchased from Bomholtsgaard, Ltd., Ry, Denmark. The colony of mice is maintained in isolation, and all food, water, cages, and bedding are sterilized. The cages are capped with sterile filter barriers (Maryland Plastics, Inc., New York, N. V.). The colony has been described in detail elsewhere (16). Breast tumor tissue from local hospitals was received in sterile F-i 2 medium supplemented with 10% human serum. A portion of each tumor was prepared for histological evaluation of tumor pathology, and a second portion was frozen for biochemical assays. The remaining tumor tissue was finely minced, rinsed for 5 mm in antibiotics (streptomycin and peni cillin), and injected with an 18-gauge needle s.c. into the flank of female nude mice. An estrogen pellet was implanted on the contralateral side[each pellet contained 25 mg of 17@-estradiol (Sigma Chemical Co., St Louis, Mo.)]. In preliminary studies, tumors were injected into mice with and without pellets; tumors routinely grew better in animals with pellets.4 It was not known if the nude mice produced enough estrogen to support those tumors which required or were growth responsive to estrogen; some 40 to 50% of human breast tumors are dependent upon ovarian hormones for growth (7). The size of the tumor was determined at biweekly intervals by caliper measurement of the s.c. tissue mass. The tumor was transplanted when it reached a size of approximately 1.5 cm. Tissue Culture. Some of the original tumor, when available, or the mouse-passaged tumor was cultured after the method originally outlined by Lasfargues and Ozzello (9). Following external sterilization of the tumor in 70% alcohol, the tissue was trimmed of fat and minced with sharp scissors. This permits the tumor cells to spill out into the medium. The following day, the supematant containing the spilled tumor cells was trans ferred to a second tissue culture dish. Any fibroblast contami nation or other cellular contaminants that were present in the tumors grown in the nude mice were of murine origin and were readily removed by treatment of the culture with rabbit anti
Breast Cancer Research and Treatment, 2010
To evaluate the extent to which each estrogen receptor (ER) subtype contributes to the stimulation or to the inhibition of mammary tumor growth, we evaluated the effects of specific agonists in MC4-L2 cells, which are stimulated by 17β-estradiol (E2), and in mammary carcinomas of the MPA mouse breast cancer model, which are inhibited by E2. Both express ERα and ERβ. In MC4-L2 cells, 4,4′,4″-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT; ERα agonist) and (4-hydroxy-phenyl)-propionitrile (DPN; ERβ agonist) stimulated cell proliferation, whereas the opposite occurred in C4-HI primary cultures. The inhibitory effect was associated with a decrease in ERα and cyclin D1 expression and an increase in progesterone receptor (PR) expression as well as in the Bax/Bcl-xl ratio. In vivo, mice carrying C4-HI or 32-2-HI tumors were treated with E2, PPT or DPN (3 mg/kg/day) or with vehicle. PPT and DPN inhibited tumor size, as did E2, during the first 72 h. After a few days, DPN-treated tumors started to grow again, while PPT-treated tumors remained quiescent for a longer period of time. A pronounced decrease in the mitotic index and an increase in the apoptotic index was associated with tumor regresion. All treated tumors showed: (a) an increase in integrin α6 and Bax expression, (b) an increased stromal laminin redistribution, and (c) a decrease in ERα, Bcl-xl and Bcl-2 expression (P
Effect of Estradiol on Nonmalignant Human Mammary Cells in Primary Culture
Annals of the New York Academy of Sciences, 1986
Most of our knowledge concerning the role of steroid hormones in human mammary carcinogenesis is based on studies of hormone-responsive metastatic breast cancer cell lines.' The in vitro approach should also be fruitful in studying the initial steps of human mammary carcinogenesis, but requires hormone-responsive primary cultures of nonmalignant human mammary cells.
Medical Principles and Practice, 2004
The aim of this work was to analyze the effect of estradiol (E 2), medroxyprogesterone and the two selective estrogen receptor modulators (SERMs) (tamoxifen (Tam) and raloxifene (Ral)) on the estrogen receptor (ER) conformers profile performed by size exclusion HPLC in relation to hormone dependence of mammary tumors. Materials and Methods: Two types of mammary tumors were studied: tumors transplanted in BALB/c mice that are medroxyprogesterone acetate (MPA)-dependent for growth, and tumors induced in Sprague-Dawley rats by intraperitoneal injection of N-nitroso-Nmethylurea (NMU). Tumors from mice treated with MPA, E 2 , Tam or Ral and NMU-treated rats were analyzed and compared to that of control. Results: The tumor conformer profiles were as follows: control and MPA-treated mice showed only one peak (oligomeric form); E 2-treated mice also showed only one peak (dimer); Tam-treated mice showed one peak corresponding to a possible proteolytic fragment, and Ral-treated mice showed two peaks (oligomeric and a possible proteolytic fragment). On the other hand, NMU-induced mammary tumors from rats showed three peaks (oligomeric, monomeric and proteolytic). Conclusion: Our findings may indicate that SERMs affect the aggregation state of ER and thereby its ability to modulate genomic transcription mechanisms related to growth rate.
in Vitro Cellular & Developmental Biology-animal, 2000
The MTW9/PL cell line was established by our laboratory in culture from the carcinogen-induced hormone-responsive MT-W9A rat mammary tumor of a Wistar-Furth (W/Fu) rat. This tumor formed estrogen, androgen, and progesterone responsive tumors in W/Fu rats (Sirbasku, D. A., Cancer Res. 38:1154–1165; 1978). It was later used to derive the MTW9/PL2 cell population which was also estrogen-responsive in vivo (Danielpour, D., et al., In Vitro Cell. Dev. Biol. 24∶42–52; 1988). In the study presented here, we describe serum-supplemented culture conditions in which the MTW9/PL2 cells demonstrate≥80-fold steroid hormone growth responses. All sera used were steroid hormone-depleted by charcoal-dextran treatment at 34°C. The studies were done with horse serum as well as serum from other mammalian species. The growth of the MTW9/PL2 cells was biphasic in response to hormone-depleted serum. Concentrations of ≤5% (v/v) promoted optimum growth. Above this concentration, serum was inhibitory. Concentrations ≥40% (v/v) inhibited growth altogether. Addition of 1.0×10−13−1.0×10−8 M 17β-estradiol (E2) reversed the inhibition completely. At 1.0×10−8 M, estrone, estriol and diethylstilbestrol promoted growth as well as E2. Testosterone and dihydrotestosterone promoted growth only at ≥10−7 M. Progesterone was effective only at≥10−6 M. Cortisol was ineffective. Labeled-hormone-binding analysis and Western immunoblotting documented that MTW9/PL2 cells had estrogen and progesterone receptors but not androgen or cortisol receptors. Estrogen treatment of MTW9/PL2 cells induced a concentration and time dependent increase in progesterone receptors. We conclude (1) the MTW9/PL2 population is the first highly steroid hormone-responsive rat mammary tumor cell line to be established in culture from a carcinogen-induced tumor, and (2) sera from a number of species including horse, rat and human contain an inhibitor which mediates estrogen sensitive MTW9/PL2 cell growth in culture.
Epidermal growth factor in NMU-induced mammary tumors in rats
Breast Cancer Research and Treatment, 1998
In this work we analyze the hypothesis that tumors induced by ip N-nitroso-N-methylurea injection express EGF-like peptides and EGF receptors which could be involved in the response to hormone manipulation. EGF receptors (EGFR) were determined in the purified membrane fraction of tumors from control and ovariectomized (OVX) animals and no significant differences were found in either maximal binding capacities (Q) or dissociation constants (Kd) between them. Neither did we observe differences between tumors that regressed (HR) or continued growing (HU) after ovariectomy. In order to test the ability of EGFR to trigger a biological response we measured the production of second messengers inositol triphosphates (IP3) and cAMP levels; we found that EGF increases IP3 production in a dose-dependent way, while cAMP levels were not affected. In addition, EGF was able to induce in vitro cell proliferation in a concentration-dependent manner when tested in primary cultures of tumor cells by the clonogenic soft agar technique. EGF/TGF-α activity was determined by a radioreceptor assay in tumor cytosols from control and OVX rats. Results showed a trend to lower values in tumors from OVX rats, but no differences between HR and HU tumors. A positive correlation was found between EGF/TGF-α activity and progesterone receptor maximal binding capacity. When we tested the action of estradiol and EGF added together to primary cultures of tumor cells we found an additive effect on cell proliferation. The study of steady state mRNA levels showed that E2 increases PgR and c-myc mRNA levels in HR but not in HU tumors. In conclusion, the autocrine loop EGFR-EGF/TGF-α present in all tumors is hormonally regulated, possibly by Pg, but is not related to the tumor response to ovariectomy.
Cancer research, 1990
The growth control of estrogen-dependent mammary cancer is very complex and only partly understood. The present study was undertaken in order to establish conditions for growth control of MCF-7 cells in monolayer culture with focus on the effect of estradiol-17 beta, fetal calf serum, and growth factors. The effect of charcoal-stripped fetal calf serum (CSFCS) on cell growth was dependent upon the presence of hormones or growth factors in the medium. In the presence of insulin (or insulin-like growth factor 1) and in the absence of estradiol-17 beta, increasing concentrations of CSFCS, 0.625-20%, produced a bell-shaped growth response curve. Serum concentrations greater than 2.5% inhibited cell growth in the absence of estradiol-17 beta, whereas CSFCS in a dose-dependent way up to 10% stimulated growth in the presence of estradiol-17 beta (5 x 10(-10) mol/liter). The growth inhibitory effect of CSFCS could not be demonstrated in the absence of insulin (or insulin-like growth factor ...
Oncology Reports, 2007
Environmental chemicals may be involved in the etiology of breast cancer. There is substantial evidence that breast cancer risk is associated with prolonged exposure to female hormones. Among these hormonal influences a leading role is attributed to the ovarian hormone estradiol, since breast cancer does not develop in the absence of ovaries. The rat mammary gland has special characteristics that make it an ideal organ for studying development, cell proliferation and transformation. In vivo and in vitro model systems for cell proliferation and mammary carcinogenesis have allowed morphological and biochemical analysis under different experimental conditions. The aim of this study was to examine the effect of eserine, an acetylcholinesterase inhibitor, as are the organophosphorous compounds malathion and parathion, and 17ß estradiol on cell proliferation and tumor formation that takes place in the rat mammary gland after in vivo and in vitro treatment. These studies showed that eserine and 17ß estradiol were capable of inducing carcinogenesis in the epithelium of rat mammary glands. It was found that there was a significant increase in the number of cells per duct of the 44-day-old rat mammary gland after the 10-day eserine treatment, compared to the control. A higher increase was observed in the animals treated for 10 days with eserine followed by 30-daily injections of estrogen in comparison to control animals. In 12 animals, two mammary tumors were directly developed in response to 17ß estradiol injected at 39 days of age with a latency period of 180 and 245 days, respectively. Such tumors were metastatic to the lung. These results suggest that terminal end buds are major targets related to rat mammary carcinogenesis and 17ß estradiol can be an initiator and promoter in this process.