Androgen sensitivity of the new human breast cancer cell line MFM-223

The mammary carcinoma cell line MFM-223 is characterized by high androgen and low estrogen and progesterone receptor levels. With the dextran charcoal method, androgen binding was determined at 160 fmol/mg protein corresponding to approximately 100,000 binding sites per cell in whole cell binding assays. The estrogen and progesterone receptor contents were between 8 and 18 fmol/mg protein. The proliferation of MFM-223 cells was significantly inhibited by doses greater than 0.01 nM dihydrotestosterone. The androgenic inhibition of cell proliferation was antagonized by the antiandrogens cyproterone acetate and hydroxyflutamide. In spite of the low estrogen receptor content, MFM-223 cell proliferation was slightly enhanced by 10 nM 17 beta-estradiol. Treatment with 17 beta-estradiol or dihydrotestosterone failed to provoke an increase of the progesterone receptor level. MFM-223 cells have characteristic patterns of isoenzyme polymorphism and of karyotype alterations revealing marker chromosomes and homogeneously staining regions. In the spectrum of human mammary carcinoma cell lines, MFM-223 cells offer a unique model to investigate molecular mechanisms of androgen receptor action.     

The effects of steroid hormones on a human colon cancer cell line in vitro.

Estrogen analogues, moxestrol (10(-8)-10(-5) M) and ethinyl estradiol (10(-8)-10(-6) M), produced a 30% and 15% inhibition of LoVo cell growth, respectively, in serum-free Ham's F-10 medium. Under the same conditions, no growth effects were observed on these cells following the addition of progesterone or testosterone (10(-8)-10(-6) M); however, metribolone (10(-8)-10(-6) M), a synthetic androgen with glucocorticoid receptor-binding properties, moderately stimulated cell growth (18%). The synthetic antiandrogen, RU 23908 (10(-6) M), did not reduce metribolone effects, and hydrocortisone (10(-9)-10(-7) M) stimulated LoVo cell growth by 31% in serum-free medium. In medium containing 10% charcoal-treated fetal bovine serum, the inhibitory effects of estrogens were not observed, and the lower concentrations (10(-11) M) of moxestrol and ethinyl estradiol facilitated cell growth (10 to 15%). The other steroid hormones produced the same results as observed with serum-free medium. These data suggest that estrogen and glucocorticoid hormones may play an important role in the growth of colon carcinoma cells. Androgen and progesterone hormones appear to be less significant in this regard. Serum factors alter the effects of estrogen, but not of glucocorticoids.     

Negative controls of cell proliferation: human prostate cancer cells and androgens

LNCaP cells represent a useful tool to explore the mechanism of sex hormone action on cell proliferation in an "in culture-in animal" model. Results indicated that: (a) these cells were inhibited from proliferating for extended periods (up to 30 days) when placed in charcoal-dextran-stripped sera; they remained, however, viable because they proliferated when sex hormones were added to this medium; (b) the inhibitory effect of sera was reversed by the addition of 5 alpha-dihydrotestosterone at 3 x 10(-10) M, 17 beta-estradiol at 3 x 10(-8) M and higher concentrations, and progesterone at 3 x 10(-10) M and higher concentrations; (c) while the dose response to androgens was biphasic (i.e., 5 alpha-dihydrotestosterone at concentrations higher than 3 x 10(-10) M resulted in progressively lower cell yields), estrogens and progestagens exhibited a monophasic pattern; (d) these cells were exceedingly sensitive to the nutritional environment in which they grew; (e) while these cells have androgen receptors (68 fmol/mg protein; Kd = 2 x 10(-9) M), estrogen and progestagen receptors could not be detected by biochemical and immunocytochemical techniques; (f) tumors grew at the site of inoculation in castrated nude mice carrying 17 beta-estradiol and progesterone pellets and in intact male nude mice implanted with placebo pellets, while tumors did not grow in castrated nude mice implanted with a 5 alpha-dihydrotestosterone pellet. Taken together the data collected are compatible with the following conclusions: (a) the proliferative response in LNCaP cells seems not to be directly mediated by their intracellular androgen receptors; (b) plasma-borne trypsin-sensitive inhibitors of the proliferation of these cells (androcolyone I) appear to play a significant role in the proliferative event; (c) natural and synthetic androgens, estrogens, and progestagens cancelled the inhibition by charcoal-dextran-stripped human sera; (d) only androgens were able to trigger an inhibition of cell proliferation (shutoff effect) at concentrations higher than those that affected maximal cell yields (direct negative hypothesis); and (e) a faulty shutoff response is probably a crucial event for the tumorigenesis of these human prostate cells.     

Proliferation kinetics of a human breast cancer line in vitro following treatment with 17beta-estradiol and 1-beta-D-arabinofuranosylcytosine

The effect of 17beta-estradiol on an estrogen receptor-positive human breast cancer cell line (MCF-7) was studied. Low concentrations (10(-9) M) of 17beta-estradiol enhanced the rate of cell proliferation; the overall cell cycle time was shortened; and the proportion of cells in the S phase increased. Higher concentrations (10(-7) M) suppressed proliferation and slightly decreased the proportion of the cells in DNA synthesis. When combined with 1-beta-D-arabinofuranosylcytosine, an S-phase-specific chemotherapeutic agent, 10(-9) M 17beta-estradiol enhanced cell killing. This enhancement was not observed with 10(-7) M 17beta-estradiol. Kinetic changes caused by hormones have profound implications in clinical therapy, since the efficacy of cycle active agents may be altered.     

Hormone dependency of human breast neoplasms cultured in vitro in the stem cell assay

The stem cell soft agar culture system provides a biological tool with which to study tumor clones derived from heterogeneous tumor cell populations. For testing the feasibility of this approach in identifying hormone-responsive clones of mammary tumors, a study was done to determine whether estrogen deprivation or supplementation would alter colony formation in sequential breast neoplasms received in the Cell Kinetics Laboratory, The Pennsylvania State University. Single-cell suspensions obtained from 22 freshly excised breast neoplasms (20 malignant, 2 benign) were plated in soft agar with and without tamoxifen (Tam) (10(-7) M) with the use of serum-supplemented media and with and without 17 beta-estradiol (E2) (10(-8) M] under serum-free medium conditions. Twenty tumors successfully grew, producing 42 +/- 8 (mean +/- SEM) colonies in control dishes in the presence of serum and 27 +/- 5 in its absence (P less than .001). Tam inhibited colony formation in 78% of malignant tumors, whereas a stimulatory effect was observed with E2 in 94%. An increase in colony formation was induced by Tam in 4 tumors (2 benign and 2 malignant). The effects of E2 and Tam on tumor growth were not influenced by the estrogen and progesterone receptor status of the tumor. These preliminary results suggest that all tumors, irrespective of their receptor status, may contain clones of hormone-responsive cells.     

Effect of androgen on proliferation of estrogen-responsive transformed mouse Leydig cells in serum-free culture

We examined the effects of steroid hormones on the proliferation of transformed mouse Leydig cells (B-1) in serum-free culture condition. Among hormones examined, androgen as well as estrogen enhanced the cell proliferation rate. Hormone binding studies revealed that B-1 cells contained both androgen and estrogen receptors. In addition, androgen-enhanced cell growth was inhibited by antiandrogen, but not by antiestrogen, while estrogen-stimulated cell growth was suppressed by antiestrogen. However, the simultaneous addition of androgen and estrogen did not show an additive effect. Dose-response study on androgen-dependent cell growth revealed that relatively high concentrations (10(-7)-10(-6) M) of dihydrotestosterone were required to obtain the maximum response. This was at least partly explained by the finding that B-1 cells could metabolize dihydrotestosterone into the less active steroids. Finally, B-1 cells were found to grow more rapidly in normal than in castrated male mice. These results clearly indicate that the proliferation of B-1 cells is stimulated by both androgen and estrogen, which utilize the different receptor systems.     

Influence of tamoxifen and estradiol on the growth of human breast cancer cells in vitro

Cells obtained from freshly resected human breast cancer were grown in vitro utilizing the soft agar technique. The effects of adding an antiestrogen (tamoxifen, TAM) and 17 beta-estradiol alone or simultaneously on cell growth were assessed. The addition of TAM (10(-6) M) to the medium resulted in a significant decrease in cell growth in 26 of 36 (72%) estrogen receptor (ER)-positive tumors and in one of 5 ER-negative tumors (20%). The degree of inhibition caused by TAM was significantly higher in the ER-positive tumors that also contain the progesterone receptor (PgR) as compared to those that lacked that receptor (i.e., PgR negative) (46.2 +/- 2% versus 36.2 +/- 1.2% inhibition, P less than 0.01). The simultaneous addition of 17 beta-estradiol (10(-8) M) neutralized the inhibitory effect of TAM (10(-6) M) in the majority of tumors. With the presence of serum in the medium, the addition of 17 beta-estradiol alone resulted in an enhancement of cell growth in 6 of 17 tumors. However, because of the confounding effects of serum in the medium, we studied the individual effect of 17 beta-estradiol (10(-8) M) when added alone under serum-free conditions. Of 20 tumors studied, 17 beta-estradiol significantly enhanced cell growth in 12. There was a 67.8 +/- 12.6% increase in the number of colonies formed in these 12 responding tumors. One of these 12 responding tumors was ER negative as well as PgR negative, while the rest were all ER positive. These in vitro studies demonstrate that this approach can provide valuable information on endocrine mechanisms controlling the growth of human breast cancer.     

Expression of gonadotropin receptor and growth responses to key reproductive hormones in normal and malignant human ovarian surface epithelial cells

Epidemiological data have implicated reproductive hormones as probable risk factors for ovarian cancer (OCa) development. Although pituitary and sex hormones have been reported to regulate OCa cell growth, no information is available regarding whether and how they influence normal ovarian surface epithelial (OSE) cell proliferation. To fill this data gap, this study has compared cell growth responses to gonadotropins and sex steroids in primary cultures of human OSE (HOSE) cells with those observed in immortalized, nontumorigenic HOSE cells and in OCa cell lines. Both malignant and normal cell lines/cultures responded equally well to the stimulatory actions of luteinizing hormone and follicle-stimulating hormone and to 17beta-estradiol and estrone, although the latter estrogen has a much lower affinity for estrogen receptor than does the former estrogen. In normal HOSE cell cultures/lines, 5alpha-dihydrotestosterone was found to be more effective than testosterone in stimulating cell growth, but in OCa cell lines, 5alpha-dihydrotestosterone and testosterone are equally potent. One OCa cell line, OVCA 433, was found to be nonresponsive to androgen stimulation. In general, primary cultures of normal HOSE cells exhibited the greatest hormone-stimulated growth responses (>10-fold enhancement), followed by immortalized HOSE cell lines (4-5-fold enhancement) and by OCa cell lines (2-4-fold enhancement). Interestingly, progesterone (P4), at low concentrations (10(-11) to 10(-10) M), was stimulatory to HOSE and OCa cell growth, but at high doses (10(-8) to 10(-6) M), P4 exerted marked inhibitory effects. In all cases, cotreatment of a cell culture/line with a hormone and its specific antagonist blocked the effect of the hormone, confirming specificity of the hormonal action. Taken together, these data support the hypothesis that reproductive states associated with rising levels of gonadotropins, estrogen, and/or androgen promote cell proliferation in the normal OSE, which favors neoplastic transformation. Conversely, those states attended by high levels of circulating P4, such as that seen during pregnancy, induce OSE cell loss and offer protection against ovarian carcinogenesis.     

Control of cell proliferation: evidence for negative control on estrogen-sensitive T47D human breast cancer cells

The human breast tumor cloned cell lines T47D-A8 and All are estrogen dependent for cell proliferation in the nude mouse model. In contrast, these cells multiplied at similar rates when grown in serum-free cultures, regardless of the presence of 17 beta-estradiol (3 X 10(-11) to 3 X 10(-8) M estradiol). Addition of 10% charcoal-dextran stripped human female serum to the culture medium resulted in a marked inhibition of cell proliferation. The addition of 3 X 10(-11) M estradiol overcame the inhibitory effect of serum. Similar results were obtained with the human breast tumor C7MCF7 cell line. Both cell lines contain similar estrophilin levels. The Kd of the estrophilin-estradiol complex was 0.39 X 10(-10) M for C7MCF7 cells and 4.4 X 10(-10) M for T47D-A11 cells. Maximal cell yields were achieved at 5 X 10(-12) M free estradiol levels in 10% charcoal-dextran stripped serum supplemented medium. These data are compatible with the following interpretation: (a) estradiol-sensitive cells are inhibited from proliferating by a serum-borne factor; and (b) estradiol neutralizes this inhibitory effect. This mechanism seems not to be mediated by estradiol binding to the cellular estrophilins because (a) the free estradiol levels needed for maximal response are significantly lower than the estrophilin Kds, and (b) maximal proliferation rates occur at similar estradiol concentrations for these three cell lines, regardless of the binding properties of their estrophilins.     

Growth-stimulating effect of pharmacological doses of estrogen on androgen-dependent Shionogi carcinoma 115 in vivo but not in cell culture

Shionogi carcinoma 115 (SC115) had been accepted for 20 years as an androgen-dependent mouse mammary tumor, the growth of which is stimulated only by androgen. However, we very recently found that the growth of SC115 tumors in vivo is stimulated not only by physiological doses of androgen but also by pharmacological doses of estrogen through the estrogen receptor system. In the present study, the growth-stimulative effect of estrogen on an androgen-dependent cloned cell line (SC-3) derived from SC115 cells, which showed androgen- and estrogen-dependent growth in vivo, was examined in vitro. In serum-supplemented medium (Eagle's minimum essential medium containing 2% steroid-free fetal calf serum), testosterone or 5 alpha-dihydrotestosterone (10(-9)-10(-6) M) significantly stimulated the growth of SC-3 cells (3.2-fold increase in cell number at day 10 in culture containing 10(-8) M androgens) and changed the shape of SC-3 cells from epithelial to spindle (fibroblast-like), whereas 17 beta-estradiol (10(-12)-10(-6) M) even in high concentrations had no such effects on SC-3 cells. Contrary to the effect of 17 beta-estradiol in vivo, 17 beta-estradiol as well as cyproterone acetate (10(-8)-10(-6) M) inhibited the growth-stimulative effect of testosterone (10(-8) M) on SC-3 cells in a dose-dependent manner in the serum-supplemented medium. The anti-androgen and 17 beta-estradiol also showed comparable competitive effects on [3H]testosterone binding to androgen receptor in SC-3 cells. In serum-free medium [Ham's F-12:Eagle's minimum essential medium (1:1, v/v) containing 0.2% bovine serum albumin], testosterone [10(-8) M] also markedly stimulated the growth of spindle-shaped SC-3 cells, and epidermal growth factor (1 ng/ml) enhanced the growth-stimulative effect of testosterone, whereas 17 beta-estradiol (10(-8)-10(-6) M) in the absence or presence of epidermal growth factor had no growth-stimulative effect on SC-3 cells. We conclude that the growth of SC115 cells is stimulated by either physiological doses of androgen or pharmacological doses of estrogen in vivo but only by androgen in cell culture.     

Long-term cultivation of a human breast cancer cell line, MCF-7, in a chemically defined medium. Effect of estradiol

The human breast cancer cell line, MCF-7, has been adapted to long-term growth in chemically defined medium without loss of estrogen and progesterone receptors or tumorigenicity in athymic mice. An estrogen reversible inhibition of cell proliferation is exerted by newborn calf serum (NCS) 10%, athymic mouse serum (AMS) 2% or tamoxifen 0.1 to 1.0 microM. The mamma-related hormones, hydrocortisone, progesterone, prolactin, and insulin could not mimic this growth inhibitory effect, and estradiol alone or combined with these hormones did not stimulate cell proliferation in chemically defined medium.     

Characterization of estrogen and progesterone receptors and the dissociated regulation of growth and progesterone receptor stimulation by estrogen in MDA-MB-134 human breast cancer cells

We have examined the properties of the estrogen receptor and progesterone receptor in MDA-MB-134 human breast cells and have evaluated the effects of estrogen on cell proliferation and progesterone receptor levels in these cells as indices of hormonal sensitivity. These cells contain high levels of estrogen receptor (approximately 1.5 pmol/mg DNA) and low levels of progesterone receptor (0.15 pmol/mg DNA). More than 80% of the estrogen receptor is found in the nuclear fraction in the absence of estrogen, and the Kd of the receptor for estradiol is approximately 1.5 X 10(-10) M. Upon exposure to estradiol, the receptors become occupied, but there is no processing or apparent decrease in either nuclear or total cellular estrogen receptor content, as can be seen in MCF-7 human breast cancer cells. The nuclear estrogen receptor sediments as a 4.6 S species on high salt sucrose gradients, and it can be detected on sodium dodecyl sulfate-polyacrylamide gel immunoblot analysis as a species of molecular weight 65,000, identical to that of the MCF-7 estrogen receptor, using the monoclonal antibodies D75P3 gamma and H222Sp gamma prepared against the MCF-7 estrogen receptor. The estrogen receptor shows binding selectivity for estrogens and antiestrogens, and its affinity for ligands follows the order diethylstilbestrol (190%) greater than estradiol (100%) greater than estriol (13%) greater than tamoxifen (3%), as expected for estrogen receptor. Hence the receptor appears normal in many of its physicochemical properties and in terms of its binding affinity and specificity for estrogens and antiestrogens. Control cells contain low levels of progesterone receptor that display high affinity (Kd = 6 X 10(-9) M) for the synthetic progestin R5020, but exposure to estradiol (10(-11)-10(-7)M) fails to increase cellular progesterone receptor levels. In contrast, estradiol markedly stimulates the rate of cell proliferation, while tamoxifen suppresses the growth of control and of estradiol treated cells. Hence, our data show that these cells, which contain substantial levels of estrogen receptor, respond to estrogen with enhanced cell proliferation but fail to have their progesterone receptor level modulated by estradiol. These cells represent an interesting and unusual situation in which estrogenic regulation of proliferation and the stimulation of progesterone receptor are dissociated. These cells should prove useful in further evaluation of estrogenic regulation of cell proliferation and specific protein synthesis in human breast cancer.     

In vitro effects of estradiol,dydrogesterone, tamoxifen and cyclophosphamide on proliferation vs. death in human breast cancer cells

The effects of 17 beta-estradiol, dihydrodydrogesterone, tamoxifen and cyclophosphamide upon parameters of cell maturation (Mucine1 expression), cell proliferation (Cyclin D1 expression) and apoptosis (loss of nuclear DNA) were studied in estrogen receptor positive (ER+) and negative (ER-) human breast cancer cells. Tamoxifen was the most potent inducer of apoptosis in ER+ and ER- breast cancer cells. 17 beta-estradiol in a concentration of 10(-6) M induced proliferation in ER+ cells after 144 h. incubation, while equimolar co-incubation with dihydrodydrogesterone prevented this effect and even induced a significant increase of cell death. It is speculated that the continuous use of combined 17 beta-estradiol plus dihydrodydrogesterone might be given as hormone replacement therapy without increased risk of breast cancer and even may reduce the relapse rate in breast cancer patients.     

Growth-inhibitory action of an estrogen-chlorambucil conjugate (KM2210) in human breast cancer cell line MCF-7: its relation to reduction of estrogen receptor and transforming growth factor-alpha secretion.

We investigated the effects of a benzoate of an estradiol-chlorambucil conjugate (KM2210) and chlorambucil on growth, estrogen receptor, and secretion of transforming growth factor (TGF)-alpha in the hormone-dependent human breast cancer cell line MCF-7. In the presence of 10(-10)-10(-6) M KM2210, the estrogen-induced growth of MCF-7 was completely inhibited. Inhibited growth of MCF-7 treated with 10(-8) or 10(-6) M KM2210 for 4 days was not rescued by removal of the drug and the addition of estradiol. By treatment of MCF-7 with KM2210 for 4 days, estrogen receptor-binding sites were decreased at 10(-8) M and were not detected at 10(-6) M but were unaltered by 10(-8) M chlorambucil. Moreover, estrogen receptor immunoreactivity and the level of estrogen receptor mRNA were decreased through treatment with 10(-6) M KM2210 for 4 days. These suppressions occurred prior to the onset of inhibitory action on MCF-7 growth. Secretion of TGF-alpha from MCF-7 was decreased by 4 days of treatment with 10(-8) and 10(-6) M KM2210 but not with chlorambucil. The addition of exogenous TGF-alpha generally restored the growth of MCF-7 treated with 10(-8) M KM2210. We concluded that KM2210 has irreversible or at least long-standing inhibitory effect on estrogen-dependent growth of MCF-7. It is conceivable that the decrease of estrogen receptor renders the cell unable to respond to estrogen with increased TGF-alpha secretion and succeeding cell growth.     

Biological characteristics and estrogen and progesterone receptors in mammary carcinoma induced in male Sprague-Dawley rats by a series of intragastric intubations of 7,12-dimethylbenz(a)anthracene

Biological characteristics and estrogen (ER) and progesterone (PR) receptors were studied in male mammary carcinomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) in male inbred Sprague-Dawley rats (MM). DMBA-induced carcinomas in females (MF) were used as controls. In 36 of 44 female rats given 20 mg DMBA once by gastric intubation at 50 days of age, MF with microscopic characteristics of cystic papillary adenocarcinoma developed 124 +/- 49 (S.D.) days after challenge. In all of the 42 male rats given 10 mg DMBA at 14-day intervals for 14 weeks starting from 28 days of age. MM with microscopic characteristics of medullary adenocarcinoma developed 106 +/- 21 days after the first intubation of DMBA. The growth of primary MM was unaffected by orchiectomy or estrogen. Eighty to 100% of the MM transplanted in the four groups could grow in intact female rats, ovariectomized female rats, intact male rats, and castrated male rats, while the transplanted MF could grow only in intact female rats. The histology of MM was unchanged in primary and transplanted tumors under various hormonal conditions. ER were present in almost all of the hormone-independent primary and transplanted MM, although the levels for cytosol ER in MM were significantly lower than those in MF. Injection of 10 micrograms 17 beta-estradiol induced marked synthesis of PR in primary and transplanted MM, even 24 and 48 hr after the 17 beta-estradiol injection. These findings show that MM are hormone independent but, like hormone-dependent female tumors, contain ER and estrogen-dependent PR.     

Hormonal modulation of plasminogen activator: an approach to prediction of human breast tumor responsiveness

We have determined that the primary reason for the frequently encountered poor survival of human scirrhous breast carcinomas in short-term (4 days) organ culture is mechanical injury to the tumor tissue during explant preparation. It was possible to minimize this injury by preparing 0.5-mm-thick slices using very sharp blades. This resulted in much improved preservation of tissue structure and function, as assessed by histology, DNA content, and enzyme synthesis and secretion. With the exception of insulin, which was always present in the culture medium, exogenous hormones, including estrogen, or serum did not further improve explant preservation. In rodent mammary tumors, growth in vivo and production of the serine protease plasminogen activator (PA) in organ culture are coordinately regulated by hormones, suggesting that PA may be a valuable indicator of tumor hormone responsiveness. We have now tested the effect of estrogen and other hormones on PA secretion in organ cultures of primary human breast carcinomas. We found that: modulation of PA by 17-beta-estradiol (10-8) M) occurred only in carcinomas which were positive for both estrogen and progesterone receptors; of 21 such tumors, 11 (52%) were responsive. Plasminogen activator was not modulated by estradiol in any of the 22 tumors which were negative for one or both receptors; hydrocortisone (10(-7) M) effectively inhibited, and 3,5,3'-L-triiodothyronine (10(-8) M) and adenylate cyclase activators effectively stimulated PA in most breast tumors, regardless of their estrogen and progesterone receptor status. Prolactin (5 micrograms/ml) had no effect when tested alone; urokinase-type PA was found to be the principal PA produced by human breast tumors. Changes in its rate of synthesis and secretion and not in the content of PA inhibitors appeared to be the prevailing mechanism of enzyme regulation by hormones. In summary, short-term organ culture coupled with the use of PA as an index of response appears to be a promising approach to the study of hormone sensitivity of primary human breast carcinomas.     

Growth inhibition by progestins in a human endometrial cancer cell line with estrogen-independent progesterone receptors

The presence of estrogen-independent progesterone receptors (PgR) was demonstrated in a subline of a human endometrial cancer cell line, Ishikawa cells, although the original Ishikawa cells contained estrogen-inducible PgR. Scatchard plot analysis of cytoplasmic binding data in our subline (IK-90) revealed a high affinity binding site for R5020 (Kd, 1.0 nM) with maximum binding sites of 158 fmol/mg protein. Competition experiments showed a binding specificity similar to that of typical PgR. By low-salt sucrose gradient centrifugation, radioactive 8S and 4S peaks were found. The addition of 1 microM progesterone in culture medium resulted in a rapid nuclear translocation of cytoplasmic PgR. In contrast to the original cells, estrogen receptors could not be detected in IK-90 cells, and an addition of 17 beta-estradiol (10 nM) to culture medium failed to increase PgR. Accumulation of glycogen in cytoplasm of IK-90 cells in response to R5020 (0.1-1 microM) was observed by periodic acid-Schiff staining. The addition of R5020 to culture medium (0.1-1 microM) also caused a marked decrease in the growth of IK-90 cells, whereas the other steroids including 17 beta-estradiol, tamoxifen, testosterone, and cortisol had no significant effects. These results demonstrate for the first time the presence of a progestin-responsive human endometrial cancer cell line that contains estrogen-independent functional PgR. IK-90 cells appear to be an ideal model for studying the mechanism of the antiproliferative effect of progestin on endometrial cancer cells.     

On the mechanism of hormone action in 7,12 dimethylbenz(a)anthracene-induced mammary tumor. I. Prolactin and progesterone effects on estrogen receptor in vitro.

The presence of ER in DMBA-tumors was demonstrated by the use of dextran-charcoal assay, sephadex chromatography, sucrose gradient sedimentation, and organ culture techniques. It was found that tumors have binding sites ranging from 10-13 to 10-15 moles/mg protein, and a dissociation constant of ER 10-9 to 10-10 M. In experiments with tumor explants, prolactin-insulin significantly stimulated ER binding capacity, as compared with control without prolactin. This stimulation was tissue-specific and inhibited by progesterone. Insulin had a synergistic effect on prolactin stimulation of ER. Our results presents a plausible explanation for tumor responses to these hormones in vivo. This interaction of prolactin, estrogen, and progesterone may be a common phenomenon for all estrogen-responsive tissues.     

Endocrine-responsive pancreatic carcinoma: steroid binding and cytotoxicity studies in human tumor cell lines

We have begun to investigate the steroid responsiveness of pancreatic cancer by comparing human (MiaPaCa, Colo-357, RWP-1, RWP-2) and rodent (AR42j) pancreatic tumor cell lines with cultured estrogen receptor-positive breast cancer cells (MCF-7, T47-D). The four human pancreatic tumors contain measurable levels of specific estradiol binding sites with dissociation constants (Kd) that range from 1 to 9 nM, in contrast to the higher-affinity binding sites measured in the breast cancer cells (Kd less than or equal to 1 nM). Growth of one pancreatic tumor line (MiaPaCa) is stimulated 40% above control by exposure to nanomolar concentrations of estradiol, suggesting that the estrogen receptor in these cells is functioning like that in MCF-7 and T47-D cells. Glucocorticoids (dexamethasone, hydrocortisone) and androgen (fluoxymesterone) stimulate proliferation of Colo-357 cells by as much as 30%. Paradoxically, glucocorticoids inhibit AR42j cells to less than 50% of control growth. Micromolar exposures of estrogen (17 beta-estradiol), antiestrogen (tamoxifen), antiandrogen (dehydroxyflutamide), progestins (progesterone, R5020, medroxyprogesterone acetate), and inhibitors of steroid-metabolizing enzymes (17 beta-N,N-diethylcarbamyl-4-methyl-4-aza-5 alpha-androstan-3-one, danazol) impair growth of these pancreatic tumors to varying degrees, and with little relationship to estrogen receptor content. In general, progestins are slightly more growth inhibiting to these pancreatic tumor lines than the other endocrine agents tested, including tamoxifen. Only the RWP-2 cells appear completely resistant to steroidal therapy, showing less than 25% growth inhibition with exposure to therapeutic concentrations (less than or equal to 2.5 microM) of these agents. Colo-357, MiaPaCa, and AR42j cells are most responsive to these endocrine agents, and their overall pattern of sensitivity suggests that the steroid-dependent growth-inhibitory mechanisms of some pancreatic carcinomas may involve both receptor antagonism and direct inhibition of steroidal oxidoreductases. 17 beta-N,N-Diethylcarbamyl-4-methyl-4-aza-5 alpha-androstan-3-one, a potent inhibitor of 5 alpha-reductase with minimal affinity for androgen receptor, inhibits growth of Colo-357 cells to less than 40% of control and also inhibits AR42j and MiaPaCa cells. Dehydroxyflutamide, a potent androgen receptor antagonist with no direct influence on 5 alpha-reductase activity, inhibits growth of MiaPaCa and AR42j cells but has no affect on Colo-357 growth.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture.

We have established or characterized six lines of human breast cancer maintained in long-term tissue culture for at least 1 year and have examined these lines for estrogen responsiveness. One of these cell lines, MCF-7, shows marked stimulation of macromolecular synthesis and cell division with physiological concentrations of estradiol. Antiestrogens are strongly inhibitory, and at concentrations greater than 3 X 10(-7) M they kill cells. Antiestrogen effects are prevented by simultaneous treatment with estradiol or reversed by addition of estradiol to cells incubated in antiestrogen. Responsive cell lines contain high-affinity specific estradiol receptors. Antiestrogens compete with estradiol for these receptors but have a lower apparent affinity for the receptor than estrogens. Stimulation of cells by estrogens is biphasic, with inhibition and cell death at concentrations of 17beta-estradiol or diethylstilbestrol exceeding 10(-7) M. Killing by high concentrations of estrogen is probably a nonspecific effect in that we observe this response with 17alpha-estradiol at equivalent concentrations and in the otherwise unresponsive cells that contain no estrogen receptor sites.