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.     

Effect of estrogen and antiestrogen on the human breast cancer cell line MCF-7 adapted to growth at low serum concentration

The human breast cancer cell line MCF-7 has been adapted to long-term growth at 0.5 and 0.05% fetal bovine serum. Free cytoplasmic and filled nuclear estrogen receptors were found in cells grown at 5, 0.5, and 0.05% fetal bovine serum. Cells grown in medium with 0.05% dextran-charcoal-treated serum contained cytoplasmic receptors but no filled nuclear receptors, indicating that this medium did not contain biologically active concentrations of estrogen. Addition of estradiol to the medium translocated the cytoplasmic receptor to the nucleus and stimulated progesterone receptor synthesis but did not increase the growth rate. The antiestrogen tamoxifen (TAM) inhibited cell growth at all serum concentrations investigated, at least in part by a reduction of the growth fraction. The sensitivity to TAM was highest at low serum concentrations. The effect of TAM could be reversed by estradiol at TAM concentrations of 10(-6) M or lower. It is concluded that estradiol does not have a direct growth-stimulatory effect on our MCF-7 cells in monolayer cultures although the cells contain functional estrogen receptors and growth of the cells in athymic mice requires estrogens. TAM has an estrogen-competitive, inhibitory effect on the growth of MCF-7 cells at concentrations lower than 10(-6) M. At higher concentrations, the growth inhibition is unspecific and noncompetitive by estradiol.     

Effect of triamcinolone acetonide on the growth of NEL-M1 human melanoma cells cultured in the presence and absence of growth stimulatory agents

The human melanoma cell line NEL-M1 proliferates in Ham's F-12 medium in the absence of serum, hormones, and growth factors. Culturing NEL-M1 cells in defined medium supplemented with insulin (5 micrograms/ml) and transferrin (5 micrograms/ml) results in a 94% increase in [3H]thymidine incorporation after 24 h and a 6- to 8-fold increase in cell number after 5 days. The addition of 17 beta-estradiol, testosterone, and progesterone to defined medium, either as single agents or in combination with insulin and transferrin, had no effect on cell growth. No specific estrogen, androgen, or progesterone receptor proteins were detected in NEL-M1 cells. In contrast, the synthetic glucocorticoid triamcinolone acetonide (10 nM) inhibited growth of NEL-M1 cells in serum-free Ham's F-12 medium by 50%. The 6- to 8-fold stimulation of cell growth by insulin and transferrin was reduced to 1.75-fold when triamcinolone acetonide (10 nM) was added to the medium. Additional studies show that medium conditioned by NEL-M1 cells stimulated [3H]thymidine incorporation into cellular DNA of NEL-M1 cells and increased the growth of NEL-M1 cells 3-fold over cells maintained in defined medium. The addition of triamcinolone acetonide (10 nM) to defined medium supplemented with conditioned medium resulted in only a 1.48-fold increase in cell number. These results show that triamcinolone acetonide inhibits growth of NEL-M1 human melanoma cells in serum-free defined medium, medium supplemented with insulin and transferrin, and medium supplemented with an endogenous growth stimulatory factor(s). Thus, NEL-M1 cells are an excellent model system to study the mechanism of action of glucocorticoids and also the interplay between exogenous hormones and growth factors and endogenous growth factors.     

Proliferation, hormonal responsiveness, and estrogen receptor content of MCF-7 human breast cancer cells grown in the short-term and long-term absence of estrogens

We have examined the effect of short-term and long-term growth in the absence of estrogens on the proliferation rate and estrogen and antiestrogen responsiveness of MCF-7 human breast cancer cells. The removal of phenol red, the pH indicator in tissue culture medium that is weakly estrogenic (Y. Berthois et al., Proc. Natl. Acad. Sci. USA, 83:2496-2500, 1986), immediately slows the cell proliferation rate, and MCF-7 cells grown in phenol red-free medium with charcoal dextran-treated serum for periods up to 1 mo maintain this reduced rate of cell proliferation. In these short-term phenol red-withdrawn cells, estradiol stimulates proliferation markedly and reproducibly, and antiestrogens inhibit estrogen-stimulated proliferation. Antiestrogens by themselves appear as partial agonists/antagonists; at low concentrations they stimulate proliferation weakly, but they show no stimulation at the high concentrations where they fully inhibit estrogen-stimulated proliferation. In contrast to the short-term phenol red-withdrawn cells, cells maintained for several months (5 to 6 mo) in the apparently complete absence of estrogens (no phenol red, with charcoal dextran-treated calf serum) show a markedly increased basal rate of cell proliferation; estradiol is unable to increase this rate of proliferation further, but antiestrogens are able to decrease proliferation. This change in growth pattern is associated with a 3-fold increase in cellular estrogen receptor levels. Despite their differing basal growth rates, cells grown in either the short-term (less than 1 mo) or long-term (greater than 6 mo) absence of estrogens both have progesterone receptor levels that are very low and, in both cases, estradiol increases progesterone receptor levels markedly. Thus, under long-term estrogen-free conditions, there is a dissociation between the stimulation of cell proliferation and of specific protein synthesis (progesterone receptor) by estrogen. The increase in the cell proliferation rate observed in cells grown in the long-term absence of estrogen may reflect altered regulation of growth factor production or altered sensitivity to growth factors in the medium or produced by the cells themselves. Hence, these breast cancer cells adapt significantly to long-term growth in estrogen-free conditions, an observation that may be relevant to understanding the growth of hormone-responsive human breast cancers in vivo.     

The human squamous cervical carcinoma cell line, HOG-1, is responsive to steroid hormones.

Growth of the human squamous cervical carcinoma cell line, HOG-I, was stimulated in response to oestradiol in serum-containing and chemically defined medium. The oestradiol-stimulated growth could be inhibited by 4-OH tamoxifen, progesterone and medroxyprogesterone acetate; the last 2 compounds also inhibited basal cell growth in serum-containing and chemically defined media. The data are consistent with the sensitivity of human squamous cervical cancer to sex-steroid hormones and suggest that endocrine therapies may be of benefit in this disease.     

Establishment and characterization of the human cholangiocarcinoma cell line HChol-Y1 in a serum-free, chemically defined medium

A human cholangiocarcinoma cell line, designated as HChol-Y1, was established in a protein-free, chemically defined medium after a very short period of primary culture in 0.1% fetal bovine serum (FBS)-containing medium. The cell line has been propagated in this medium for 2 years. The cells grew as a monolayer and the doubling time was about 52 hours. Addition of FBS did not stimulate cell growth (population-doubling time = 50 hr) or increase saturation density. The cells grown in a protein-free medium secreted small amounts of carcinoembryonic antigen (CEA) and large amounts of carbohydrate antigen (CA) 19/9 (CEA: 12.5 +/- 2.1 ng/10(6) cells/48 hr; CA 19/9: 760 +/- 52 IU/10(6) cells/48 hr); these tumor markers were immunohistochemically demonstrated in HChol-Y1 cells. Addition of FBS slightly stimulated the production of CEA and CA 19/9. The HChol-Y1 cell line was xenotransplantable in athymic nude mice and increased the serum CEA and CA 19/9 levels in the tumor-bearing nude mice. For determination as to whether a human carcinoma cell line can proliferate and secrete CEA and CA 19/9 in synthetic medium without any protein supplements, the cells were cultivated long term (2 yr) in a protein-free, chemically defined medium. When this method of cultivation is used, it is easy to purify these substances from spent medium, because contaminating antigens such as FBS or other substances usually added to cultures are absent.     

Inhibition of T47D human breast cancer cell growth by the synthetic progestin R5020: effects of serum,estradiol, insulin,and EGF

Summary The mechanism of the antiproliferative effects of progestins on human breast cancer cells is not known. In view of the ability of estrogen to stimulate human breast cancer cell production of peptide growth factors, and since previous studies have suggested that the inhibitory action of progestins is dependent on estrogen-stimulated growth, the present study examined the interaction of growth factors and the synthetic progestin R5020 on the proliferation of T47D human breast cancer cells. In this study, the concentrations of estradiol, insulin, and EGF for optimal stimulation of T47D cell growth in 3% dextran-charcoal treated fetal bovine serum (DCC-FBS) were determined to be 1 nM, 100 nM, and 1 nM, respectively. Furthermore, incubation with these optimal concentrations of estradiol, insulin, and EGF in various combinations produced additive effects on T47D cell proliferation, suggesting that these agents act, at least in part, by different mechanisms. In contrast, in a chemically defined medium (DM), both estradiol and EGF were unable to stimulate T47D cell proliferation. In the case of estradiol, the inability to demonstrate stimulation of T47D cell growth in DM was not due to down-regulation of the estrogen receptor.R5020 inhibited the growth of T47D cells, although its effect was more marked in the presence of 3% DCC-FBS than in DM. Stimulation of T47D cell growth by either estradiol or insulin in 3% DCC-FBS was effectively inhibited by R5020. In contrast, growth of T47D cells stimulated by EGF in the absence of estradiol was not markedly inhibited by R5020, the growth being comparable to that of untreated control cells. These findings suggest that the inhibitory effect of R5020 on T47D cell proliferation is dominant over the action of some, but not all, breast cancer mitogens.     

Regulation of DNA synthesis and growth of cells derived from primary human meningiomas

We have studied the effects of insulin, epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor, and steroid hormones (estradiol, progesterone, and cortisol) on human meningioma cell proliferation and DNA synthesis in a serum-free culture system. The growth factors, particularly EGF and FGF, increased DNA synthesis in a dose-dependent manner as measured by [3H]thymidine incorporation, and they stimulated submaximal cell proliferation. No individual factor or combination of factors was able to successfully reproduce the effects of 10% fetal calf serum (FCS) on cell growth, although a combination of platelet-derived growth factor (5 units/ml) and EGF (10 ng/ml) synergistically stimulated DNA synthesis to near maximal levels. In addition, serum dependency was observed in studies involving the mitogenic effects of insulin, EGF, or FGF. Both EGF and FGF (10 ng/ml) maximally stimulated cell growth in the presence of 5% FCS. The effects of steroid hormones on cell proliferation, individually or in combination with growth factors or charcoal-treated FCS, were also evaluated. Estradiol (100 nM) significantly increased cell number over control values only in the presence of charcoal-treated FCS; no effects of progesterone or cortisol on cell proliferation were observed. In conclusion, both EGF and FGF stimulated cell proliferation and DNA synthesis in human meningioma cultures in a serum-free system, whereas steroid hormones were without effect. It appears that additional serum components are required for both estradiol-stimulated growth and for maximal proliferation of human meningioma cells under serum-free conditions.     

A new human breast carcinoma cell line (PMC42) with stem cell characteristics. III. Hormone receptor status and responsiveness

PMC42 is a new human breast carcinoma cell line. In this report the content of estrogen, progesterone, and glucocorticoid receptors in PMC42 has been determined. The estrogen receptor content (1,750 cytoplasmic sites and 350 nuclear sites) was lower than that described for MCF7 and T47D. The cells would not proliferate in serum-free medium without the addition of beta-estradiol (optimum concentration 10(-8) M) or progesterone (5 X 10(-8) M). The addition of both hormones induced a more than additive increase in proliferation (P less than .005, n = 18). Similarly, addition of insulin or hydrocortisone induced proliferation; however, in this case, the effect of the hormones together was only additive. The addition of tamoxifen (10(-6) M) led to a significant decrease in cell numbers and inhibited the stimulatory effects of 10(-8) M beta-estradiol.     

Influence of hormones on N-(4-hydroxyphenyl) retinamide inhibition of 7,12-dimethylbenz[alpha]anthracene transformation of mammary cells in organ culture

Influence of estrogen and progesterone on the inhibitory action of N-(4-hydroxyphenyl) retinamide (4-HPR) was examined during the promotional stage of 7,12-dimethylbenz[alpha]anthracene (DMBA) transformation of the epithelial cells in culture of the whole mammary organs of BALB/c mice. In medium containing insulin, prolactin, hydrocortisone, and aldosterone, 4-HPR caused 68% inhibition of transformation as determined by the presence of nodule-like alveolar structures in the glands exposed to DMBA in vitro. Addition of estrogen and progesterone to the medium reduced this pronounced inhibitory action of 4-HPR to only 15%. While the medium containing insulin, prolactin, growth hormone, estrogen and progesterone was highly conducive to DMBA transformation, 4-HPR inhibition of transformation was limited to only 21%. The antagonistic action of the ovarian steroid hormones was present also at the level of frequency of nodule-like alveolar lesions (NLAL) per gland. Although both ovarian hormones reduced the inhibitory action of 4-HPR, on mammary cell transformation, the antagonistic action of estrogen was noticeably more pronounced.     

Estrogen induced growth of human breast cancer cells (MCF-7) in athymic nude mice is enhanced by secretions from a transplantable pituitary tumor

MCF-7, a human breast carcinoma cell line, was maintained s.c. in female athymic nude mice for a period of 5–6 weeks. Administration of estrogen (s.c. pellet of 17β-estradiol and estrone in drinking water, 0.5 mg/l) to these mice resulted in sustained (P < 0.001) growth of MCF-7 tumors. Grafting of a prolactin and growth hormone secreting rat pituitary tumor to the estrogen-treated mice resulted in an increased (P < 0.05) rate of MCF-7 tumor growth. MCF-7 did not grow in athymic nude mice grafted with rat pituitary tumor alone or in mice without hormone treatment (controls). Thus, secretions of pituitary hormones alone are not capable of promoting in vivo growth of MCF-7 although such secretions significantly enhance estrogen-induced growth of this cell line. A synergism between pituitary hormones and estrogen for in vivo growth of a human breast carcinoma has been demonstrated in this study.     

Evidence for a novel pituitary factor that potentiates the mitogenic effect of estrogen in human breast cancer cells

Estrogen, prolactin, and other tissue-derived factors are implicated in the etiology and pathophysiology of human breast cancer (HBC). In a previous study, we demonstrated that a factor(s) secreted by rat pituitary tumor cells (GH3) synergizes with estrogen to induce growth of HBC cells (T-47D) transplanted into athymic nude mice. The present studies were carried out to characterize further this pituitary growth factor. Pituitary tumor cell lines (GH3, GH1, 235-1, and AtT-20) and normal rat pituitaries were transplanted s.c. into estrogen-treated (estradiol valerate injection, 500 micrograms/14 days) athymic nude mice which also received T-47D cells. The influence of the presence of these normal and tumorous pituitary cells on growth (size and weight) of T-47D tumors was monitored for 49 to 56 days. The results indicate that factor(s) from normal rat pituitary glands as well as from the GH1 and GH3 but not 235-1 and AtT-20 pituitary tumor cells were able to potentiate the growth of T-47D tumors in estrogenized mice. To ascertain whether or not prolactin and/or growth hormone are responsible for the growth-promoting activity, purified human and ovine growth hormone and ovine prolactin were administered to estrogenized athymic nude mice either by daily s.c. injection (100 micrograms/day) or by constant infusion using Alzat osmotic minipumps (1.25 and 5.0 micrograms/h) for 29 to 56 days. None of these treatments stimulated the growth of the T-47D tumors, suggesting that prolactin, growth hormone, and their intermediates may not be directly involved. We further determined whether the factor from pituitary tumor cells was present in serum-free conditioned medium and could stimulate the growth of HBC cells in vitro. Conditioned medium from GH3 and GH1 but not from 235-1 and AtT-20 pituitary cells significantly stimulated growth of T-47D cells in the presence of estradiol (10(-10) M) after 12 days of culture in a serum-free medium (Dulbecco's modified Eagle's medium containing bovine serum albumin, 0.5 mg/ml). Optimal serum-free growth of T-47D cells (2-fold above control) was observed in the presence of estradiol (10(-10) M) and conditioned medium (30% v/v) from 48-h cultures of GH3 cells. The bovine serum albumin concentration of the serum-free medium (Dulbecco's modified Eagle's medium) was also important: optimal T-47D cell proliferation was observed with BSA between 0.5 and 2.0 mg/ml. Conditioned medium preparations from serum-pretreated flasks (without cells) from GH3 cell monolayers for zero time and from actinomycin D plus cycloheximide-inhibited GH3 cells were inactive.(ABSTRACT TRUNCATED AT 400 WORDS)     

Loss of mammary epithelial prolactin receptor delays tumor formation by reducing cell proliferation in low-grade preinvasive lesions

Top quartile serum prolactin levels confer a twofold increase in the relative risk of developing breast cancer. Prolactin exerts this effect at an ill defined point in the carcinogenic process, via mechanisms involving direct action via prolactin receptors within mammary epithelium and/or indirect action through regulation of other hormones such as estrogen and progesterone. We have addressed these questions by examining mammary carcinogenesis in transplants of mouse mammary epithelium expressing the SV40T oncogene, with or without the prolactin receptor, using host animals with a normal endocrine system. In prolactin receptor knockout transplants the area of neoplasia was significantly smaller (7 versus 17%; P < 0.001 at 22 weeks and 7 versus 14%; P = 0.009 at 32 weeks). Low-grade neoplastic lesions displayed reduced BrdU incorporation rate (11.3 versus 17% P = 0.003) but no change in apoptosis rate. Tumor latency increased (289 days versus 236 days, P < 0.001). Tumor frequency, growth rate, morphology, cell proliferation and apoptosis were not altered. Thus, prolactin acts directly on the mammary epithelial cells to increase cell proliferation in preinvasive lesions, resulting in more neoplasia and acceleration of the transition to invasive carcinoma. Targeting of mammary prolactin signaling thus provides a strategy to prevent the early progression of neoplasia to invasive carcinoma.     

Transplantation of human breast epithelia to mammary-gland-free fat-pads of athymic nude mice: influence of mammotrophic hormones on growth of breast epithelia

Normal human breast tissue was enzymatically dissociated and the cells were injected into the gland-free fat-pads of athymic nude mice. Within 30 days, small, spherical, duct-like epithelial elements (organoids) formed in 68% of the fat-pads inoculated (0-23 organoids/fat-pad). Short-term (30-day) treatment of the host mice with mammotrophic hormones [secretions from a chorionic, soamto-mammotrophin-secreting transplantable human choriocarcinoma (JEG-3), secretions from a prolactin- and growth-hormone-secreting transplantable rat pituitary tumor (GH3), estrogen and/or progesterone] and/or cAMP inducers (cholera toxin) significantly (p less than 0.05) increased the size of the human breast organoids but did not increase organoid number or induce extensive and expansive growth (extensive duct elongation and branching) of these structures. Such treatments induced intense proliferation of the host mouse mammae resembling that which occurs during late pregnancy. The results of this study, therefore, provide evidence that normal human breast epithelium can be readily accepted by and maintained in the gland-free fat-pad of the athymic nude mouse, and the epithelium, within 30 days, forms spherical duct-like structures (organoids). The human breast organoids are hormone-responsive, as they respond to a mammotrophic growth stimulus by an increase in size. The failure of the human breast organoids to grow expansively in the gland-free fat-pad of this immunologically deficient mouse does not appear to be due to the absence of an appropriate hormonal growth stimulus.     

Characterization of a receptor-negative, hormone-nonresponsive clone derived from a T47D human breast cancer cell line kept under estrogen-free conditions

We have established an estrogen receptor- and progesterone receptor-negative, hormone-nonresponsive breast cancer cell line from a receptor-positive, hormone-responsive line grown under estrogen-free conditions. T47D breast cancer cells were cultured under estrogenized conditions (in phenol red-containing medium supplemented with whole fetal bovine serum) and cloned to produce line T47D:A18. The parental T47D line was also estrogen deprived (in phenol red-free medium supplemented with dextran-coated charcoal-treated fetal bovine serum) for more than 1 year and subsequently clone T47D:C4 was established. T47D:A18 was estrogen receptor and progesterone receptor positive as determined by both ligand binding assay analysis and enzyme immunoassay analysis. T47D:C4 cells were estrogen receptor and progesterone receptor negative and mRNA for these receptors was not detected. Incubation of hormone-responsive T47D:A18 cells with 17 beta-estradiol caused a 3-fold increase in cell growth over 8 days when compared to control. This stimulation of growth was completely inhibited by the anti-estrogens 4-hydroxytamoxifen (0.1 microM) and ICI 164,384 (1.0 microM). Receptor-negative T47D:C4 cells were refractory to the effects of both 17 beta-estradiol and the antiestrogens. T47D:A18 cells grown under both estrogen-containing and estrogen-free conditions expressed low levels of transforming growth factor (TGF)-alpha and epidermal growth factor receptor mRNA. In the presence of estrogen, high levels of TGF-beta 1 mRNA were detected in T47D:A18 cells. These levels decreased when T47D:A18 cells were grown in estrogen-free media. Conversely, TGF-beta 2 mRNA was not detected in T47D:A18 cells cultured under estrogenic conditions; however, message was detected after the cells were cultured under estrogen-free conditions. T47D:C4 cells expressed low levels of TGF-alpha, epidermal growth factor receptor, TGF-beta 1, and TGF-beta 2 mRNA. These studies characterize a novel hormone-nonresponsive cell line which has been established from a hormone-responsive cell line grown under estrogen-free and drug-free conditions. Further analysis of these lines should provide valuable information concerning the development of antiestrogen-resistant breast cancer.     

Hormone sensitivity in vitro and in vivo of v-ras-transfected MCF-7 cell derivatives

Human mammary carcinoma cell lines (MCF-7) were analysed for their hormone sensitivity before and after transfection with a v-Ha-ras oncogene or with a neomycin-resistance gene followed by selection in vitro or in vivo. Our aim was to test how the expression of the ras oncogene would influence the estradiol sensitivity of MCF-7 cells. In culture, MCF-7 cells expressing the viral p21 oncogene product, as compared to parental MCF-7 cells and their control derivatives, showed lower levels of a 67-kDa estrogen receptor. Progesterone receptors, however, remained sensitive to up-regulation by estrogens. The oncogene-expressing cells were less sensitive than all controls to stimulation of proliferation by 10(-8)M estradiol or to inhibition of proliferation by 2-CH3-4-OH tamoxifen, and this was not dependent upon the type of culture medium used. After s.c. or i.p. injection into female athymic nude mice, ovariectomized or left intact, the growth of MCF-7 cells expressing the ras oncogene product and of all control cells was sensitive to stimulation by estrogen supplementation. Conversely, cell lines derived from tumors generated with long latency in untreated athymic nude mice by v-ras-expressing MCF-7 cells showed efficient formation of quickly growing tumors in the absence of estrogen supplementation. No differences were observed in invasion and metastasis of the different MCF-7 cell types injected into athymic nude mice that were supplemented with estrogens or not.     

A new triphenylethylene derivative,TAT-59; hormone receptors; insulin-like growth factor 1; and growth suppression of hormone-dependent MCF-7 tumors in athymic mice

TAT-59 {(E)-4-[1-[4-[2-(dimethylamino)ethoxy]-phenyl]-2-(4-isopropyl)phenyl-1-butenyl]-phenyl-monophosphate} treatment was performed on hormone-dependent MCF-7 tumors in athymic mice. TAT-59 given at 1, 5, and 20 mg/kg inhibited the estrogen-stimulated growth of MCF-7 tumors in athymic mice in a dose-dependent fashion. The most clear decrease in tumor growth was shown in the TAT-59 alone group, although it was not dramatic. Average serum concentrations of DP-TAT-59 {(Z)-[1-[4-[2-(dimethylamino)-ethoxy]phenyl]-2-(4-isopropyl)phenyl-1-butenyl]-4-hydroxybenzene} and DM-DP-TAT-59(desmethyl-DP-TAT-manner. Much higher levels of DP-TAT-59 and DM-DP-TAT-59 wer shown in tumors (target tissues of estrogen) as compared with muscles (nontarget tissues of estrogen) or serum. A serum concentration of DP-TAT-59 or DM-DP-TAT-59 corresponding to the physiologic levels of serum estradiol in premenopausal women was sufficient to inhibit the estrogen-stimulated growth of MCF-7 tumors in mice. TAT-59 induced a dose-dependent increase in estrogen receptor levels in the MCF-7 tumors. In contrast, it prevented the estradiol (E₂)-induced increase in progesterone receptor levels in a dose-dependent manner. Insulin-like growth factor 1 levels measured in the MCF-7 tumors significantly decreased in the TAT-59 alone group and in the no treatment group as compared with the E₂ alone group. These results show the pronounced antiestrogenic action of TAT-59 on hormone-dependent MCF-7 tumors in athymic mice.     

Breast cancer chemoprevention phase I evaluation of biomarker modulation by arzoxifene, a third generation selective estrogen receptor modulator.

PURPOSE: Arzoxifene, a new selective estrogen receptor modulator with strong breast antiestrogen activity and absence of uterine agonist activity, was explored as a potential chemoprevention agent. We performed a multi-institutional evaluation of arzoxifene in women with newly diagnosed ductal carcinoma in situ or T1/T2 invasive cancer. EXPERIMENTAL DESIGN: In a Phase IA trial, 50 pre- or postmenopausal women were randomized to 10, 20, or 50 mg of arzoxifene daily in the interval between biopsy and re-excision or were enrolled as no-treatment controls. In a Phase IB trial, 76 postmenopausal women were randomized to 20 mg of arzoxifene versus matched placebo. Serum specimens collected at entry and at re-excision were assayed for various hormones and growth factors. Tissue from biopsies (estrogen receptor + and/or progesterone receptor +) and re-excision specimens was evaluated immunohistochemically for proliferation (Ki-67 by MIB-1 and proliferating cell nuclear antigen) and other biomarkers. RESULTS: In both trials, increases in serum sex hormone binding globulin were noted, as were decreases in insulin-like growth factor (IGF)-I and the IGF-I:IGF binding protein-3 ratio (P < 0.007 versus control/placebo). For 45 evaluable women in Phase IA, decreases in proliferation indices were more prevalent for arzoxifene (particularly 20 mg) than for controls. For 58 evaluable women in Phase IB, a decrease in estrogen receptor expression for arzoxifene was observed compared with no change with placebo (P = 0.0068). However, decreases in proliferation indices for arzoxifene were not statistically different from placebo, perhaps due to a confounding effect of stopping hormone replacement therapy before entry. CONCLUSION: Given the favorable side effect profile and the biomarker modulations reported here, arzoxifene remains a reasonable candidate for additional study as a breast cancer chemoprevention agent.     

Clonal proliferation of rat tracheal epithelial cells in serum-free medium and their responses to hormones, growth factors and carcinogens

A serum- and feeder cell-free medium has been developed forthe proliferation of rat tracheal epithelial (RTE) cells atclonal density. In this medium, RTE cells continue to proliferatefor several weeks after cells in serum containing medium onfeeder cells have begun to differentiate. The responsivenessof RTE cells to selected hormones and growth factors was determinedusing a clonal growth assay. The colony-forming efficiency (CFE)of RTE cells was reduced >85% when bovine pituitary extractor bovine serum albumin were deleted from the medium and 45– 70% reductions in CFE were observed when insulin, hydrocortisone,epidermal growth factor or cholera toxin were deleted. RTE cellsalso require high concentrations of Ca²⁺ (0.8 mM) for maximalclonal proliferation in this medium. The induction by carcinogensof preneoplastic RTE cell variants resistant to serum-mediatedsquamous differentiation was compared in serum-free medium andin serum-containing medium on feeder cells. N-methyl-N'-nitro-N-nitrosoguanidmewas considerably more cytotoxk and effective as a transformingagent on an equivalent dose basis for RTE cells in serum-freemedium. In contrast, (±)-7B, 8-dihydroxy-9, 10-epoxy-7,8, 9, 10-tetrahydrobenzo[a]pyrene was equally cytotoxic andtransforming under both culture conditions. This serum-freeculture system for primary RTE cells will be useful in studieson the control of normal epithelial cell proliferation and differentiationby defined growth factors and in studies on the cellular changesinvolved in carcinogenesis.     

Inhibition of human breast cancer cell proliferation in tissue culture by the neuroleptic agents pimozide and thioridazine

Permanent cell culture lines derived from human breast cancer tissue are important experimental models in the study of human breast cancer cell proliferation. In the present work, pimozide, thioridazine, W-13, and W-12 were shown to inhibit MCF-7 human breast cancer cell growth. The 50% inhibition concentration values determined in two proliferation assays, [3H]thymidine incorporation and cell number, were in close agreement for each compound tested. The order of potency for growth inhibition in the presence of 2% stripped calf serum was pimozide (Ki 2 microM) greater than thioridazine (Ki 5 microM) greater than W-13 (Ki 15 microM) greater than W-12 (Ki 39 microM). Similar concentrations of these compounds blocked estradiol-induced growth of MCF-7 cells, but estrogen receptor (ER) interactions do not seem to be involved. Pimozide and thioridazine had no effect on the estradiol binding properties of the MCF-7 ER, nor did pimozide interfere with the induction of progesterone receptors by estradiol. Furthermore, pimozide also inhibited incorporation of [3H]thymidine into MCF-7 cells stimulated by polypeptide hormones in serum-free medium. The Ki for pimozide in serum-free medium alone, 0.46 microM, was similar to that determined in the presence of insulin (0.42 microM), insulin-like growth factor I (0.54 microM), and epidermal growth factor (0.43 microM). The effects of pimozide on breast cancer cell growth were not limited to the MCF-7 cell line. Pimozide also blocked cell growth and [3H]thymidine incorporation into the ER-positive T47D and ZR75-1B human breast cancer cell lines and the ER-negative human breast cancer cell line, MDA-MB-231. Although numerous mechanisms of action of pimozide and thioridazine have been identified, both drugs are calmodulin antagonists at drug concentrations that inhibit breast cancer cell growth in vitro. Inhibition of MCF-7 cell growth by the selective calmodulin antagonists W-13 and W-12 is consistent with a role for calmodulin antagonism in the broad growth-inhibitory properties of pimozide. We conclude that pimozide and thioridazine may be useful in the control of estradiol- and polypeptide hormone-induced growth of ER-positive and ER-negative human breast tumors.