Acquired resistance to selective estrogen receptor modulators (SERMs) in clinical practice (tamoxifen & raloxifene) by selection pressure in breast cancer cell populations

Tamoxifen, a pioneering selective estrogen receptor modulator (SERM), has long been a therapeutic choice for all stages of estrogen receptor (ER)-positive breast cancer. The clinical application of long-term adjuvant antihormone therapy for the breast cancer has significantly improved breast cancer survival. However, acquired resistance to SERM remains a significant challenge in breast cancer treatment. The evolution of acquired resistance to SERMs treatment was primarily discovered using MCF-7 tumors transplanted in athymic mice to mimic years of adjuvant treatment in patients. Acquired resistance to tamoxifen is unique because the growth of resistant tumors is dependent on SERMs. It appears that acquired resistance to SERM is initially able to utilize either E₂ or a SERM as the growth stimulus in the SERM-resistant breast tumors. Mechanistic studies reveal that SERMs continuously suppress nuclear ER-target genes even during resistance, whereas they function as agonists to activate multiple membrane-associated molecules to promote cell growth. Laboratory observations in vivo further show that three phases of acquired SERM-resistance exists, depending on the length of SERMs exposure. Tumors with Phase I resistance are stimulated by both SERMs and estrogen. Tumors with Phase II resistance are stimulated by SERMs, but are inhibited by estrogen due to apoptosis. The laboratory models suggest a new treatment strategy, in which limited-duration, low-dose estrogen can be used to purge Phase II-resistant breast cancer cells. This discovery provides an invaluable insight into the evolution of drug resistance to SERMs, and this knowledge is now being used to justify clinical trials of estrogen therapy following long-term antihormone therapy. All of these results suggest that cell populations that have acquired resistance are in constant evolution depending upon selection pressure. The limited availability of growth stimuli in any new environment enhances population plasticity in the trial and error search for survival.     

Pre- and postnatal development studies of lasofoxifene, a selective estrogen receptor modulator (SERM), in Sprague-Dawley rats

BACKGROUND: Lasofoxifene is a nonsteroidal selective estrogen receptor modulator (SERM) developed for the treatment of postmenopausal osteoporosis. The purpose of these studies was to evaluate the effects of lasofoxifene on the postnatal development, behavior, and reproductive performance of offspring of female rats given lasofoxifene during organogenesis and lactation. METHODS: Two range-finding studies were conducted to determine the effects of lasofoxifene at doses from 0.01-10 mg/kg on parturition and lactation in pregnant rats and on the early postnatal development of the offspring, and to optimize the dosing regimen. Maternal milk and plasma were sampled for concentrations of lasofoxifene on Lactation Days 4, 7, and 14. In the pre- and postnatal development study, lasofoxifene was administered to pregnant and lactating rats by oral gavage at dose levels of 0.01, 0.03, and 0.1 mg/kg on Gestation Days 6-17 and Lactation Days 1-20. Maternal body weight and food consumption were measured throughout pregnancy, and body weight was measured throughout lactation. Parturition was monitored closely. The F1 offspring were measured for viability, body weight, anogenital distance, the appearance of postnatal developmental indices and reflex behaviors, sensory function, in an age-appropriate functional observational battery, motor activity, auditory startle, passive avoidance, and the Cincinnati Water Maze. The F1 generation was assessed for reproductive function, and the F2 offspring were measured for body weight and viability throughout the lactation period. RESULTS: In the range-finding studies, indications of maternal toxicity included decreased body weight and food consumption, increased length of gestation, prolonged parturition, dystocia, and increased offspring mortality at birth. Concentrations of lasofoxifene in maternal plasma were similar to those in milk, increased with increasing dose, and remained consistent over a 10-day period. In the pre- and postnatal development study, maternal body weights and food consumption were decreased in all treated groups during gestation. Length of gestation was increased, parturition was prolonged, and dystocia was noted in the dams in the 0.1 mg/kg group. There was increased pup mortality in the F1 litters in the 0.1 mg/kg group and all treated groups had decreased offspring body weights beginning at 1 week of age, continuing into the postweaning period and, for the F1 males, into adulthood. Female F1 offspring in the 0.03 and 0.1 mg/kg groups had increased body weights as adults. There were delays in the age of appearance of preputial separation in the males in the 0.1 mg/kg group and vaginal opening in the females in all treated groups. Body temperature was decreased by <0.5 degrees C after weaning for male and female offspring in the 0.1 mg/kg group. The sensory, behavioral, and functional measures, including the tests of learning and memory, were unaffected by treatment. Mating success was lower for the F1 animals in the 0.1 mg/kg group, but there were no effects on the reproductive parameters. Mating, reproduction, and maternal behavior of the F1 animals in the 0.01 and 0.03 mg/kg groups and the survival and body weights of the F2 offspring in all treated groups through Postnatal Day 21 were unaffected by treatment. CONCLUSION: The maternal findings in this study were related to the pharmacologic activity of lasofoxifene. Inhibition of growth of the F1 offspring after perinatal exposure to lasofoxifene was observed, but there were no significant effects on the sensory, behavioral, or functional measures, including learning and memory. There were no effects on the F2 generation. The findings are consistent with those reported for at least one other SERM. The findings of this study do not suggest increased risk for the primary indication of use in postmenopausal women.     

The selective estrogen receptor modulators, tamoxifen and raloxifene, impair dendritic cell differentiation and activation

Most immune cells, including myeloid progenitors and terminally differentiated dendritic cells (DC), express estrogen receptors (ER) making these cells sensitive to estrogens. Our laboratory recently demonstrated that 17-beta-estradiol (E2) promotes the GM-CSF-mediated development of CD11c+ CD11b(int) DC from murine bone marrow precursors. We tested whether the therapeutic selective estrogen receptor modulators (SERM), raloxifene and tamoxifen, can perturb DC development and activation. SERM, used in treatment of breast cancer and osteoporosis, bind to ER and mediate tissue-specific agonistic or antagonistic effects. Raloxifene and tamoxifen inhibited the differentiation of estrogen-dependent DC from bone marrow precursors ex vivo in competition experiments with physiological levels of E2. DC differentiated in the presence of SERM were assessed for their capacity to internalize fluoresceinated Ags as well as respond to inflammatory stimuli by increasing surface expression of molecules important for APC function. Although SERM-exposed DC exhibited increased ability to internalize Ags, they were hyporesponsive to bacterial LPS: relative to control DC, they less efficiently up-regulated the expression of MHC class II, CD86, and to a lesser extent, CD80 and CD40. This phenotype indicates that these SERM act to maintain DC in an immature state by inhibiting DC responsiveness to inflammatory stimuli. Thus, raloxifene and tamoxifen impair E2-promoted DC differentiation and reduce the immunostimulatory capacity of DC. These observations suggest that SERM may depress immunity when given to healthy individuals for the prevention of osteoporosis and breast cancer and may interfere with immunotherapeutic strategies to improve antitumor immunity in breast cancer patients.     

Embryo/fetal toxicity assessment of lasofoxifene, a selective estrogen receptor modulator (SERM), in rats and rabbits

BACKGROUND: The purpose of this study was to evaluate the effects of lasofoxifene, a selective estrogen receptor modulator (SERM), on rat and rabbit fetal development. METHODS: Lasofoxifene was administered orally to rats (1, 10, 100 mg/kg) between gestation days (GD) 6-17, and in rabbits (0.1, 1, 3 mg/kg) between GD 6-18. Maternal body weight and food consumption were monitored throughout pregnancy. Fetuses were delivered by Cesarean section on GD 21 in rats, and GD 28 in rabbits, to evaluate fetal viability, weight, and morphology. Drug concentrations in maternal plasma were measured in a separate cohort of animals at several time points commencing on GD 17 (rats) and 18 (rabbits). On GD 18 (rat) and GD 19 (rabbit) drug concentrations were measured in maternal plasma and in fetal tissue 2 hr post dosing to determine the fetal to maternal drug ratio. RESULTS: In rats, there were dose-related declines in maternal weight gain and food consumption. Post implantation loss was significantly increased at dosages of 10 and 100 mg/kg, and the number of viable fetuses was decreased at 100 mg/kg. The placental weights increased, whereas fetal weights decreased in a dose-dependent manner. Lasofoxifene-related teratologic findings were noted at 10 and 100 mg/kg and included imperforate anus with hypoplastic tails, dilatation of the ureters and renal pelvis, misaligned sternebrae, hypoflexion of hindpaw, wavy ribs, and absent ossification of sternebrae. In rabbits, neither maternal weight gain nor food consumption were affected during treatment. Between GD 26-28, there was a dose-dependent increased incidence of red discharge beneath the cages. At 1 and 3 mg/kg, resorptions and post-implantation loss increased. There were no significant external or visceral effects, but 3 mg/kg there was an increased incidence of supernumerary ribs. Although the maternal plasma Cmax and AUC(0-24) were dose-dependent, the exposures in the rat were many orders of magnitude greater than in the rabbit even for the same 1 mg/kg dose. The single time point fetal/maternal drug ratio was higher in the rat (1.3-0.78) than in the rabbit (0.21-0.16). CONCLUSION: In general, both maternal and fetal effects of lasofoxifene were similar to those reported with other SERMs. Although the incidence or severity of these effects was, in some instances, greater in the rat than in the rabbit, the doses and the resultant maternal and fetal exposures were many orders of magnitude higher in the rat, suggesting the rabbit to be more sensitive to the toxicological effects of lasofoxifene.     

Design,synthesis and biological evaluation of novel indole-xanthendione hybrids as selective estrogen receptor modulators

Ground breaking clinical therapeutic advances in the treatment of breast cancer (BC) is the introduction of selective estrogen receptor modulators (SERMs). We have expeditiously designed and synthesized indole-xanthendione hybrids by coalescing the indole nucleus with xanthendione. All the compounds were first screened for anti-proliferative activity, cytotoxicity and ER-α binding affinity by utilizing ER-α dominant T47D BC cell lines, PBMCs and ER-α competitor assay kit. From this study, two representative compounds 6e and 6f showing most promising activity were advanced for gene expression studies for targeting ER-α. Cell imaging experiment undoubtedly indicate that both the compounds were able to cross cellular bio membrane and accumulate thus instigating cytotoxicity. RT-PCR and Western blotting experiments further strengthened that both compounds altered the expression of mRNA and receptor protein of ER-α, thereby forestalling downstream transactivation and signalling pathway in T47D cells line. Structural investigation from induced fit simulation study suggest that indole moiety of the compounds 6e and 6f helps in the anchoring of the xanthendione moiety in the hydrophobic region of the cavity thus enabling the compound to bind in antagonistic conformation similar to bazedoxifene by extensive hydrogen bonding and Van der Waals forces. All these finding collectively imply that compound 6e and 6f represents a novel potent ER-α antagonist and in the development of SERMs for the management of BC.     

Endometrial effects of selective estrogen receptor modulators (SERMs) on estradiol-responsive gene expression are gene and cell-specific

Three selective estrogen receptor modulator (SERM) drugs which included 4-OH-tamoxifen (Tam), EM-800 (EM) and GW 5638 (GW) were investigated to determine their ability to inhibit estradiol-responsive gene expression in sheep endometrium. The uteri of ovariectomized ewes (10 ewes per SERM group) were infused with 10⁻⁷ M SERMs for 24 h prior to hysterectomy. Five ewes from each group received 50 μg 17β-estradiol (E2) and the remaining five ewes received vehicle 18 h prior to hysterectomy. Northern blot analyses and in situ hybridization demonstrated that E2 treatment increased estrogen receptor (ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and cyclophilin (CYC) mRNA levels in most endometrial cells examined. Tam and GW exhibited characteristics similar to E2 by increasing ER gene expression, but they antagonized the E2-induced increases in PR and CYC mRNA levels. EM acted as an E2-agonist of GAPDH gene expression, but antagonized the E2 up-regulation of ER, PR and CYC gene expression in most endometrial cells. Immunohistochemistry determined that EM decreased ER protein levels in the glandular epithelium, and the SERMs investigated antagonized increases in PR protein levels in endometrium. In conclusion, GW and EM exhibit fewer agonist effects than Tam on endometrial gene expression. EM demonstrated the greatest antagonism of E2-enhanced levels of ER, PR and CYC, likely due to the inhibition of ER gene expression at both mRNA and protein levels.     

Reproductive toxicity assessment of lasofoxifene, a selective estrogen receptor modulator (SERM), in female rats

BACKGROUND: Lasofoxifene is a nonsteroidal selective estrogen receptor modulator (SERM). With high affinity to the alpha and beta human estrogen receptors and greater potency than other SERMs, lasofoxifene is potentially a superior treatment for postmenopausal osteoporosis. In light of the known effects of estrogen-modulating compounds on female reproductive indices, two studies were conducted to evaluate the effects of lasofoxifene on female rat cyclicity, reproduction, and parturition. METHODS: One study evaluated effects of lasofoxifene on estrous cyclicity, and the second study assessed effects on implantation and parturition. In the cyclicity study, lasofoxifene was administered to female rats at doses of 0.1, 0.3, and 1.0 mg/kg/day for 14 consecutive days. After treatment, there was a 3-week reversibility phase followed by a mating phase. In the implantation study, lasofoxifene was administered to pregnant female rats at doses of 0.01, 0.03, and 0.1 mg/kg/day for 7 consecutive days (gestation day [GD] 0-6). Some animals were euthanized on GD 21, and the remainder of the group was allowed to deliver the F1 generation. Several developmental indices were evaluated in the F1 pups through post-natal day (PND) 21. RESULTS: In the cyclicity study, all lasofoxifene-treated females were anestrous by Study Day 7 (1.0 mg/kg) or 9 (0.3 and 0.1 mg/kg). The reversibility phase resulted in restoration of normal estrous cycles by the end of 1 (0.1 mg/kg) or 2 weeks (0.3 and 1.0 mg/kg). During the mating phase, no adverse effects occurred in pregnancy success or reproductive parameters. In the implantation study, all doses of lasofoxifene increased pre- and post-implantation losses, increased gestation length, and reduced litter size. None of the developmental parameters measured on the F1 generation was adversely affected. CONCLUSION: Lasofoxifene reversibly altered the estrous cycle and inhibited implantation, consistent with what would be expected from a member of the SERM class.     

Myometrial effects of selective estrogen receptor modulators on estradiol-responsive gene expression are gene and cell-specific

We examined in vivo effects of selective estrogen receptor modulators (SERMs) 4-OH-tamoxifen (Tam), GW 5638 (GW) and EM-800 (EM) on myometrial gene expression. The uteri of ovariectomized ewes were infused with 10⁻⁷ M of one SERM via indwelling catheters for 24 h preceding hysterectomy. Half of the ewes in each SERM group received an intramuscular injection of 50 μg 17β-estradiol (E2) 18 h prior to hysterectomy. Northern blot analysis and in situ hybridization demonstrated that E2 increased estrogen receptor (ER), progesterone receptor (PR) and cyclophilin (CYC) gene expression in the cells of both inner layer of myometrium (IM) and outer layer of myometrium (OM) as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in OM. Tam also increased ER mRNA levels in OM. EM appeared to increase ER gene expression, but antagonized E2’s up-regulation of PR and CYC gene expression in both IM and OM. Tam and GW also antagonized E2 up-regulation of PR gene expression in OM but not IM. No SERM affected GAPDH gene expression with or without E2. Immunohistochemistry indicated that E2 increased nuclear ER and PR protein levels in both IM and OM. EM was unique in up-regulating ER protein levels, opposite to its effects in endometrial cells. All SERMs tested antagonized this increase in PR immunostaining preferentially in OM compared to the IM layer. These results illustrate gene and cell layer-specific effects of SERMs in sheep myometrium.     

Comparative study of the short-term effects of a novel selective estrogen receptor modulator, ospemifene, and raloxifene and tamoxifen on rat uterus

To investigate the differential short-term effects of selective estrogen receptor (ER) modulators (SERMs) on uterus, we treated adult ovariectomized rats with a novel SERM, ospemifene (Osp), two previously established SERMs (tamoxifen and raloxifene (Ral)) and estradiol. The expression of two estrogen-regulated early response genes c-fos and vascular endothelial growth factor (VEGF), and DNA synthesis were analysed at 1-24 h after treatment of ovariectomized rats. Induction of c-fos mRNA by each of the SERMs showed a biphasic pattern with peaks at 3 and 20 h, respectively. The maximum level of VEGF mRNA was observed at 1 h after raloxifene and 6 h after tamoxifen or ospemifene treatment. Maximum levels of the c-fos and VEGF mRNA after raloxifene treatment were higher than those seen after treatments with E2 or a corresponding dose of tamoxifen or ospemifene. DNA synthesis was significantly increased by ospemifene, tamoxifen and raloxifene both in luminal and glandular epithelium. The stimulation was transient, peaking at 16 h. In comparison, the maximum level observed at 16 h after E2 treatment sustained at least until 24 h. DNA synthesis in stromal cells was increased by the SERMs but not by E2 at 24 h. When treated together with E2, the SERMs were able to antagonise E2-stimulated DNA synthesis at 16 h. Our results demonstrate that the initial response of uterus to ospemifene, raloxifene and tamoxifen includes activation of early response genes and even transient stimulation of DNA synthesis in spite of their different long-term effects. However, the early stimulatory events may be mediated by different mechanisms leading to diverging pathways in various tissue compartments and development of differential SERM-specific long-term responses of uterus.     

ER和PRmRNAs在内异症子宫内膜表达的变化

目的 :探讨雌激素受体 (ER)和孕激素受体 (PR)在子宫内膜异位症 (内异症 )子宫内膜的表达。方法 :利用大鼠内异症动物模型 ,采用逆转录聚合酶链反应 (RT PCR)技术 ,检测子宫内膜ER和PRmRNAs的表达情况。结果 :内异症模型组大鼠异位内膜ER、PRmRNAs的表达低于在位内膜和对照组正常子宫内膜 ,与后两者比较差异有显著性意义 (P <0 .0 1) ;而模型组在位内膜ER、PRmRNAs的表达与正常对照组比较差异无显著性意义 (P >0 .0 5 )。内异症模型组异位内膜ER/PRmRNA比值大于在位内膜和正常子宫内膜ER/PRmRNA比值 (P <0 .0 1)。结论 :内异症大鼠异位内膜ERmRNA表达的相对增高在内异症的发生与发展中起着一定的作用。     

Novel nonsteroidal selective estrogen receptor modulators. Carbon and heteroatom replacement of oxygen in the ethoxypiperidine region of raloxifene

Compounds were synthesized where oxygen in the ethoxypiperidine region of raloxifene is replaced with carbon, sulfur, or nitrogen linkages. Thia- and aza-substituted compounds were prepared by novel methodology. The compounds were evaluated in vitro as selective estrogen receptor modulators (SERMs). Calculations suggested the compounds exhibit an ER-alpha binding affinity/conformational energy relationship.     

The effects of estradiol and selective estrogen receptor modulators on gene expression and messenger RNA stability in immortalized sheep endometrial stromal cells and human endometrial adenocarcinoma cells

The purpose of this study was to identify an endometrial cell line that maintained the E2 up-regulation of estrogen receptor (ER) mRNA by enhanced message stability and to assess its dependence on ER protein. Estradiol (E2) effects on gene expression were measured in three cell lines: one immortalized from sheep endometrial stroma (ST) and two from human endometrial adenocarcinomas (Ishikawa and ECC-1). E2 up-regulated ER mRNA levels in ST and Ishikawa cells, but down-regulated ER mRNA levels in ECC-1 cells. E2 up-regulated progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and transforming growth factor-alpha (TGF-alpha) in both Ishikawa and ECC-1 cells. The selective estrogen receptor modulator ICI 182,780 antagonized the E2-induced up-regulation of ER and/or PR mRNA levels in all three cells, while another, GW 5638, antagonized the up-regulation of PR mRNA in Ishikawa and ECC-1 cells. In mechanistic studies, E2 had no effect on ER mRNA stability in ST cells and it destabilized ER mRNA in ECC-1 cells. Thus, Ishikawa cells appear to be the most physiologically relevant cell line in which to study the up-regulation of ER mRNA levels by enhanced mRNA stability. Its antagonism by ICI 182,780 reveals that ER protein is involved in this E2 response.     

Histologic changes in ovary, uterus, vagina, and mammary gland of mature beagle dogs treated with the SERM idoxifene

BACKGROUND: Idoxifene is a selective estrogen receptor modulator similar to tamoxifen but is no longer in pharmaceutical development due to adverse genitourinary effects in the clinic. Histologic observations of the reproductive system and mammary glands are presented from female dogs treated with idoxifene for up to 12 months. METHODS: Studies were conducted as part of regulatory requirements to support clinical development. Idoxifene was given orally by capsule, once daily, for 1, 6, or 12 months to female Beagle dogs (n = 3 or 4/group) aged 11-14 months (start of dosing) at dosages 0, 0.03, 0.3, 1.5, or 3 mg/kg/day. Evaluations included the following: clinical observations, hematology, hemostasis, chemistry, toxicokinetics, and histology. RESULTS: Dose- and time-dependent findings were present in dogs given > or = 0.03 mg/kg/day and included abnormal vaginal discharge, minor increases of platelet and neutrophil counts, and microscopic observations in the ovary (atrophy and mesothelial [ovarian surface epithelium] hyperplasia), endometrium (edema, inflammation, glandular atrophy, squamous metaplasia, increased collagen), myometrium (edema, increased collagen), vagina (squamous hyperplasia, keratinization), and mammary gland (atrophy). CONCLUSION: Dogs given idoxifene exhibited estrogenic effects in ovary, uterus, and vagina but antiestrogenic effects in endometrial and mammary glands consistent with several observations in clinical trials in post-menopausal women treated with triphenylethylenes.     

Effects of raloxifene, a selective estrogen receptor modulator, on thymus, T cell reactivity, and inflammation in mice

Raloxifene is a selective estrogen receptor modulator approved for prevention of osteoporosis in postmenopausal women. It is selective by virtue of having estrogen agonistic effects in bone, vessels, and blood lipids, while it is antagonistic with mammary and uterine tissue. The aim of the study was to examine whether the raloxifene analogue LY117018 (LY) has estrogenic effects on the thymus, T cell responsiveness, and inflammation. Oophorectomized normal mice were treated with subcutaneous injections of equipotent antiosteoporotic doses of LY (3 mg/kg) and 17beta-estradiol (E2) (0.1 mg/kg) or vehicle as controls. Effects on thymus were studied by analyses of thymus weight, cellularity, and CD4 and CD8 phenotype expression and histology, while inflammation was determined as T-cell-mediated delayed-type hypersensitivity (DTH) and granulocyte-mediated footpad swelling. LY lacked the suppressive properties of E2 on DTH and granulocyte-mediated inflammation. Furthermore, LY induced only minor thymus atrophy compared with E2 and did not, in contrast to E2, alter the thymic CD4/CD8 phenotypes. These results clearly demonstrate that raloxifene principally lacks the modulatory effects of estrogen on T cell responsiveness and inflammation. Our data are discussed in the context of recent findings in estrogen receptor biology and also with respect to estrogen-mediated alteration of autoimmune rheumatic diseases.     

Nuclear estrogen receptor II (nER-II) is involved in the estrogen-dependent ribonucleoprotein transport in the goat uterus I. Localization of nER-II in snRNP.

Exposure of goat uterine nuclei to estradiol in vitro results in an immediate exit of ribonucleoproteins (RNP) from the nuclei to the medium. This RNP exit appears to be mediated by an estrogen receptor localized in small nuclear ribonucleoproteins containing U1 and U2 snRNA. Available evidence indicates that the estrogen receptor involved is not the ERalpha, but an alternative form, which is also a 66 kDa protein. This is the nuclear estrogen receptor II (nER-II) that has no DNA-binding capacity. The transport is estrogen-specific since non-estrogenic steroids do not stimulate the transport of the RNP where the receptor is localized.     

Conditionally immortal ovarian cell lines for investigating the influence of ovarian stroma on the estrogen sensitivity and tumorigenicity of ovarian surface epithelial cells

The tendency of the ovarian surface epithelium (OSE) to undergo metaplastic and morphogenetic changes during the life cycle, at variance with the adjacent peritoneal mesothelial cells, suggests that its biology may be regulated by underlying ovarian stromal cues. However, little is known about the role that the ovarian stroma plays in the pathobiology of the OSE, largely because of the lack of a suitable in vitro model. Here, we describe the establishment and characterization of conditionally immortalized ovarian stromal and surface epithelial cell lines from H-2K(b)-tsA58 transgenic mice that carry the thermolabile mutant of SV-40 large T antigen under the control of an interferon-gamma (IFN-gamma)-inducible promoter. These cells express functional T antigens, grow continuously under permissive conditions at 33 degrees C in the presence of IFN-gamma, and stop dividing when the activity and expression of the tumor antigen is suppressed by restrictive conditions without IFN-gamma at 39 degrees C. Morphological, immunohistochemical, and ultrastructural analyses show that conditionally immortal OSE cells form cobblestone-like monolayers, express cytokeratin and vimentin, contain several microvilli, and develop tight junctions, whereas stromal cells are spindle-like, express vimentin but not cytokeratin, and contain rare microvilli, thus exhibiting epithelial and stromal phenotypes, respectively. At variance with the reported behavior of rat epithelial cells, conditionally immortal mouse epithelial cells are not spontaneously transformed after continuous culture in vitro. More importantly, conditioned media from stromal cells cultured under permissive conditions increase the specific activity of the endogenous estrogen receptor in BG-1 human ovarian epithelial cancer cells and promote these cells' anchorage-independent growth, suggesting the paracrine influence of a stromal factor. In addition, stromal cells cultured under restrictive conditions retain this growth-stimulatory activity, which, therefore, appears to be independent of T antigen expression. These established cell lines should provide a useful in vitro model system for studying the role of cellular interactions in OSE cell growth and tumorigenesis.