Effects of a non-IGF binding mutant of IGFBP-5 on cell death in human breast cancer cells

We have demonstrated previously that IGFBP-5 alone had no effect on cell death but modulated ceramide-induced apoptosis in Hs578T IGF non-responsive cells. To investigate if IGFBP-5 maintains its intrinsic ability to modulate apoptosis in IGF-responsive cells, we used a non-IGF binding mutant of IGFBP-5. In Hs578T cells, non-glycosylated, glycosylated or mutant IGFBP-5 alone each had no effect on cell death, whereas all forms inhibited ceramide-induced apoptosis. In IGF-responsive MCF-7 cells, each wild type form reduced ceramide-induced cell death but mutant IGFBP-5 was without effect. In the presence of mutant IGFBP-5, however, IGF-I no longer conferred survival and in the presence of wild type IGFBP-5, long R3 IGF-I was also unable to confer survival. In summary, all forms of IGFBP-5 modulated ceramide-induced apoptosis in Hs578T cells. In MCF-7 cells, IGF-I-induced survival could be facilitated by IGFBP-5, but also blocked by IGFBP-5 if association with IGFBP-5 was prevented.     

Differential insulin-like growth factor (IGF)-independent interactions of IGF binding protein-3 and IGF binding protein-5 on apoptosis in human breast cancer cells. Involvement of the mitochondria

We have demonstrated previously in Hs578T cells that insulin-like growth factor binding protein (IGFBP)-3 can significantly accentuate ceramide (C2)-induced apoptosis, but has no effect on cell death induced by integrin detachment [using an arginine-glycine-aspartic acid (RGD)-containing peptide]. In contrast we found that IGFBP-5 could inhibit apoptosis induced by either C2 or integrin detachment. It is now clear that the mitochondria not only provide the energy required for cell viability, but can also play an important role during the commitment phase to apoptosis. We used a mitochondrial respiratory chain inhibitor, antimycin A, at both apoptotic and nonapoptotic doses to further investigate the IGF-independent actions of IGFBP-3 and IGFBP-5 on C2 and RGD-induced apoptosis in the Hs578T cells. Hs578T cells had one of three treatments. 1: They were incubated with increasing doses of antimycin A for 24 h. 2: They were coincubated with an apoptotic dose of either C2 or RGD together with a nonapoptotic dose of antimycin A for 24 h. 3: They were incubated with a binding protein (100 ng/ml) for 24 h followed by coincubation of the binding protein with an apoptotic dose of antimycin A for a further 24 h. Cell viability was assessed by trypan blue dye exclusion and MTT assay, and apoptosis was confirmed and measured by morphologic assessment and flow cytometry. We found that antimycin A initiated apoptosis at 10 micromol/L and above. We also demonstrated that a nonapoptotic dose of antimycin A (0.1 micromol/L) significantly inhibited C2-induced apoptosis, whereas it significantly accentuated RGD-induced cell death. In addition, we found that cell death induced by antimycin A can be accentuated by IGFBP-3 but is not affected by IGFBP-5. These data indicate that IGFBP-3 can directly enhance apoptosis triggered via the mitochondria; either directly by a mitochondrial inhibitor or by C2 (which we demonstrate to act via effects on the mitochondria in this model). IGFBP-5, however, appears to confer survival effects via a distinct pathway not involving the mitochondria.     

Oncogenic ras causes resistance to the growth inhibitor insulin-like growth factor binding protein-3 (IGFBP-3) in breast cancer cells.

Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits proliferation and promotes apoptosis in normal and malignant cells. In MCF-10A human mammary epithelial cells, 30 ng/ml human plasma-derived IGFBP-3 inhibited DNA synthesis to 70% of control. This inhibition appeared IGF-independent, since neither an IGF-receptor antibody nor IGFBP-6 inhibited DNA synthesis. Malignant transformation of MCF-10A cells by transfection with Ha-ras oncogene abolished the inhibitory effect of IGFBP-3, concomitant with an increase in IGFBP-3 secretion and cell association of approximately 60 and 300%, respectively. When mitogen-activated protein (MAP) kinase activation was partially inhibited using PD 98059, IGFBP-3 sensitivity in ras-transfected cells was restored, with a significant inhibitory effect at 10 ng/ml IGFBP-3. PD 98059 had no effect on IGFBP-3 secretion or cell association by ras-transfected or parent MCF-10A cells. Hs578T, a tumor-derived breast cancer cell line that expresses activated Ha-ras, similarly has a high level of secreted and cell-associated IGFBP-3. In the absence of PD 98059, DNA synthesis by Hs578T cells was reduced to 70% of control by 1000 ng/ml IGFBP-3. PD 98059 increased sensitivity to IGFBP-3, so that this level of inhibition was achieved with 100 ng/ml IGFBP-3. These results suggest that MAP kinase activation by oncogenic ras expression causes IGFBP-3 resistance, a possible factor in the dysregulation of breast cancer cell growth.     

Differential IGF-independent effects of insulin-like growth factor binding proteins (1-6) on apoptosis of breast epithelial cells

We have demonstrated previously that insulin-like growth factor binding protein (IGFBP)-3 alone has little growth inhibitory effect on Hs578T human breast cancer cells, but that it can dramatically accentuate the apoptotic response to the physiological trigger, ceramide, in an IGF-independent manner. We have now studied the potential of other IGFBPs (1-6) to interact with apoptotic signalling pathways. Hs578T cells were preincubated with a binding protein (100 ng/ml) for 24 h, followed by co-incubation of the binding protein with an apoptotic dose of ceramide or RGD-containing peptide for a further 24 h. Apoptosis was assessed using flow cytometry, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue) assay and morphological assessment. Binding protein profiles were determined using ligand and immunoblotting techniques. Each of the IGFBPs (1-6) alone had no significant (P > 0. 05) growth inhibitory effects relative to control cells. In contrast to IGFBP-3, which significantly (P < 0.05) accentuated C2-induced apoptosis, IGFBP-1, -2, and -6 had no effect, whereas IGFBP-4 and -5 each caused marked (P < 0.01) inhibition of ceramide-induced programmed cell death. Apoptosis induced by RGD was also significantly (P < 0.05) reduced by IGFBP-5, whereas IGFBP-3 had no effect. These data provide evidence to suggest that individual IGFBPs have specific IGF-independent effects and act differentially on apoptotic signalling pathways.     

Signalling pathways involved in the direct effects of IGFBP-5 on breast epithelial cell attachment and survival

We have demonstrated previously that IGFBP-5 can confer survival against apoptosis induced by ceramide, C2, or a small synthetic arginine-glycine-aspartic acid (RGD)-containing peptide in a direct manner. The endogenous ceramide-induced pathway is normally counter-balanced by survival signals mediated by sphingosine kinase (SK) and protein kinase C (PKC). In order to investigate whether these pathways are involved in the IGFBP-5 survival effect, we have used inhibitors of SK (N, N-di-methyl sphingosine, DMS) and PKC (chelerythrine chloride, CC). The effect of pre-incubating Hs578T breast cancer cells with IGFBP-5 on cell adhesion or on subsequent cell death induced by C2 or RGD was investigated with and without the presence of DMS or CC. Cell death was determined by trypan blue cell counts and apoptosis confirmed by morphological assessment and flow cytometry. Cell attachment was determined by a cell adhesion assay. The presence of IGFBP-5 significantly inhibited cell death induced by C2 or RGD, compared to the triggers of apoptosis alone (P<0.01 in both cases). In the presence of either IGFBP-5, CC or DMS, there was no significant effect on cell death compared to the control. IGFBP-5 in the presence of either inhibitor resulted in a significant increase in cell death; IGFBP-5 also lost its ability to confer survival on C2 and RGD-induced apoptosis and in contrast significantly increased cell death. In the cell adhesion assay, IGFBP-5 significantly increased cell attachment over basal levels. In the presence of either inhibitor the IGFBP-5 effect on cell adhesion was reversed and cell attachment was reduced to below basal levels. These data suggest that IGFBP-5 promotes the attachment and survival of Hs578T cells by modulating the balance between ceramide and opposing survival signals.     

Interaction of vitamin D analogs with signaling pathways leading to active cell death in breast cancer cells

Induction of apoptosis is a feature of the anti-tumor effects of certain vitamin D analogs. The aim of this study was to identify if common effectors are involved in cell death mediated by serum starvation, vitamin D analogs and tumor necrosis factor (TNF) alpha in 3 human breast cancer cell lines: MCF-7, T47-D and Hs578T. Incubation of cells in serum-free medium induced apoptosis as assessed by loss of cell viability and increased DNA fragmentation. Addition of IGF-I (30 ng/ml) protected against loss of cell viability in MCF-7 cells and co-treatment with two synthetic analogs (CB1093 and EB1089, 50 nM for 4 days) prevented these anti-apoptotic effects of IGF-I. Pretreatment of MCF-7 and Hs578T cells with the vitamin D analogs substantially potentiated the cytotoxic effects of TNFalpha. This cytokine was not cytotoxic for T47-D cells but co-incubation with CB1093 led to loss of cell viability. Potentiation by CB1093 of TNFalpha-induced apoptosis in MCF-7 cells was accompanied by increased activation of cytosolic phospholipase A2 and arachidonic acid release, which was partially inhibited by AACOCF3, a specific cPLA2 inhibitor. The broad-spectrum caspase inhibitor z-VAD-fmk prevented TNFalpha but not CB1093 mediated cell death and activation of cPLA2. Serum starvation induced apoptosis was accompanied by cPLA2 activation, which was inhibited by IGF-I and by z-VAD-fmk. However, the ability of these agents to suppress cPLA2 activation was abrogated by co-treatment with CB1093, suggesting a role for arachidonic acid release in the caspase-independent mechanism by which vitamin D analogs prevent the protective effects of IGF-I on breast cancer cell survival.     

In vitro anti-proliferative activities of ellagic acid

The potential cytotoxic and anti-proliferative activities of ellagic acid (a naturally occurring bioactive compound in berries, grapes, and nuts) was evaluated using human umbilical vein endothelial cells (HUVEC), normal human lung fibroblast cells HEL 299, Caco-2 colon, MCF-7 breast, Hs 578T breast, and DU 145 human prostatic cancer cells. Ellagic acid at concentration in the range 10-100 micromol/L did not affect the viability of normal fibroblast cells during a 24-hour incubation. An increase in adenosine triphosphate (ATP) bioluminescence of approximately 18-21% was observed in normal cells incubated with ellagic acid. In contrast, ellagic acid at 1-100 micromol/L dose-dependently inhibited HUVEC tube formation and proliferation on a reconstituted extracellular matrix and showed strong anti-proliferative activity against the colon, breast, and prostatic cancer cell lines investigated. The most sensitive cells were the Caco-2, and the most resistant were the breast cancer cells. Ellagic acid induced cancer cell death by apoptosis as shown by the microscopic examination of cell gross morphology. Ellagic acid induced reduced cancer cell viability as shown by decreased ATP levels of the cancer cells. After 24 hours incubation of 100 micromol/L of ellagic acid with Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells, ellagic acid suppressed fetal bovine serum (FBS) stimulation of cell migration. The apoptosis induction was accompanied by a decreased in the levels of pro-matrix metalloproteinase-2 (pro-MMP-2 or gelatinase A), pro-matrix metalloproteinase-9 (pro-MMP-9 or gelatinase B), and vascular endothelial growth factor (VEGF(165)) in conditioned media. The results suggest that ellagic acid expressed a selective cytotoxicity and anti-proliferative activity, and induced apoptosis in Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells without any toxic effect on the viability of normal human lung fibroblast cells. It was also observed that the mechanism of apoptosis induction in ellagic acid-treated cancer cells was associated with decreased ATP production, which is crucial for the viability of cancer cells.     

Identification of the insulin-like growth factor binding proteins 5 and 6 (IGFBP-5 and 6) in human breast cancer cells.

Four estrogen receptor-positive (ER+) [MCF-7, T47D, ZR75 and BT474] and 3 ER- [Hs578T, MDA-MB-468 and MDA-MB-231] human breast cancer cell lines were examined for expression of the IGFBP-5 and IGFBP-6 genes. Northern blot analysis revealed that all cell lines, except MDA-MB-231, expressed IGFBP-5 mRNA. IGFBP-6 mRNA, however, was expressed only by the ER- cell lines. Western immunoblotting indicated that the previously unidentified 31-kDa and 32-kDa IGF binding species secreted by these cell lines are IGFBP-5. The levels of IGFBP-4 and IGFBP-5 were increased in MCF-7 cells by estradiol and IGF-I, respectively, indicating that these BPs may contribute to the growth stimulatory response to these mitogens.     

IGFBP-3 Can Either Inhibit or Enhance EGF-mediated Growth of Breast Epithelial Cells Dependent upon the Presence of Fibronectin

Progression of breast cancer is associated with remodeling of the extracellular matrix, often involving a switch from estrogen dependence to a dependence on EGF receptor (EGFR)/HER-2 and is accompanied by increased expression of the main binding protein for insulin-like growth factors (IGFBP-3). We have examined the effects of IGFBP-3 on EGF responses of breast epithelial cells in the context of changes in the extracellular matrix. On plastic and laminin with MCF-10A normal breast epithelial cells, EGF and IGFBP-3 each increased cell growth and together produced a synergistic response, whereas with T47D breast cancer cells IGFBP-3 alone had no effect, but the ability of EGF to increase cell proliferation was markedly inhibited in the presence of IGFBP-3. In contrast on fibronectin with MCF-10A cells, IGFBP-3 alone inhibited cell growth and blocked EGF-induced proliferation. With the cancer cells, IGFBP-3 alone had no effect but enhanced the EGF-induced increase in cell growth. The insulin-like growth factor-independent effects of IGFBP-3 alone on cell proliferation were completely abrogated in the presence of an EGFR, tyrosine kinase inhibitor, Iressa. Although IGFBP-3 did not affect EGFR phosphorylation [Tyr¹⁰⁶⁸], it was found to modulate receptor internalization and was associated with activation of Rho and subsequent changes in MAPK phosphorylation. The levels of fibronectin and IGFBP-3 within breast tumors may determine their dependence on EGFR and their response to therapies targeting this receptor.     

Differential insulin‐like growth factor (IGF)‐independent interactions of IGF binding protein‐3 and IGF binding protein‐5 on apoptosis in human breast cancer cells. Involvement of the mitochondria.

We have demonstrated previously in Hs578T cells that insulin‐like growth factor binding protein (IGFBP)‐3 can significantly accentuate ceramide (C2)‐induced apoptosis, but has no effect on cell death induced by integrin detachment [using an arginine‐glycine‐aspartic acid (RGD)‐containing peptide]. In contrast we found that IGFBP‐5 could inhibit apoptosis induced by either C2 or integrin detachment. It is now clear that the mitochondria not only provide the energy required for cell viability, but can also play an important role during the commitment phase to apoptosis. We used a mitochondrial respiratory chain inhibitor, antimycin A, at both apoptotic and nonapoptotic doses to further investigate the IGF‐independent actions of IGFBP‐3 and IGFBP‐5 on C2 and RGD‐induced apoptosis in the Hs578T cells. Hs578T cells had one of three treatments. 1: They were incubated with increasing doses of antimycin A for 24 h. 2: They were coincubated with an apoptotic dose of either C2 or RGD together with a nonapoptotic dose of antimycin A for 24 h. 3: They were incubated with a binding protein (100 ng/ml) for 24 h followed by coincubation of the binding protein with an apoptotic dose of antimycin A for a further 24 h. Cell viability was assessed by trypan blue dye exclusion and MTT assay, and apoptosis was confirmed and measured by morphologic assessment and flow cytometry. We found that antimycin A initiated apoptosis at 10 μmol/L and above. We also demonstrated that a nonapoptotic dose of antimycin A (0.1 μmol/L) significantly inhibited C2‐induced apoptosis, whereas it significantly accentuated RGD‐induced cell death. In addition, we found that cell death induced by antimycin A can be accentuated by IGFBP‐3 but is not affected by IGFBP‐5. These data indicate that IGFBP‐3 can directly enhance apoptosis triggered via the mitochondria; either directly by a mitochondrial inhibitor or by C2 (which we demonstrate to act via effects on the mitochondria in this model). IGFBP‐5, however, appears to confer survival effects via a distinct pathway not involving the mitochondria. J. Cell. Biochem. 80:248–258, 2000. © 2000 Wiley‐Liss, Inc.     

Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G(1) arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G(1) arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.     

Dual functions of autophagy in the response of breast tumor cells to radiation: cytoprotective autophagy with radiation alone and cytotoxic autophagy in radiosensitization by vitamin D 3

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D 3, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D 3 enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D 3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D 3 was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D 3 with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D 3). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.     

Insulin-like growth factor binding protein-3 prevents retinoid receptor heterodimerization: implications for retinoic acid-sensitivity in human breast cancer cells

Insulin-like growth factor binding protein-3 (IGFBP-3) has both IGF-dependent and -independent effects on cell growth, which are frequently growth-inhibitory. Interestingly, the development of a more aggressive phenotype in breast cancer cells (BCCs) correlates positively with elevated expression of IGFBP-3 and is often associated with all-trans-retinoic acid (atRA)-resistance. IGFBP-3 was previously demonstrated to interact directly with retinoid X receptor (RXR). In this study we have shown that IGFBP-5 also interacts with RXR and that both IGFBPs interact with retinoic acid receptor (RAR). To investigate whether the presence of IGFBP-3 regulates breast cancer cell responsiveness to atRA, we immuno-neutralized the IGFBP-3 expressed by the atRA-resistant Hs578T and MDA-MB-231 BCCs (which express IGFBP-3 constitutively) and showed that they become more sensitive to the growth-inhibitory effects of atRA. Similarly, in Hs578T cells expressing a reporter gene under the control of an RAR response element (RARE), depletion of IGFBP-3 resulted in the induction of reporter gene expression in response to atRA. In investigating possible mechanisms for IGFBP-3 regulation of atRA-sensitivity, we found that IGFBP-3 blocked the formation of RAR:RXR heterodimers and disrupted the ligand-inducible receptor complex. Thus, IGFBP-3 has the potential to reduce the RARE-mediated transactivation of target genes and modulate the atRA-response in BCCs.     

Identification of a Novel Cell Death Receptor Mediating IGFBP-3-induced Anti-tumor Effects in Breast and Prostate Cancer

Insulin-like growth factor-binding protein-3 (IGFBP-3), a major regulator of endocrine actions of IGFs, is a p53-regulated potent apoptotic factor and is significantly suppressed in a variety of cancers. Recent epidemiologic studies suggest that IGFBP-3 contributes to cancer risk protection in a variety of cancers, and a polymorphic variation of IGFBP-3 influences cancer risk, although other studies vary in their conclusions. Some antiproliferative actions of IGFBP-3 have been reported to be independent of IGFs, but the precise biochemical/molecular mechanisms of IGF-independent, antiproliferative actions of IGFBP-3 are largely unknown. Here we report a new cell death receptor, IGFBP-3R, that is a single-span membrane protein and binds specifically to IGFBP-3 but not other IGFBP species. Expression analysis of IGFBP-3 and IGFBP-3R indicates that the IGFBP-3/IGFBP-3R axis is impaired in breast and prostate cancer. We also provide evidence for anti-tumor effect of IGFBP-3R in vivo using prostate and breast cancer xenografts in athymic nude mice. Further in vitro studies demonstrate that IGFBP-3R mediates IGFBP-3-induced caspase-8-dependent apoptosis in various cancer cells. Knockdown of IGFBP-3R attenuated IGFBP-3-induced caspase activities and apoptosis, whereas overexpression of IGFBP-3R enhanced IGFBP-3 biological effects. IGFBP-3R physically interacts and activates caspase-8, and knockdown of caspase-8 expression or activity inhibited IGFBP-3/IGFBP-3R-induced apoptosis. Here, we propose that IGFBP-3R represents a novel cell death receptor and is essential for the IGFBP-3-induced apoptosis and tumor suppression. Thus, the IGFBP-3/IGFBP-3R axis may provide therapeutic and prognostic value for the treatment of cancer.     

Dual functions of autophagy in the response of breast tumor cells to radiation

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D₃, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D₃ enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D₃ failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D₃ was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D₃ with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D₃). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.     

Expression of estrogen and progesterone receptors and estrogen metabolizing enzymes in different breast cancer cell lines

Prolonged exposure to estrogens is a significant risk factor for the development of breast cancer. Estrogens exert carcinogenic effects by stimulating cell proliferation or through oxidative metabolism that forms DNA-damaging species. In the present study, we aimed to provide a better understanding of estrogen metabolism and actions in breast cancer, and to characterize model breast cancer cell lines. We determined the expression profiles of the genes for the estrogen and progesterone receptors, and for 18 estrogen-metabolizing enzymes in eight cell lines: MCF-7, MCF-10A, T47D, SKBR3, MDA-MB-231, MDA-MB-361, Hs-578T and Hs-578Bst cells. Similar gene expression profiles of these receptors and enzymes for the formation of estradiol via the aromatase and sulfatase pathways were observed in the MCF-7 and T47D metastatic cell lines. The MDA-MB-361 cells expressed ESR1, ESR2 and PGR as well, but differed in expression of the estrogen-metabolizing enzymes. In the MDA-MB-231 and SKBR3 cells, all of these estrogen-forming enzymes were expressed, although the lack of ESR1 and the low levels of ESR2 expression suggested that the estrogens can only act via non-ER mediated pathways. In the non-tumorigenic MCF-10A cell line, the key enzymes of the aromatase pathway were not expressed, and the sulfatase pathway also had a marginal role. The comparison between gene expression profiles of the non-tumorigenic Hs-578Bst cells and the cancerous Hs-578T cells revealed that they can both form estrogens via the sulfatase pathway, while the aromatase pathway is less important in the Hs-578Bst cells. The Hs-578T cells showed low levels of ESR1, ESR2 and PGR expression, while only ESR1 and ESR2 expression was detected in the Hs-578Bst cells. Our data show that the cell lines examined provide the full range of model systems and should further be compared with the expression profiles of breast cancer specimens.