While stimulating the growth of fibroblasts, transforming growth factor beta 1 (TGF-beta 1) inhib... more While stimulating the growth of fibroblasts, transforming growth factor beta 1 (TGF-beta 1) inhibits the growth of various normal and malignant cell lines in vitro. We studied the effects of TGF-beta 1 in vivo. The level of TGF-beta 1 in serum was maximally elevated 2 h after injecting 1 muCi of 125I-TGF-beta 1 into the peritoneal cavity of nude mice. Five h after the i.p. administration of 10 micrograms of unlabeled TGF-beta 1, 20 ng/ml of TGF-beta-like material in serum were detected by a radioreceptor assay on A549 lung carcinoma cells. Trichloracetic acid-precipitable 125I-TGF-beta 1 was taken up by liver, spleen, lungs, kidneys, and tumor tissue but not by the brain. At doses exceeding 2 micrograms/day, TGF-beta 1 induced a generalized interstitial fibrosis and a cachexia, which was not mediated by elevated serum levels of tumor necrosis factor alpha as determined by Western blot analysis and enzyme-linked immunosorbent assay. A total of 200,000 cells of the estrogen receptor-negative human breast cancer line MDA-MB-231, which had been shown to be maximally growth inhibited in vitro by 40 pM TGF-beta 1 and to have high-affinity receptors (9, 11, 12), were injected into the mammary fat pad of each nude mouse. The duration of treatment was 16 days with ten animals in the control group and five animals in the treated groups. The dose ranged from 1 to 4 micrograms per animal daily. The treatment was started 24 h after the injection of the tumor cells. Tumor growth was not significantly affected at either nontoxic or toxic doses of TGF-beta 1. Thus, we have demonstrated that TGF-beta 1, apart from being a local growth factor, has systemic effects, such as cachexia and multiple fibrosis. Its role as an antitumor agent may be limited.
An estrogen-independent variant (R3) was selected by cloning wild type MCF-'7 cells in soft agar ... more An estrogen-independent variant (R3) was selected by cloning wild type MCF-'7 cells in soft agar in the presence of tamoxifen. R3 has a slightly slower growth rate under optimal growth conditions. Estradiol and tamoxifen have minimal effects on growth rate and thymidine incorporation as compared to wild type MCF-7 cells. R3 contains both cytoplasmic receptor and a receptor derived from crude nuclear extract with slightly higher numbers of cytosol receptor than wild type MCF-7. The same dissociation constant (K d) and the same molecular weight (estimated by Sephadex chromatography) and sucrose density behavior for both cytoplasmic and nuclear estrogen receptor are seen in R3 and MCF-7. Estrogen receptor complexes were activated by salt and nucleotide and translocated to the nucleus equivalently in R3 and MCF-7. Relative binding affinities and extent of competition by estradiol and tamoxifen for cytoplasmic estrogen receptor are the same for MCF-7 and R3. Induction of progesterone receptor and nuclear estrogen receptor processing following treatment with estradiol in R3 is minimal compared with MCF-7. DNA affinity (as assessed by DNA cellulose chromatography) of estrogen receptor complexes from R3 and MCF" 7 is different. R3 appears to represent a cell with a defect expressed in receptor function apart from the initial hormone-binding step.
Previously, we described FGF-1or FGF-4-transfected MCF-7 breast carcinoma cells which are tumorig... more Previously, we described FGF-1or FGF-4-transfected MCF-7 breast carcinoma cells which are tumorigenic and metastatic in untreated or tamoxifen-treated ovariectomised nude mice. In this study, we have assessed the effects of AGM-1470, an antiangiogenic agent, and pentosan polysulphate (PPS), an agent that abrogates the effects of FGFs, on tumour growth and metastasis produced by these FGF-transfected MCF-7 cells. Untreated or tamoxifen-treated ovariectomised mice were injected with FGF-transfected cells, treated with AGM-1470 or PPS, and tumour growth and metastasis analysed. The sensitivity of FGF-transfected and parental MCF-7 cells to AGM-1470 or PPS was also determined in vitro. Both AGM-1470 and PPS inhibited tumour growth in otherwise untreated or tamoxifen-treated mice injected with either FGFor FGF-4transfected MCF-7 cells. This effect was more reliably seen in tamoxifen-treated animals. AGM-1470 was about 105 times less potent in inhibiting the anchorage-dependent growth of parental MCF-7 or FGF-transfected MCF-7 cells than in inhibiting the growth of human umbilical vein endothelial cells. PPS did not affect the in vitro growth of the transfectants or parental cells. Thus, the growth-inhibitory effect on tumours was in excess of the effect of either drug on the same cells in tissue culture, implying that stromal elements are important determinants of the effects of these drugs. There was a positive correlation between tumour size and the extent of proximal lymph node metastasis. However, neither drug had a significant effect on the extent of metastasis to proximal or distal lymph nodes or lungs. AGM-1470 or PPS may be helpful in cases of breast carcinoma in which angiogenesis is due to expression of FGFs by the tumour cells and may be more effective when combined with tamoxifen.
Transactions of the American Clinical and Climatological Association, 2008
We have used data derived from hormonal responses in human breast cancer cell lines to develop a ... more We have used data derived from hormonal responses in human breast cancer cell lines to develop a panel of 36 genes which can robustly identify patients who will or will not benefit from tamoxifen treatment. These genes had the following characteristics: 1) induction by 17beta-estradiol (E2) in estrogen receptor (ER)-positive breast cancer cell lines in vitro, 2) under-expression in ER-negative breast tumors, and 3) correlation with PR mRNA expression in breast tumors. The average expression of these 36 genes was used as a "risk index" for assessing disease-specific survival in two independent tumor profile datasets of 60 and 67 patients treated with tamoxifen (these data not having been used to initially select the 36 genes), with a high risk group in each dataset defined as those with the bottom 25% of risk index values. This combined biological and informatic analysis is potentially applicable to many other cancer therapeutics.
We recently established transfectants of MCF-7 human breast cancer cells with fibroblast growth f... more We recently established transfectants of MCF-7 human breast cancer cells with fibroblast growth factor 4 (fgf-4) that showed rapid growth and spontaneous metastasis in ovariectomized and tamoxifen-treated nude mice. To establish a spontaneous metastatic model of human breast cancer cells in nude mice with a sensitive marker for detection of micrometastasis, the transfection of fgf-4 was combined with transfection of the bacterial lacZ gene encoding beta-galactosidase. MKL-4 cells, a lacZ transfectant of an fgf-4-transfected cell line, showed the same level of fgf-4 expression as parental cells and expressed a high level of beta-galactosidase activity. When MKL-4 cells were injected s.c. into female nude mice, rapidly growing tumors developed. Whole organ staining for beta-galactosidase activity was able to detect even small numbers of metastatic tumor cells. Micrometastases in lymph nodes, lung, and brain were detected 3 weeks after the tumor cell injections, the first time point te...
The MCF-7 human breast cancer cell line and four derived variant sublines (R27, R3, R3-12, and R3... more The MCF-7 human breast cancer cell line and four derived variant sublines (R27, R3, R3-12, and R3-98), which were all estrogen receptor positive, as well as the receptor-deficient line, MDA-MB-231, were compared both in vitro and as heterotransplants into athymic nude mice. The cell lines and heterotransplanted tumors were evaluated in terms of growth, receptor status, morphology by light and electron microscopy, karyotype, and allozyme phenotype analyses. The R3 and R3-12 variant lines exhibited markedly retarded growth rates in vitro as compared with those of the MCF-7 parent line. The R3-12 line, which had the slowest growth rate in vitro as compared to those of the other cell lines, failed to produce tumors in nude mice after many attempts using various concentrations of tumor cell inocula. The MCF-7 and derived lines would not grow in oophorectomized animals without 170estradiol replacement, with one rare exception, while the MDA-MB-231 receptor-deficient line was able to grow in oophorecto mized mice with and without 17/8-estradiol replacement. Com pared to the parent MCF-7 and R27 line in vivo and in vitro, the R3 line and its subclones had reduced progesterone receptor markedly. By light microscopy, all of the cell lines in vivo and in vitro could be identified as adenocarcinoma. Using electron micros copy, the MCF and variant lines showed better tissue organiza tion and more squamous features in vivo than in vitro where glandular features were more prominent. The R27 line in vivo and in vitro showed the most cellular polarity and differentiation as compared to the MCF or R3 lines (R3, R3-12, and R3-98), while the R3 lines were the least differentiated of all the lines. The MDA-MB-231 line in vivo and in vitro had the most pro nounced glandular features of all the lines and was not affected by changes in media additions of fetal calf serum, 170-estradiol, or charcoal-treated calf serum as were the MCF-7-and MCF-7derived lines. All the tumors in vivo and in vitro showed a human karyotype, and the MCF-7 and each of the derived lines showed both common and unique chromosome markers. The original cell line (MCF-7), the derived sublines (R27 and R3), and tumors derived in nude mice from these lines were typed for species identity using standard isozyme procedures and were found to be human. The allozyme phenotype at seven polymorphic human loci (allozyme genetic signature) demon strated that the MCF-7 line and its derivatives were derived from the same individual and were distinct from the signatures of HeLa and a variety of other human breast cancer lines. This multifaceted study, using both in vivo and in vitro systems, may provide a model for better understanding the nature of tumor heterogeneity and its implications in therapeutic designs.
To investigate if the estrogen control of the tumorigenic phenotype of breast cancer cells was me... more To investigate if the estrogen control of the tumorigenic phenotype of breast cancer cells was mediated through activation of the c-fos protooncogene, we examined the expression of this oncogene in MCF-7 cells. In cells synchronized by double thymidine blockade, the peptide growth factors transforming growth factor alpha and epidermal growth factor increased c-fos mRNA levels 6-fold above controls after 30 min of treatment. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, increased c-fos mRNA levels 4- to 5-fold above control. 17 beta-Estradiol, a growth stimulator, increased c-fos mRNA levels less than 2-fold above control levels, while progesterone, vitamin D3, dihydrotestosterone, and dexamethasone had little effect on c-fos mRNA levels. In contrast, 17 beta-estradiol treatment initially diminished the c-myc RNA level after 30 min of treatment and resulted in an elevation of c-myc by 2.5 h after initiation of treatment. We conclude that c-fos induction in these cells is g...
Insulin-like growth factor II is a growth factor important in fetal development. Several cancer t... more Insulin-like growth factor II is a growth factor important in fetal development. Several cancer tissues and cell lines have been reported to express IGF-II and rat IGF-II is mitogenic for breast cancer cell lines. Using Northern analysis and ribonuclease protection assays, IGF-II mRNA was detected in normal fibroblasts and in the established breast cancer cell line, T47D. In this cell line, steady state levels of IGF-II message were increased by treatment with estradiol. 10 nM IGF-II, purified from human serum, was mitogenic for breast cancer cell lines. In vitro, IGF-II may act as an autocrine growth factor for some cell lines. RNA derived from breast cancer, pathologically normal breast tissue, and benign breast disease also contained IGF-II mRNA. When paired samples of normal and cancer tissue were obtained from the breast of the same patient, the level of IGF-II mRNA expression in the normal tissue was at least that found in the cancer. This is consistent with previous observati...
Estrogen increases the ability of the estrogen-dependent MCF-7 human breast cancer cell line to b... more Estrogen increases the ability of the estrogen-dependent MCF-7 human breast cancer cell line to both proliferate and invade through an artificial basement membrane. In studying the response of MCF-7 cells to various antiestrogens, we found that 4-hydroxytamoxifen and tamoxifen inhibited cell proliferation but increased their invasiveness. In contrast, the structurally unrelated benzothiophene antiestrogens, LY117018 and LY156758, were potent antiproliferative agents which did not stimulate invasiveness. The differential effects of these antiestrogenic agents on invasion correlated with changes in production of collagenase IV, while no significant change was seen in the chemotactic activity of the cells. Invasiveness was increased by 17 beta-estradiol or 4-hydroxytamoxifen after a few hours of treatment and was rapidly lost when 17 beta-estradiol was withdrawn. Stimulation of invasiveness with 17 beta-estradiol was blocked by the antiestrogen, LY117018. Cells from the MDA-MB-231 line...
Estrogen is known to stimulate the proliferation and basement membrane invasiveness of the MCF-7 ... more Estrogen is known to stimulate the proliferation and basement membrane invasiveness of the MCF-7 human breast cancer cell line. We have compared the new steroidal antiestrogen ICI 164,384, the triphenylethylene 4-hydroxytamoxifen (OHT), and the benzothiophene LY 117018, for their effects on the proliferation and invasiveness of the MCF-7 cell line and its antiestrogen-resistant variant LY-2. While all three antiestrogens blocked the proliferative effects of 17 beta-estradiol on MCF-7 cells, OHT and LY 117018, but not ICI 164,384 stimulated their proliferation in the absence of estrogen. The proliferative effects of OHT and LY 117018 were blocked by ICI 164,384. Basement membrane invasiveness of MCF-7 cells was stimulated by 17 beta-estradiol and OHT, but not LY 117018 or ICI 164,384. Both ICI 164,384 and LY 117018 were able to block the invasiveness induced by either 17 beta-estradiol or OHT. The LY-2 antiestrogen-resistant variant of the MCF-7 cell line showed increased basal proli...
Amplification and mRNA expression of the erbB-2 gene was analyzed in 61 samples of primary human ... more Amplification and mRNA expression of the erbB-2 gene was analyzed in 61 samples of primary human breast carcinoma. In the 57 samples where RNA could be isolated four different expression level groups were identified. Comparison of hybridization signal with that for beta-actin revealed that erbB-2 mRNA could not be detected in 6 of 57 samples (11%), was detected at normal levels in 32 of 57 samples (56%), showed 4- to 8-fold overexpression in 8 of 57 samples (14%), and showed 16- to 128-fold overexpression in 11 of 57 samples (19%). Examination of the DNA of the same set of samples revealed 6 of 61 samples (10%) with distinct gene amplification and 6 of 61 samples (10%) with possible gene amplification. The highest levels of erbB-2 overexpression were associated with gene amplification. Samples with 4- to 16-fold overexpression of the erbB-2 mRNA occurred without evident gene abnormalities. There was no association of erbB-2 expression or gene amplification with clinical stage of bre...
Glucocorticoids, at physiological concentration, inhibit cell division and thymidine incorporatio... more Glucocorticoids, at physiological concentration, inhibit cell division and thymidine incorporation in three lines of human breast cancer maintained in long-term tissue culture. At steroid concentrations sufficient to inhibit thymidine incorporation 50%, little or no effect is seen on protein synthesis 48 hr after hormone addition. All three of these lines are shown to have glucocorticoid receptors demonstrable by competitive protein binding assays. Receptors are extensively characterized in one line by sucrose density gradient analysis and binding specificity studies. Good correlation between receptor-binding specificity and biological activity is found except for progesterone, which binds to glucocorticoid receptor but is noninhibitory. Cross-competition and quantification studies demonstrate a separate receptor for progesterone. This receptor has limited binding specificities restricted largely to progestational agents, whereas the glucocorticoid receptor bound both glucocorticoid...
The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture
Cancer research, 1976
We have established or characterized six lines of human breast cancer maintained in long-term tis... more 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 pr...
Conditions are described under which prolonged estradiol retention and estrogenic activity are ob... more Conditions are described under which prolonged estradiol retention and estrogenic activity are observed in human breast cancer cells in tissue culture. The cells were incubated for three hr with a physiological concentration of [3H]estradiol (3 to 5 nM) and then were washed with 3 successive exchanges of medium 3, 17, and 24 or 48 hr following incubation with [3H]estradiol. The total wash period was 78 hr. The following parameters were monitored to assess the duration of estrogen action in MCF-7 human breast cancer cells in tissue culture; (a) the concentration of [3H]estradiol and [3H]estradiol metabolites in the media washes; (b) the intracellular concentration of [3H]estradiol and [3H]estradiol metabolites; and (c) the time course of estradiol-enhanced rates of radiolabeled thymidine incorporation. The [3H]estradiol concentration in the final medium wash was approximately 0.05 nM. The total intracellular concentration of tritium was about 50 nM prior to wash and 9 nM following 78...
Estrogen plays a central role in breast cancer pathogenesis. Although many studies have character... more Estrogen plays a central role in breast cancer pathogenesis. Although many studies have characterized the estrogen regulation of genes using in vitro cell culture models by global mRNA expression profiling, it is not clear whether these genes are similarly regulated in vivo or how they might be coordinately expressed in primary human tumors. We generated DNA microarray-based gene expression profiles from three estrogen receptor alpha (ERalpha)-positive breast cancer cell lines stimulated by 17beta-estradiol (E2) in vitro over a time course, as well as from MCF-7 cells grown as xenografts in ovariectomized athymic nude mice with E2 supplementation and after its withdrawal. When the patterns of genes regulated by E2 in vitro were compared to those obtained from xenografts, we found a remarkable overlap (over 40%) of genes regulated by E2 in both contexts. These patterns were compared to those obtained from published clinical data sets. We show that, as a group, E2-regulated genes from...
Angiopoietin-1 (Ang1) has been shown to act as an angiogenic promoter in embryonic angiogenesis b... more Angiopoietin-1 (Ang1) has been shown to act as an angiogenic promoter in embryonic angiogenesis by promoting vascular branching, pericyte recruitment and endothelial survival. We have investigated the role of Ang1 in tumour neovascularization under clinical conditions and in animal models. The expression of Ang1 in clinical breast cancer specimens was analysed by using laser-capture microdissection and reverse transcriptase-linked polymerase chain reaction (RT-PCR) on RNA isolated from the samples. Despite the expression of Ang1 in many human breast cancer cell lines, the gene was expressed in only three of 21 breast cancer clinical specimens, even though its receptor, Tie2, is abundant in the vasculature of all of these tumours. Ang1 was then overexpressed in a human breast cancer cell line (MCF-7) on its own and in conjunction with FGF1, an angiogenic factor shown to be able to increase the tumorigenicity of MCF-7 cells. High concentrations of Ang1 were produced in the conditioned...
Proceedings of the National Academy of Sciences, 1987
The MCF-7 human breast cancer cell line responds to estrogen stimulation in vitro by increased se... more The MCF-7 human breast cancer cell line responds to estrogen stimulation in vitro by increased secretion of growth factors and proliferation and in vivo by tumor formation in the nude mouse. To test a possible role of growth factor secretion in expression of the tumorigenic phenotype, we stably transfected MCF-7 cells with the v-Ha-ras oncogene to produce the MCF-7ras cell line. The MCF-7ras cell line was tumorigenic in the absence of estrogens and secreted 3- to 5-fold elevated levels of a high molecular weight form of a type alpha transforming growth factor-like growth factor, type beta transforming growth factor, and insulin-like growth factor I. MCF-7ras cells, in contrast to MCF-7, were less sensitive to further growth stimulation by estrogen, type alpha transforming growth factor, and insulin-like growth factor I and showed little change in receptor levels for these hormones. Conditioned medium from MCF-7ras cells as well as two of its component growth factors (insulin-like gr...
Proceedings of the National Academy of Sciences, 1987
We report that human breast cancer cells secrete a growth factor that is biologically and immunol... more We report that human breast cancer cells secrete a growth factor that is biologically and immunologically similar to platelet-derived growth factor (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or "competence" activity that is capable of inducing incorporation of [3H]thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. In addition, the conditioned medium contains an activity that competes with 125I-labeled PDGF for binding to PDGF receptors on normal human fibroblasts. The secretion of PDGF-like activity by the hormone-responsive cell line MCF-7 is stimulated by 17 beta-estradiol. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (95 degrees C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of approximately equal to 30 kDa on NaDodSO4/polyacrylamide gels. Immunop...
It has been proposed that the insulin-like growth factors (IGFs) can act as autocrine and/or para... more It has been proposed that the insulin-like growth factors (IGFs) can act as autocrine and/or paracrine growth promoters in breast cancer. To investigate this hypothesis, we infected early passage MCF-7 cells with a retroviral vector containing the coding sequence for the IGF-II preprohormone along with a constitutive cytomegalovirus promoter sequence. These cells do not normally express IGF-I or IGF-II. After infection with the retroviral vector, several single cell clones were analyzed. Seven of nine isolated clones expressed very high levels of IGF-II mRNA. Biologically active IGF-II protein was easily detectable in the medium conditioned by the IGF-ll-expressing clones, and IGF receptors were down-regulated in these. All IGF-II-expressing clones showed marked morphological changes in anchorage-dependent culture, growing in large clumps and as free-floating colonies. The cells also cloned in soft agar in the absence of estrogen, while the wild-type MCF-7 cells and control cells infected with an irrelevant DNA sequence showed none of these properties. alR-3, an antibody that blocks the type I IGF receptor, inhibited the growth of IGF-II-expressing clones in serum-free medium. This model demonstrates that IGF-II can serve as an autocrine growth stimulant in breast cancer epithelial cells and that IGF-II overexpression may be capable of mediating malignant progression in human breast cancer. (Molecular Endocrinology 6: 91-100, 1992) 91
While stimulating the growth of fibroblasts, transforming growth factor beta 1 (TGF-beta 1) inhib... more While stimulating the growth of fibroblasts, transforming growth factor beta 1 (TGF-beta 1) inhibits the growth of various normal and malignant cell lines in vitro. We studied the effects of TGF-beta 1 in vivo. The level of TGF-beta 1 in serum was maximally elevated 2 h after injecting 1 muCi of 125I-TGF-beta 1 into the peritoneal cavity of nude mice. Five h after the i.p. administration of 10 micrograms of unlabeled TGF-beta 1, 20 ng/ml of TGF-beta-like material in serum were detected by a radioreceptor assay on A549 lung carcinoma cells. Trichloracetic acid-precipitable 125I-TGF-beta 1 was taken up by liver, spleen, lungs, kidneys, and tumor tissue but not by the brain. At doses exceeding 2 micrograms/day, TGF-beta 1 induced a generalized interstitial fibrosis and a cachexia, which was not mediated by elevated serum levels of tumor necrosis factor alpha as determined by Western blot analysis and enzyme-linked immunosorbent assay. A total of 200,000 cells of the estrogen receptor-negative human breast cancer line MDA-MB-231, which had been shown to be maximally growth inhibited in vitro by 40 pM TGF-beta 1 and to have high-affinity receptors (9, 11, 12), were injected into the mammary fat pad of each nude mouse. The duration of treatment was 16 days with ten animals in the control group and five animals in the treated groups. The dose ranged from 1 to 4 micrograms per animal daily. The treatment was started 24 h after the injection of the tumor cells. Tumor growth was not significantly affected at either nontoxic or toxic doses of TGF-beta 1. Thus, we have demonstrated that TGF-beta 1, apart from being a local growth factor, has systemic effects, such as cachexia and multiple fibrosis. Its role as an antitumor agent may be limited.
An estrogen-independent variant (R3) was selected by cloning wild type MCF-'7 cells in soft agar ... more An estrogen-independent variant (R3) was selected by cloning wild type MCF-'7 cells in soft agar in the presence of tamoxifen. R3 has a slightly slower growth rate under optimal growth conditions. Estradiol and tamoxifen have minimal effects on growth rate and thymidine incorporation as compared to wild type MCF-7 cells. R3 contains both cytoplasmic receptor and a receptor derived from crude nuclear extract with slightly higher numbers of cytosol receptor than wild type MCF-7. The same dissociation constant (K d) and the same molecular weight (estimated by Sephadex chromatography) and sucrose density behavior for both cytoplasmic and nuclear estrogen receptor are seen in R3 and MCF-7. Estrogen receptor complexes were activated by salt and nucleotide and translocated to the nucleus equivalently in R3 and MCF-7. Relative binding affinities and extent of competition by estradiol and tamoxifen for cytoplasmic estrogen receptor are the same for MCF-7 and R3. Induction of progesterone receptor and nuclear estrogen receptor processing following treatment with estradiol in R3 is minimal compared with MCF-7. DNA affinity (as assessed by DNA cellulose chromatography) of estrogen receptor complexes from R3 and MCF" 7 is different. R3 appears to represent a cell with a defect expressed in receptor function apart from the initial hormone-binding step.
Previously, we described FGF-1or FGF-4-transfected MCF-7 breast carcinoma cells which are tumorig... more Previously, we described FGF-1or FGF-4-transfected MCF-7 breast carcinoma cells which are tumorigenic and metastatic in untreated or tamoxifen-treated ovariectomised nude mice. In this study, we have assessed the effects of AGM-1470, an antiangiogenic agent, and pentosan polysulphate (PPS), an agent that abrogates the effects of FGFs, on tumour growth and metastasis produced by these FGF-transfected MCF-7 cells. Untreated or tamoxifen-treated ovariectomised mice were injected with FGF-transfected cells, treated with AGM-1470 or PPS, and tumour growth and metastasis analysed. The sensitivity of FGF-transfected and parental MCF-7 cells to AGM-1470 or PPS was also determined in vitro. Both AGM-1470 and PPS inhibited tumour growth in otherwise untreated or tamoxifen-treated mice injected with either FGFor FGF-4transfected MCF-7 cells. This effect was more reliably seen in tamoxifen-treated animals. AGM-1470 was about 105 times less potent in inhibiting the anchorage-dependent growth of parental MCF-7 or FGF-transfected MCF-7 cells than in inhibiting the growth of human umbilical vein endothelial cells. PPS did not affect the in vitro growth of the transfectants or parental cells. Thus, the growth-inhibitory effect on tumours was in excess of the effect of either drug on the same cells in tissue culture, implying that stromal elements are important determinants of the effects of these drugs. There was a positive correlation between tumour size and the extent of proximal lymph node metastasis. However, neither drug had a significant effect on the extent of metastasis to proximal or distal lymph nodes or lungs. AGM-1470 or PPS may be helpful in cases of breast carcinoma in which angiogenesis is due to expression of FGFs by the tumour cells and may be more effective when combined with tamoxifen.
Transactions of the American Clinical and Climatological Association, 2008
We have used data derived from hormonal responses in human breast cancer cell lines to develop a ... more We have used data derived from hormonal responses in human breast cancer cell lines to develop a panel of 36 genes which can robustly identify patients who will or will not benefit from tamoxifen treatment. These genes had the following characteristics: 1) induction by 17beta-estradiol (E2) in estrogen receptor (ER)-positive breast cancer cell lines in vitro, 2) under-expression in ER-negative breast tumors, and 3) correlation with PR mRNA expression in breast tumors. The average expression of these 36 genes was used as a "risk index" for assessing disease-specific survival in two independent tumor profile datasets of 60 and 67 patients treated with tamoxifen (these data not having been used to initially select the 36 genes), with a high risk group in each dataset defined as those with the bottom 25% of risk index values. This combined biological and informatic analysis is potentially applicable to many other cancer therapeutics.
We recently established transfectants of MCF-7 human breast cancer cells with fibroblast growth f... more We recently established transfectants of MCF-7 human breast cancer cells with fibroblast growth factor 4 (fgf-4) that showed rapid growth and spontaneous metastasis in ovariectomized and tamoxifen-treated nude mice. To establish a spontaneous metastatic model of human breast cancer cells in nude mice with a sensitive marker for detection of micrometastasis, the transfection of fgf-4 was combined with transfection of the bacterial lacZ gene encoding beta-galactosidase. MKL-4 cells, a lacZ transfectant of an fgf-4-transfected cell line, showed the same level of fgf-4 expression as parental cells and expressed a high level of beta-galactosidase activity. When MKL-4 cells were injected s.c. into female nude mice, rapidly growing tumors developed. Whole organ staining for beta-galactosidase activity was able to detect even small numbers of metastatic tumor cells. Micrometastases in lymph nodes, lung, and brain were detected 3 weeks after the tumor cell injections, the first time point te...
The MCF-7 human breast cancer cell line and four derived variant sublines (R27, R3, R3-12, and R3... more The MCF-7 human breast cancer cell line and four derived variant sublines (R27, R3, R3-12, and R3-98), which were all estrogen receptor positive, as well as the receptor-deficient line, MDA-MB-231, were compared both in vitro and as heterotransplants into athymic nude mice. The cell lines and heterotransplanted tumors were evaluated in terms of growth, receptor status, morphology by light and electron microscopy, karyotype, and allozyme phenotype analyses. The R3 and R3-12 variant lines exhibited markedly retarded growth rates in vitro as compared with those of the MCF-7 parent line. The R3-12 line, which had the slowest growth rate in vitro as compared to those of the other cell lines, failed to produce tumors in nude mice after many attempts using various concentrations of tumor cell inocula. The MCF-7 and derived lines would not grow in oophorectomized animals without 170estradiol replacement, with one rare exception, while the MDA-MB-231 receptor-deficient line was able to grow in oophorecto mized mice with and without 17/8-estradiol replacement. Com pared to the parent MCF-7 and R27 line in vivo and in vitro, the R3 line and its subclones had reduced progesterone receptor markedly. By light microscopy, all of the cell lines in vivo and in vitro could be identified as adenocarcinoma. Using electron micros copy, the MCF and variant lines showed better tissue organiza tion and more squamous features in vivo than in vitro where glandular features were more prominent. The R27 line in vivo and in vitro showed the most cellular polarity and differentiation as compared to the MCF or R3 lines (R3, R3-12, and R3-98), while the R3 lines were the least differentiated of all the lines. The MDA-MB-231 line in vivo and in vitro had the most pro nounced glandular features of all the lines and was not affected by changes in media additions of fetal calf serum, 170-estradiol, or charcoal-treated calf serum as were the MCF-7-and MCF-7derived lines. All the tumors in vivo and in vitro showed a human karyotype, and the MCF-7 and each of the derived lines showed both common and unique chromosome markers. The original cell line (MCF-7), the derived sublines (R27 and R3), and tumors derived in nude mice from these lines were typed for species identity using standard isozyme procedures and were found to be human. The allozyme phenotype at seven polymorphic human loci (allozyme genetic signature) demon strated that the MCF-7 line and its derivatives were derived from the same individual and were distinct from the signatures of HeLa and a variety of other human breast cancer lines. This multifaceted study, using both in vivo and in vitro systems, may provide a model for better understanding the nature of tumor heterogeneity and its implications in therapeutic designs.
To investigate if the estrogen control of the tumorigenic phenotype of breast cancer cells was me... more To investigate if the estrogen control of the tumorigenic phenotype of breast cancer cells was mediated through activation of the c-fos protooncogene, we examined the expression of this oncogene in MCF-7 cells. In cells synchronized by double thymidine blockade, the peptide growth factors transforming growth factor alpha and epidermal growth factor increased c-fos mRNA levels 6-fold above controls after 30 min of treatment. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, increased c-fos mRNA levels 4- to 5-fold above control. 17 beta-Estradiol, a growth stimulator, increased c-fos mRNA levels less than 2-fold above control levels, while progesterone, vitamin D3, dihydrotestosterone, and dexamethasone had little effect on c-fos mRNA levels. In contrast, 17 beta-estradiol treatment initially diminished the c-myc RNA level after 30 min of treatment and resulted in an elevation of c-myc by 2.5 h after initiation of treatment. We conclude that c-fos induction in these cells is g...
Insulin-like growth factor II is a growth factor important in fetal development. Several cancer t... more Insulin-like growth factor II is a growth factor important in fetal development. Several cancer tissues and cell lines have been reported to express IGF-II and rat IGF-II is mitogenic for breast cancer cell lines. Using Northern analysis and ribonuclease protection assays, IGF-II mRNA was detected in normal fibroblasts and in the established breast cancer cell line, T47D. In this cell line, steady state levels of IGF-II message were increased by treatment with estradiol. 10 nM IGF-II, purified from human serum, was mitogenic for breast cancer cell lines. In vitro, IGF-II may act as an autocrine growth factor for some cell lines. RNA derived from breast cancer, pathologically normal breast tissue, and benign breast disease also contained IGF-II mRNA. When paired samples of normal and cancer tissue were obtained from the breast of the same patient, the level of IGF-II mRNA expression in the normal tissue was at least that found in the cancer. This is consistent with previous observati...
Estrogen increases the ability of the estrogen-dependent MCF-7 human breast cancer cell line to b... more Estrogen increases the ability of the estrogen-dependent MCF-7 human breast cancer cell line to both proliferate and invade through an artificial basement membrane. In studying the response of MCF-7 cells to various antiestrogens, we found that 4-hydroxytamoxifen and tamoxifen inhibited cell proliferation but increased their invasiveness. In contrast, the structurally unrelated benzothiophene antiestrogens, LY117018 and LY156758, were potent antiproliferative agents which did not stimulate invasiveness. The differential effects of these antiestrogenic agents on invasion correlated with changes in production of collagenase IV, while no significant change was seen in the chemotactic activity of the cells. Invasiveness was increased by 17 beta-estradiol or 4-hydroxytamoxifen after a few hours of treatment and was rapidly lost when 17 beta-estradiol was withdrawn. Stimulation of invasiveness with 17 beta-estradiol was blocked by the antiestrogen, LY117018. Cells from the MDA-MB-231 line...
Estrogen is known to stimulate the proliferation and basement membrane invasiveness of the MCF-7 ... more Estrogen is known to stimulate the proliferation and basement membrane invasiveness of the MCF-7 human breast cancer cell line. We have compared the new steroidal antiestrogen ICI 164,384, the triphenylethylene 4-hydroxytamoxifen (OHT), and the benzothiophene LY 117018, for their effects on the proliferation and invasiveness of the MCF-7 cell line and its antiestrogen-resistant variant LY-2. While all three antiestrogens blocked the proliferative effects of 17 beta-estradiol on MCF-7 cells, OHT and LY 117018, but not ICI 164,384 stimulated their proliferation in the absence of estrogen. The proliferative effects of OHT and LY 117018 were blocked by ICI 164,384. Basement membrane invasiveness of MCF-7 cells was stimulated by 17 beta-estradiol and OHT, but not LY 117018 or ICI 164,384. Both ICI 164,384 and LY 117018 were able to block the invasiveness induced by either 17 beta-estradiol or OHT. The LY-2 antiestrogen-resistant variant of the MCF-7 cell line showed increased basal proli...
Amplification and mRNA expression of the erbB-2 gene was analyzed in 61 samples of primary human ... more Amplification and mRNA expression of the erbB-2 gene was analyzed in 61 samples of primary human breast carcinoma. In the 57 samples where RNA could be isolated four different expression level groups were identified. Comparison of hybridization signal with that for beta-actin revealed that erbB-2 mRNA could not be detected in 6 of 57 samples (11%), was detected at normal levels in 32 of 57 samples (56%), showed 4- to 8-fold overexpression in 8 of 57 samples (14%), and showed 16- to 128-fold overexpression in 11 of 57 samples (19%). Examination of the DNA of the same set of samples revealed 6 of 61 samples (10%) with distinct gene amplification and 6 of 61 samples (10%) with possible gene amplification. The highest levels of erbB-2 overexpression were associated with gene amplification. Samples with 4- to 16-fold overexpression of the erbB-2 mRNA occurred without evident gene abnormalities. There was no association of erbB-2 expression or gene amplification with clinical stage of bre...
Glucocorticoids, at physiological concentration, inhibit cell division and thymidine incorporatio... more Glucocorticoids, at physiological concentration, inhibit cell division and thymidine incorporation in three lines of human breast cancer maintained in long-term tissue culture. At steroid concentrations sufficient to inhibit thymidine incorporation 50%, little or no effect is seen on protein synthesis 48 hr after hormone addition. All three of these lines are shown to have glucocorticoid receptors demonstrable by competitive protein binding assays. Receptors are extensively characterized in one line by sucrose density gradient analysis and binding specificity studies. Good correlation between receptor-binding specificity and biological activity is found except for progesterone, which binds to glucocorticoid receptor but is noninhibitory. Cross-competition and quantification studies demonstrate a separate receptor for progesterone. This receptor has limited binding specificities restricted largely to progestational agents, whereas the glucocorticoid receptor bound both glucocorticoid...
The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture
Cancer research, 1976
We have established or characterized six lines of human breast cancer maintained in long-term tis... more 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 pr...
Conditions are described under which prolonged estradiol retention and estrogenic activity are ob... more Conditions are described under which prolonged estradiol retention and estrogenic activity are observed in human breast cancer cells in tissue culture. The cells were incubated for three hr with a physiological concentration of [3H]estradiol (3 to 5 nM) and then were washed with 3 successive exchanges of medium 3, 17, and 24 or 48 hr following incubation with [3H]estradiol. The total wash period was 78 hr. The following parameters were monitored to assess the duration of estrogen action in MCF-7 human breast cancer cells in tissue culture; (a) the concentration of [3H]estradiol and [3H]estradiol metabolites in the media washes; (b) the intracellular concentration of [3H]estradiol and [3H]estradiol metabolites; and (c) the time course of estradiol-enhanced rates of radiolabeled thymidine incorporation. The [3H]estradiol concentration in the final medium wash was approximately 0.05 nM. The total intracellular concentration of tritium was about 50 nM prior to wash and 9 nM following 78...
Estrogen plays a central role in breast cancer pathogenesis. Although many studies have character... more Estrogen plays a central role in breast cancer pathogenesis. Although many studies have characterized the estrogen regulation of genes using in vitro cell culture models by global mRNA expression profiling, it is not clear whether these genes are similarly regulated in vivo or how they might be coordinately expressed in primary human tumors. We generated DNA microarray-based gene expression profiles from three estrogen receptor alpha (ERalpha)-positive breast cancer cell lines stimulated by 17beta-estradiol (E2) in vitro over a time course, as well as from MCF-7 cells grown as xenografts in ovariectomized athymic nude mice with E2 supplementation and after its withdrawal. When the patterns of genes regulated by E2 in vitro were compared to those obtained from xenografts, we found a remarkable overlap (over 40%) of genes regulated by E2 in both contexts. These patterns were compared to those obtained from published clinical data sets. We show that, as a group, E2-regulated genes from...
Angiopoietin-1 (Ang1) has been shown to act as an angiogenic promoter in embryonic angiogenesis b... more Angiopoietin-1 (Ang1) has been shown to act as an angiogenic promoter in embryonic angiogenesis by promoting vascular branching, pericyte recruitment and endothelial survival. We have investigated the role of Ang1 in tumour neovascularization under clinical conditions and in animal models. The expression of Ang1 in clinical breast cancer specimens was analysed by using laser-capture microdissection and reverse transcriptase-linked polymerase chain reaction (RT-PCR) on RNA isolated from the samples. Despite the expression of Ang1 in many human breast cancer cell lines, the gene was expressed in only three of 21 breast cancer clinical specimens, even though its receptor, Tie2, is abundant in the vasculature of all of these tumours. Ang1 was then overexpressed in a human breast cancer cell line (MCF-7) on its own and in conjunction with FGF1, an angiogenic factor shown to be able to increase the tumorigenicity of MCF-7 cells. High concentrations of Ang1 were produced in the conditioned...
Proceedings of the National Academy of Sciences, 1987
The MCF-7 human breast cancer cell line responds to estrogen stimulation in vitro by increased se... more The MCF-7 human breast cancer cell line responds to estrogen stimulation in vitro by increased secretion of growth factors and proliferation and in vivo by tumor formation in the nude mouse. To test a possible role of growth factor secretion in expression of the tumorigenic phenotype, we stably transfected MCF-7 cells with the v-Ha-ras oncogene to produce the MCF-7ras cell line. The MCF-7ras cell line was tumorigenic in the absence of estrogens and secreted 3- to 5-fold elevated levels of a high molecular weight form of a type alpha transforming growth factor-like growth factor, type beta transforming growth factor, and insulin-like growth factor I. MCF-7ras cells, in contrast to MCF-7, were less sensitive to further growth stimulation by estrogen, type alpha transforming growth factor, and insulin-like growth factor I and showed little change in receptor levels for these hormones. Conditioned medium from MCF-7ras cells as well as two of its component growth factors (insulin-like gr...
Proceedings of the National Academy of Sciences, 1987
We report that human breast cancer cells secrete a growth factor that is biologically and immunol... more We report that human breast cancer cells secrete a growth factor that is biologically and immunologically similar to platelet-derived growth factor (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or "competence" activity that is capable of inducing incorporation of [3H]thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. In addition, the conditioned medium contains an activity that competes with 125I-labeled PDGF for binding to PDGF receptors on normal human fibroblasts. The secretion of PDGF-like activity by the hormone-responsive cell line MCF-7 is stimulated by 17 beta-estradiol. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (95 degrees C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of approximately equal to 30 kDa on NaDodSO4/polyacrylamide gels. Immunop...
It has been proposed that the insulin-like growth factors (IGFs) can act as autocrine and/or para... more It has been proposed that the insulin-like growth factors (IGFs) can act as autocrine and/or paracrine growth promoters in breast cancer. To investigate this hypothesis, we infected early passage MCF-7 cells with a retroviral vector containing the coding sequence for the IGF-II preprohormone along with a constitutive cytomegalovirus promoter sequence. These cells do not normally express IGF-I or IGF-II. After infection with the retroviral vector, several single cell clones were analyzed. Seven of nine isolated clones expressed very high levels of IGF-II mRNA. Biologically active IGF-II protein was easily detectable in the medium conditioned by the IGF-ll-expressing clones, and IGF receptors were down-regulated in these. All IGF-II-expressing clones showed marked morphological changes in anchorage-dependent culture, growing in large clumps and as free-floating colonies. The cells also cloned in soft agar in the absence of estrogen, while the wild-type MCF-7 cells and control cells infected with an irrelevant DNA sequence showed none of these properties. alR-3, an antibody that blocks the type I IGF receptor, inhibited the growth of IGF-II-expressing clones in serum-free medium. This model demonstrates that IGF-II can serve as an autocrine growth stimulant in breast cancer epithelial cells and that IGF-II overexpression may be capable of mediating malignant progression in human breast cancer. (Molecular Endocrinology 6: 91-100, 1992) 91
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Papers by Marc Lippman