Control of proliferation of MCF-7 breast cancer cells in a commercial preparation of charcoal-stripped adult bovine serum

Welshons, Wade V.; Grady, Leigh H.; Engler, Kathleen S.; Judy, Barbara M.

doi:10.1007/bf01831481

Cited by 17 publications

(8 citation statements)

References 28 publications

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“…MCF-7 cells were obtained from Dr. V. Craig Jordan, University of Wisconsin-Madison. The cells were cultured in maintenance medium (MEM with nonessential amino acids, 10 pg/ml phenol red, 10 mM HEPES, 6 ng/ml insulin, 100 unitslml penicillin, 100 pg/ml streptomycin, and 5% charcoal-stripped calf serum) at 37°C and 5% CO, (13,14). Because the responsiveness of MCF-7 cells that are maintained continuously in stripped calf serum can drift (15), cells were propagated in stripped calf serum for approximately 1 year and then replaced with cells derived from our primary source, MCF-7 cells that had been maintained in whole serum before storage in liquid N,.…”

mentioning

confidence: 99%

The Effective Free Fraction of Estradiol and Xenoestrogens in Human Serum Measured by Whole Cell Uptake Assays: Physiology of Delivery Modifies Estrogenic Activity

Nagel

Saal

Welshons

1998

Experimental Biology and Medicine

163

100

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The biological activity of natural estrogens is influenced by the degree to which they bind to serum proteins. To determine directly how serum affected the uptake of estradiol, we compared the whole cell uptake of [3H]estradiol in intact MCF-7 human breast cancer cells from serum-free medium with the uptake from 100% serum from adult men. In estrogen receptor saturation assays, 28.9 times more estradiol was required in serum to occupy the same number of estrogen receptors as was required in serum-free medium (SFM), suggesting that the effective free fraction of estradiol in adult male serum was 3.46% (1/28.9). Since most xenoestrogens are not available in tritium-labeled form, the cell uptake of unlabeled xenoestrogens could not be measured directly with saturation analysis. Therefore, we developed the relative binding affinity-serum modified access (RBA-SMA) assay to determine the effect of serum on the access of nonradioactive xenoestrogens to estrogen receptors within intact MCF-7 cells. Serum modified access (SMA) was calculated by dividing the relative binding affinity (RBA, relative to estradiol) measured in 100% serum, by the RBA measured in serum-free medium. An SMA > 1 indicated that the xenoestrogen had greater access to estrogen receptors than estradiol from serum. In contrast, an SMA < 1 indicated that the xenoestrogen had less access to estrogen receptors from serum than did estradiol. The synthetic estrogen diethylstilbestrol (DES) binds poorly to sex hormone binding globulin (SHBG), and DES showed enhanced access in serum, SMA = 6.2. Additional calculations through the Ki (inhibition constant) indicated that this corresponded to an effective free fraction of 26.9% for DES in serum. The phytoestrogens, coumestrol, genistein, and equol, showed substantial enhanced access in serum, over 10-fold relative to estradiol (SMA = 12.1, 10.3, and 11.3, respectively), and effective free fractions in serum of 47.8, 45.8, and 49.7%, respectively. Since most in vitro assays of xenoestrogens do not address how serum influences their bioactivity, the estrogenic activity of these phytoestrogens would be underestimated. Conversely, biochanin A showed decreased access from serum (SMA = 0.44) and had an effective free fraction of 2.4%; its estrogenic activity would be overestimated in serum-free assays.

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confidence: 99%

The Effective Free Fraction of Estradiol and Xenoestrogens in Human Serum Measured by Whole Cell Uptake Assays: Physiology of Delivery Modifies Estrogenic Activity

Nagel

Saal

Welshons

1998

Experimental Biology and Medicine

163

100

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“…Cell culture (Grady et al, 1991;Welshons et al, 1992) MCF-7, T47D, ZR-75-1, and MDA-MB-231 cells were obtained from Dr. V. Craig Jordan (University of Wisconsin-Madison). MCF-7 cells were maintained in MEM with nonessential amino acids, phenol red (10 pg/ml) 10 mM HEPES, insulin (6 ng/ml), penicillin (100 unitdml), streptomycin (100 pg/ml), and 5% charcoalstripped calf serum (maintenance medium).…”

Section: Methodsmentioning

confidence: 99%

“…Lithium has been reported to replace the 0 1995 WILEY-LISS, INC. requirement for EGF in the primary culture of normal mouse mammary epithelial cells (Imagawa et al, 1982;Tomooka et al, 1983). Since growth of the MCF-7 mammary cancer cells is strongly stimulated by EGF, we were interested in whether the growth of these cells could also be specifically stimulated by lithium; our initial studies indicated that lithium could stimulate some growth in these cells (Schaberg and Welshons, 1989;Welshons et al, 1992), although increases in cell number (proliferation) were not shown.…”

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confidence: 99%

Lithium‐stimulated proliferation and alteration of phosphoinositide metabolites in MCF‐7 human breast cancer cells

Welshons

Engler

Taylor

et al. 1995

Journal Cellular Physiology

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Lithium, which is used to treat bipolar psychiatric disorders, can stimulate proliferation of a number of cells in tissue culture. Proliferation of MCF-7 human breast cancer cells, which also respond to EGF and estrogens, was stimulated by LiCl (1-5 mM) within the concentration range that is encountered during human therapy with lithium. Stimulation of growth was specific for lithium; rubidium, potassium, and sodium showed no such effect. In the presence of antiestrogen, lithium stimulated the growth of hormone-dependent breast cancer cells MCF-7, ZR-75-1, and T47D but not hormone-independent MDA-MB-231 cells or an estrogen-independent clone of MCF-7 cells. Lithium-stimulated proliferation was limited by cytotoxicity which could be moderated by added potassium chloride (5-20 mM) in the medium. Each of the mitogens lithium, 17 beta-estradiol, and EGF increased the rate of uptake of myo-inositol into MCF-7 cells. Whether normalized to inositol lipids, to protein, or to DNA, steady-state levels of inositol phosphates were elevated by each of the mitogens including lithium, which inhibits the breakdown of inositol phosphates in the phosphoinositide signaling pathway. These data indicate that therapeutic concentrations of lithium can stimulate the proliferation of human breast cancer cells by a mechanism that may involve the phosphoinositide pathway.

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“…For proliferation assay, cells were seeded at 2,000 per well in a 96-well plate in phenol red -free MEM containing 5% charcoal-stripped fetal bovine serum as described previously (22). The cells were cultured in estrogen-free medium for 3 days and incubated in medium alone or medium containing 10 À9 mol/L E2, 10 until day 7 when the wells (four wells per treatment) were washed with HBSS and assayed for DNA content using the diphenylamine assay adapted for a 96-well tissue culture plate format (23).…”

Section: +mentioning

confidence: 99%

Estrogenic Regulation of Host Immunity against an Estrogen Receptor–Negative Human Breast Cancer

Curran¹,

Judy²,

Duru³

et al. 2006

Clinical Cancer Research

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Purpose:The risk of developing breast cancer is positively correlated with exposure to increased levels of estrogen and/or an increased duration of estrogen exposure. Many different mechanisms have been proposed to explain the association of estrogens with breast cancer risk; however, the well-documented immune modulatory properties of estrogen have received little attention. In part, this is due to a lack of suitable models for studying this relationship. Experimental Design: We have developed an animal model using estrogen receptor (ER)-negative human breast cancer cell line, MDA-MB-468, xenografted into severe combined immunodeficient (SCID) mice. We also generated the ER-a knockout (ER-aKO) mice on the SCID background and then tested the ability of 17h-estradiol to stimulate growth of xenografted ER-negative human breast cancer tumors in wild-type and ER-aKO SCID mice. We quantified vascularization of tumors, macrophage recruitment to the tumor site by immunocytochemistry, and inflammatory cytokine production. Results: We show that estrogen treatment of C57BL/6/SCID mice promotes the growth of xenografted ER-negative tumors in wild-type mice and this estrogen-induced tumor growth is abrogated in ER-aKO mice. Tumor neovascularization of estrogen-treated mice was unchanged versus control; however, estrogen treatment of the C57BL/6/SCID host suppressed macrophage recruitment to and inflammatory cytokine production at the tumor site. Conclusions: These data are consistent with estrogen modulation of the inflammatory response as a contributing factor in estrogen-stimulated growth of an ER-negative tumor.This effect on the host innate immune response was mediated by ER-a.

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Control of proliferation of MCF-7 breast cancer cells in a commercial preparation of charcoal-stripped adult bovine serum

Cited by 17 publications

References 28 publications

The Effective Free Fraction of Estradiol and Xenoestrogens in Human Serum Measured by Whole Cell Uptake Assays: Physiology of Delivery Modifies Estrogenic Activity

The Effective Free Fraction of Estradiol and Xenoestrogens in Human Serum Measured by Whole Cell Uptake Assays: Physiology of Delivery Modifies Estrogenic Activity

Lithium‐stimulated proliferation and alteration of phosphoinositide metabolites in MCF‐7 human breast cancer cells

Estrogenic Regulation of Host Immunity against an Estrogen Receptor–Negative Human Breast Cancer

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