The specific binding of estradiol and estrone and the subsequent distribution of estrogen-receptor complexes within MCF-7 human breast cancer cells

MacIndoe, John H.; Woods, Gayle; Etre, Lois A.

doi:10.1016/0039-128x(82)90145-3

Cited by 20 publications

(8 citation statements)

References 4 publications

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“…The blank due to methodological processing and the apparent translocation of hormone by boiled tissue and by receptor-negative non-target tissues was found to be small ( < 12 fmol/mg DNA per 2 h). The time of maximal nuclear labelling (1-2 h) accords well with the results of other in-vivo (King, Cowan & Inman, 1965;Nicholson, Davies & Griffiths, 1976;Clark & Peck, 1979) and in-vitro (Maclndoe, Woods & Etre, 1982) studies. Although the lowering of the incubation temperature eliminated only 85% of the apparent nuclear uptake, this is in accord with the findings of Traish, Müller & Wotiz (1979), who studied intact uterine cells.…”

Section: Discussionsupporting

confidence: 85%

Oestrogen receptor activity and intranuclear translocation of oestradiol in rat and human mammary tumours

Hawkins¹,

Scott²,

Freedman³

1983

Journal of Endocrinology

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Forty-eight rat mammary tumours and 25 human breast carcinomata were examined for (a) oestrogen receptor activity and (b) capacity for intranuclear translocation of oestrogen. Receptor activity was determined by saturation analysis using charcoal adsorption to separate free and bound hormone. The capacity for intranuclear translocation was determined by incubation of tumour slices in Krebs-Ringer bicarbonate solution containing [3H]oestradiol-17 beta at a physiological concentration, in the presence and absence of a large excess of non-radioactive oestradiol, at 37 degrees C. Saturable nuclear uptake of [3H]oestradiol (= translocation) in boiled or receptor-negative tissues was minimal, i.e. less than 12 fmol/mg DNA per 2 h and in receptor-positive tissue was reduced by 85% when the temperature was lowered to 0 degree C. In 27 ovary-independent transplantable tumours and 21 dimethylbenz(a)anthracene-induced tumours (predominantly ovary dependent) saturable nuclear uptake was strongly correlated with level of oestrogen receptor activity (Spearman's rank correlation coefficient r = +0.73, P less than 0.001). Nineteen of these 48 tumours were examined further: 60-80% of the saturable uptake was precipitable with protamine sulphate and this fraction of the total saturable uptake was also highly correlated with receptor level (Spearman's r = +0.87, P less than 0.001). In the 25 human tumours studied, saturable nuclear uptake was again correlated with receptor level (Spearman's r = +0.75, P less than 0.001). These studies suggest that saturable transfer of oestradiol from the extracellular medium through the cytoplasm to the cell nucleus is mediated by the oestrogen receptor in the rat and human tumours examined. They provide no evidence for any defect of the receptor mechanism before nuclear binding in tumours which are receptor positive but hormone insensitive.

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Section: Discussionsupporting

confidence: 85%

Oestrogen receptor activity and intranuclear translocation of oestradiol in rat and human mammary tumours

Hawkins¹,

Scott²,

Freedman³

1983

Journal of Endocrinology

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“…More detailed studies with antisense oligo 7 show that both ERa protein and 3 H-E 2 binding were decreased by 50%-60%, suggesting that both methods measured the quantity of synthesized ERa. The values for K d and B max for 3 H-E 2 binding to MCF7-K2 cell extracts were similar to that reported by MacIndoe et al (1982) but crucially different from those of others (Gyling and Leclercq, 1988;Otto, 1995). This discrepancy is probably due to the subclone of MCF7 cells used in the present studies.…”

Section: Characterization Of Mcf7-k2 Cellssupporting

confidence: 49%

Specific Inhibition of Estrogen Receptor Alpha Function by Antisense Oligodeoxyribonucleotides

Taylor¹,

Bell²,

Al‐Azzawi³

2001

Antisense and Nucleic Acid Drug Development

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We have tested the effect of a range of antisense oligodeoxyribonucleotides (ODN) directed against the human estrogen receptor alpha (ERa) on ERa protein expression and function. Antisense ERa ODN transfected into the ERa-positive human breast carcinoma cell line MCF7-K2 showed variable responses dependent on the oligo used. The most active antisense ODN (oligo 7) decreased the levels of ERa protein by 61% as measured by Western blot analysis. Exogenous 17b-estradiol (17b-E 2 ), but not 17a-E 2 , augmented this effect, with a threshold effect at 10 28 M 17b-E 2 . The inhibitory effect of antisense ERa oligo 7 was confirmed by measurement of functional ERa protein. 3H-17b-E 2 binding to MCF7 cell extracts was inhibited to approximately 40% of control values in the presence of oligo 7. Antisense-transfected MCF7-K2 cell cultures produced a further 30% binding reduction in the presence of exogenous 17b-E 2 . An inhibitory effect on 17b-E 2 -dependent cell function was confirmed by the demonstration that ERa oligo 7-transfected MCF7-K2 cells failed to exhibit 17b-E 2 -stimulated cell proliferation. Exogenous 17b-E 2 enhanced the inhibitory effect of the antisense ODN by increasing ODN transfection efficiency but without ERa catabolism via the proteosomal pathway, suggesting an effect of 17b-E 2 on the plasma membrane and the existence of different ERa degradation pathways in the MCF7-K2 cell subclone. As 17b-E 2 had no effect on ERa protein degradation, we conclude that the observed reduction of ERa protein levels is due solely to the presence of the antisense ERa ODN. Antisense ERa ODN molecules, therefore, may form the basis of effective therapies against ERa-dependent malignancies.

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“…On the other hand, the conversion of E1 to E2 is of particular interest since it may provide an intracellular mechanism for amplifying a relatively weak estrogenic signal. In fact, this phenomenon may explain the unexpected estrogenic potency of E1 within MCF-7 human breast cancer cells [17].…”

Section: Discussionmentioning

confidence: 98%

Dehydroepiandrosterone and estrone 17-ketosteroid reductases in MCF-7 human breast cancer cells

MacIndoe

Hinkhouse

Woods

1990

Breast Cancer Res Tr

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The identification of several steroid-transforming enzymes within human breast cancers has led to speculation that the growth of some hormone-responsive tumors might be mediated in part by intracellularly derived estrogens. Reports that MCF-7 human breast cancer cells can transform both estrone (E1)1 to estradiol (E2) and dehydroepiandrosterone (DHEA) to the estrogenic steroid 5-androstenediol (AED), have prompted us to investigate the 17-ketosteroid reductase activities (17-KSR's) which mediate these potentially important reactions. Enzyme assays were performed by quantifying the amounts of [3H]AED or [3H]E2 former from [3H]DHEA or [3H]E1, respectively, by various subcellular preparations from MCF-7 cells under a variety of experimental conditions. DHEA 17-KSR was found to be localized exclusively within cytosol, whereas the E1 17-KSR activity appeared to be nearly equally divided between the soluble and particulate cytoplasmic subfractions. The particulate E1 17-KSR appeared capable of utilizing NADH or NADPH, whereas both the cytosolic form of this enzyme and the soluble DHEA 17-KSR activity showed a strict requirement for NADPH. Although both of the soluble 17-KSR's also showed similar pH optima, several other features suggested that they are different enzymes in MCF-7. E1 did not inhibit the conversion of DHEA to AED, and DHEA did not interfere with the transformation of E1 to E2, indicating that major differences in substrate specificity exist between the two cytosolic activities. Furthermore, DHEA 17-KSR activity within cytosol stored at -20 degrees C deteriorated almost completely over twelve weeks of storage, whereas E1 17-KSR activity remained stable. Finally, although both enzymes were found to be subject to product inhibition, AED inhibited DHEA 17-KSR competitively, whereas cytosolic E1 17-KSR activity was inhibited by E2 in noncompetitive fashion. Studies of the oxidation of E2 to E1 by MCF-7 cells showed that this transformation is catalyzed by both soluble and particulate 17-hydroxysteroid oxidases which utilize either NAD or NADP as cofactor. Having previously reported the presence of a particulate NADP(H)-linked androstenedione (AE) 17-ketosteroid oxidoreductase in MCF-7, we now suggest that at least three different enzymes, one particulate and two soluble forms, participate in the conversion of 17-ketosteroids to their hormonally active 17-hydroxysteroid derivatives within this cell line. The restricted substrate requirements of each enzyme provide a rationale for developing selective enzyme inhibitors which could provide important investigational tools and potentially effective therapeutic agents.

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The specific binding of estradiol and estrone and the subsequent distribution of estrogen-receptor complexes within MCF-7 human breast cancer cells

Cited by 20 publications

References 4 publications

Oestrogen receptor activity and intranuclear translocation of oestradiol in rat and human mammary tumours

Oestrogen receptor activity and intranuclear translocation of oestradiol in rat and human mammary tumours

Specific Inhibition of Estrogen Receptor Alpha Function by Antisense Oligodeoxyribonucleotides

Dehydroepiandrosterone and estrone 17-ketosteroid reductases in MCF-7 human breast cancer cells

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