Expression of insulin-like growth factor binding proteins in human breast cancer correlates with estrogen receptor status

The insulin-like growth factors (IGFs) have been implicated in the growth regulation of human breast cancer. Since the IGFs are associated with specific binding proteins (IGFBPs) which may modulate receptor/ligand interactions, production of IGFBPs by breast cancer cells could alter their IGF-dependent growth. This study examined the expression of IGFBPs 4, 5, and 6 in eight breast cancer cell lines (BCCLs) using ribonuclease (RNase) protection assays. IGFBP-4 mRNA was detected in all BCCLs studied. IGFBP-5 expression was higher in estrogen receptor (ER) positive cells, while IGFBP-6 mRNA was detected in only two ER negative BCCLs. We also found that E₂ treatment enhanced the expression of IGFBPs 2, 4, and 5 in T47-D cells. We next studied IGFBP mRNA expression in 40 primary breast tumors. All tumors expressed mRNA for IGFBPs 2–6 but none expressed IGFBP-1 message. IGFBP-3 expression was higher in ER negative tumors, while that of IGFBP-4 and -5 was higher in ER positive specimens. These differences were statistically significant (P < .05). Ligand blot analysis of tumor extracts confirmed the presence of IGFBPs in breast cancer tissues. Thus, differential IGFBP expression in ER positive and negative tumors suggests an important role for this protein in breast cancer biology.     

Estrogen response in the hFOB 1.19 human fetal osteoblastic cell line stably transfected with the human estrogen receptor gene

The gene coding for the human wild-type estrogen receptor (ER) was stably transfected into the human fetal osteoblastic cell line hFOB 1.19, a clonal cell line which is conditionally immortilized with a temperature sensitive mutant of SV40 large T antigen (tsA58). Five subclones were obtained which express various levels of ER mRNA and protein. The subclone with the highest level of functional (nuclear bound) ER, hFOB/ER9, contained 3,931 (±1,341) 17β-estradiol molecules bound/nucleus as determined by the nuclear binding (NB) assay. Using the dextran coated charcoal (DCC) method, the level of total cytosolic ER measured was 204 (±2) fmol/mg protein. This subclone was examined further for estradiol (E₂) responsiveness. The ER expressed in hFOB/ER9 cells was shown to be functional using a transiently transfected ERE-TK-luciferase construct. Expression of luciferase from this construct increased ～25-fold in hFOB/ER9 cells following 10^?9M E₂ treatment. This effect on ERE-TK-luciferase expression was both dose and steroid dependant. Further, treatment of hFOB/ER9 cells with 10^?9M E₂ resulted in a 2.5–4.0-fold increase in endogenous progesterone receptor (PR) levels detected by steroid binding assays, and a noticeable increase in both the A and B forms of PR by western blot assay. The establishment of this estrogen responsive human osteoblastic cell line should provide an excellent model system for the study of estrogen action on osteoblast function. © 1995 Wiley-Liss, Inc.     

Structural organization and regulation of the chicken estrogen receptor

We have cloned the chicken estrogen receptor (ER) from a chicken oviduct lambda gt11 library using the human ER cDNA sequence. This chicken ER sequence is virtually identical to the recently published sequence. One noteable difference is an amino acid change from glutamine to arginine located toward the central region of the sequence. The size of the ER protein predicted from the 589 amino acids is approximately 66,000 which fits well with the range of molecular weights previously published for the calf uterine and human ER (65,000-70,000). We observed the size of the chicken ER mRNA to be approximately 7.8 kilobases which is in agreement with the previously published size of 7.5 kilobases. In vivo secondary stimulation of chicken oviduct total RNA with diethylstilbestrol does not induce chicken ER mRNA. A time course following the chicken ER mRNA levels after secondary stimulation with diethylstilbestrol indicated a decrease in mRNA levels 8 h after DES administration. A similar study was performed using progesterone for the secondary stimulation. An increase in the chicken ER mRNA levels was observed 24 h after stimulation with progesterone. Two regions of very high homology were delineated by analyzing the sequence of this chicken ER cDNA and comparing it to the sequences of the human ER, human glucocorticoid, and chicken progesterone receptors and the P75-erbA fusion product of the avian erythroblastosis virus. The first concensus region is 72 amino acids in length and the second region of high homology is 62 amino acids long. Detailed comparisons of these regions for the steroid hormone receptors and v-erb A are presented. Possible functions for the individual regions of high homology are discussed.     

Relationship between estrogen receptors, 17 beta-hydroxysteroid dehydrogenase and estrogen content in human breast cancer.

Estrone and estradiol levels in tumor tissue cytosols were determined in 11 premenopausal and 20 postmenopausal women at the same time that 17 beta-hydroxysteroid dehydrogenase and estrogen receptors (ER) were carried out on their breast cancers. Estrogen receptor positive tumors showed significantly higher levels of estrone and estradiol. However, all ER negative tumors contained measurable amounts of both estradiol and estrone. Higher levels of estrone were observed in ER negative tumors which correlates well with high 17 beta-hydroxysteroid dehydrogenase activity. These results suggest that false negative receptor assays in the premenopausal women is not likely to be due to occupancy of receptors by endogenous estrogens. Furthermore, the higher estrone content in the ER negative group is probably due to high 17 beta-hydroxysteroid dehydrogenase activity inherent to these tumor cells.     

Regulation of estrogen receptor protein and messenger ribonucleic acid by estradiol and progesterone in rat uterus

The objective of this study was to examine the mechanisms of estrogen receptor (ER) processing and replenishment in the uterus of ovariectomized rats after estradiol and progesterone treatment. Uterine ER binding activity, ER protein and ER mRNA were measured by receptor binding exchange assay, Western blot and slot blot, respectively. The regulation of ER levels in rat uterus by estradiol and progesterone was very dramatic. Changes in ER protein were faithfully reflected by changes in binding activity. Estradiol caused receptor “processing” within 4 h of administration followed by recovery or “replenishment” of ER levels to the initial level by 20 h. The term “processing” has previously been used to describe the loss of ER binding activity in the early phase of estradiol-action, but it was never clear whether the ligand binding site was inactivated by processing or if the receptor molecule actually disappeared. This study shows that receptor “processing” constitutes disappearance of receptor protein and the later “replenishment” phase represents new ER protein rather than recycling of “processed” receptor. Progesterone-action, on the other hand, influenced only the “replenishment” phase by blocking recovery of ER protein. ER mRNA was suppressed by estradiol at 8 h, after the receptor was “processed” and “replenishment” already initiated. Progesterone, on the other hand, did not alter the steady state level of the message. Other mechanisms, such as regulation of translation rate of existing mRNA and changes in the rate of degradation of ER proteins are more likely involved in acute regulation of ER by these ovarian steroid hormones.     

Hormonal regulation of estrogen receptor messenger ribonucleic acid in T47Dco and MCF-7 breast cancer cells

Using a combination of hormone-binding assays, immunologic techniques, and mRNA hybridizations we have measured the estrogen receptor (ER) content and studied the hormonal regulation of ER mRNA in one estrogen responsive and one estrogen unresponsive breast cancer cell line, MCF-7 and T47Dco, respectively. Estradiol binding could be detected in cytosol from MCF-7 cells but not in T47Dco cells. However, when measured by an enzyme-linked immunosorbent assay, T47Dco cells were found to contain approximately 15 fmol ER/mg cytosolic protein or 10% of the ER content in MCF-7 cells. Immunologically reactive ER in T47Dco cells was indistinguishable in size (approximately equal to 68 KD) from the ER in MCF-7 cells, as shown by Western blotting using a monoclonal antihuman ER antibody. Quantification of ER mRNA in MCF-7 and T47Dco cells indicated that T47Dco cells contained approximately 50% of the ER mRNA levels found in MCF-7 cells. This basal level of ER mRNA in T47Dco cells was not decreased by estradiol treatment, as opposed to in MCF-7 cells where estradiol caused 40-60% decrease in the ER mRNA expression. Also, estradiol did not increase the progesterone receptor (PR) mRNA levels in T47Dco cells whereas in MCF-7 cells an approximately 5-fold increase of the PR mRNA levels occurred after estradiol treatment. However, incubation of the cells with the synthetic progestin R5020 decreased the ER mRNA levels to approximately the same degree in both cell lines. In conclusion, we have shown that estrogen down-regulates ER mRNA and up-regulates PR mRNA in MCF-7 cells. Neither of these estrogenic effects were seen in T47Dco cells. It appears that the steroid-resistance in T47Dco cells does not occur as a consequence of a complete absence of ER mRNA or protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new assay of estrogen receptor by thin-layer gel filtration.

Application of a thin-layer gel filtration (TLG) method using Sephadex G-200 superfine for the assay of estrogen receptor (ER) is described. The method is based on the effective separation of specific receptor protein from nonspecific binding proteins and free hormone by TLG and subsequent determination of the radioactivity bound to specific ER. By the aid of fluorescein-labeled human γ-globulin, the position of specific ER can be visualized. The assay excludes completely the undesired fluctuation depending on the concentration of coexisting proteins and permits reliable and rapid quantitation of specific ER. Association constant and concentration of specific ER can be determined with 100 mg of mammary tumor tissue. The method has the additional advantage of not requiring expensive equipment.     

Nuclease sensitivity of estradiol-charged estrogen receptor binding sites in nuclei isolated from normal and neoplastic rat mammary tissues

The interaction of partially purified calf uterine estradiol-charged estrogen receptor ([3H]ER) with rat nuclei was studied in vitro. We previously observed a significantly greater number of [3H]ER binding sites (at saturation) in nuclei of R3230AC mammary tumors from intact vs ovariectomized (ovex) rats with no difference in the affinity of [3H]ER binding for these nuclei. We now report on the nuclease sensitivity of [3H]ER binding sites in nuclei from these tumors and from normal rat tissues. Digestion of tumor nuclei with deoxyribonuclease I (DNase I) prior to incubation with [3H]ER in vitro resulted in a progressive loss of [3H]ER binding capacity, which was not accompanied by alterations in the affinity of [3H]ER for the nuclei (Kd = 1-3 nM). A significantly lower concentration (P less than 0.005) of DNase I eliminated 50% of the [3H]ER binding sites in nuclei of tumors from intact hosts (8 unit.min/ml) compared to tumors from ovex hosts (22 unit.min/ml). These results indicate that DNA regions capable of binding ER are more susceptible to DNase I digestion in tumors from intact rats than those from ovex hosts, suggesting that the endogenous hormonal milieu is responsible, at least in part, for maintenance of nuclease-sensitive DNA conformations in this hormone-responsive mammary tumor. The amount of DNase I required to eliminate 50% of [3H]ER binding to nuclei from lactating mammary gland, liver, and kidney ranged from 14 to 56 unit.min/ml. Therefore, accessibility of [3H]ER binding sites to nuclease digestion in normal rat tissue is generally less than that of R3230AC tumors.     

Regulation of estrogen receptor messenger ribonucleic acid and protein levels in human breast cancer cell lines by sex steroid hormones, their antagonists, and growth factors

Since sex steroid hormones and growth factors are known to modulate the proliferation of breast tumors, we have studied the effects of estrogen and progestin, their antagonists, and growth factors on the regulation of estrogen receptor (ER) mRNA and protein levels in T47D breast cancer cells, which contain low levels of ER, and in two sublines of MCF-7 cells which contain high ER levels. The mRNA levels were measured by Northern blot analysis using lambda OR8, a cDNA probe for ER, and protein levels were measured by hormone binding or Western blot analysis. Treatment of T47D cells with estradiol (E2) caused a 2.5-fold increase in ER mRNA (6.6 kilobases) levels after 48 h. The progestin R5020 evoked a marked decrease in ER mRNA and protein levels to 20% of control values, while the antiprogestin RU38,486 caused no change in ER. In MCF-7 cells, the effect of E2 on ER levels was dependent on the prior growth history of the cells. In cells grown in low estrogen [5% charcoal-dextran-treated calf serum with phenol red for 8 yr (MCF-7-K2)], which are still E2 responsive, treatment with E2, the antiestrogen LY117018, or both produced little change in ER mRNA or protein; in contrast, ER mRNA and protein were reduced by E2 to 40% and 50% of control levels, respectively, in MCF-7 cells (denoted MCF-7-K1) which had been maintained routinely in medium containing 5% calf serum. This decrease in ER mRNA was dose dependent; 10(-11) E2 reduced levels to 60%, and 10(-10) M E2 evoked the maximal drop to 40% of the control level in 2 days. LY117018 alone did not alter ER mRNA levels in these cells, but it completely prevented the down-regulation of ER by E2. Administration of progestin, but not antiprogestin, along with E2 partially prevented the decrease in ER evoked by E2. Addition of epidermal growth factor or insulin-like growth factor-I to MCF-7-K1 cells, which increased cell proliferation, had no detectable effect on ER levels. Treatment with transforming growth factor-beta, which decreased cell proliferation, reduced ER by about 20%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tissue-specific regulation of rat estrogen receptor mRNAs

The estrogen receptor (ER) is present in a wide variety of mammalian tissues and is required for physiological estrogen responses, including estrogen-induced tissue-specific changes in gene expression. We studied the estrogen regulation of the mRNAs encoding the ER in rat uterus, liver, and pituitary. Ovariectomized (21-28 day post surgery) female CD-1 rats were injected daily with 17 beta-estradiol (E2, 10 micrograms/100 g BW) for 0, 1, or 4 h, 1, 3, or 7 days and compared with intact controls. Steady-state levels of ER mRNA were quantified using a human ER cDNA probe. Only one hybridizing species of approximately 6.2 kilobase (kb) was detected in uterine and liver RNA, similar to that observed in MCF7 human breast cancer cells. However, the ER mRNA regulation by E2 differed in direction depending on the tissue examined. In uterus, ER mRNA increased 3- to 6-fold after ovariectomy, and returned to intact levels within 24 h of E2 replacement. In contrast, liver ER mRNA declined 1.5- to 3-fold after ovariectomy and returned to intact levels after 1-3 days of E2. In pituitary tissue two hybridizing forms of ER mRNA were observed, with one species migrating at 6.2 kb, equivalent to the form in other tissues, and a second smaller species at approximately 5.5 kb. The lower molecular weight species varied somewhat in abundance from animal to animal, averaging about 20% of the intensity of the 6.2 kb band. The ER mRNA forms were regulated positively with E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Expression of genes coding for pS2, c-erbB2, estrogen receptor and the H23 breast tumor-associated antigen. A comparative analysis in breast cancer

Expression of the gene coding for a new breast tumor-associated antigen, H23, was compared to expression of genes coding for pS2, c-erbB2 and estrogen receptor (ER). Comparison involved mRNA expression in normal and malignant breast tissues as well as in non-breast tumors. Results obtained by RNA dot blot and Northern hybridizations showed that expression of the H23 antigen coding gene is a discriminatory marker in human breast cancer. It is expressed in 92% of breast tumors whereas 69%, 62% and 56% of breast tumors demonstrate significant mRNA levels of c-erbB2, ER and pS2, respectively. Non-malignant or normal breast tissue expresses much lower levels of the H23 antigen mRNA. From the comparative analysis presented here it is concluded that the gene coding for H23 antigen furnishes a most useful marker for human breast cancer.