Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells

Dual fluoroimmunohistochemical staining of estrogen receptor (ER) and bromodeoxyuridine was performed in a human osteoblastic osteosarcoma cell line, HOS TE85 cells. ER immunoreactivity was observed preferentially in the nuclei of the cells that were bromodeoxyuridine positive. ER expression at various phases of the cell cycle was investigated in HOS TE85 cells, which were synchronized at the G1/S phase boundary by intermittent exposure to thymidine and hydroxyurea. ER immunoreactivity became detectable in the S phase, decreased in the G2/M and G1 phases, and then reappeared in the S phase of the next cell cycle. Western blot analysis also showed that ER protein exists in these cells and increases in the S phase. Moreover, Northern blot analysis demonstrated that the expression of ER messenger RNA increases in the early S phase, gradually decreases during the progress of the cell cycle, and increases again in the S phase of the subsequent cell cycle. Interestingly, 17 beta-estradiol (10(-8) M) increased cell number and [3H]thymidine incorporation into DNA in the synchronized HOS TE85 cells, whereas this effect was not observed in the nonsynchronized HOS TE85 cells. The present studies suggest that the cell cycle-dependent regulation may contribute to the heterogeneity of ER expression in osteoblastic cells.     

Overexpression of the human type-1 insulin-like growth factor receptor in rat L6 myoblasts induces ligand-dependent cell proliferation and inhibition of differentiation

The human type-1 insulin-like growth factor receptor was overexpressed in rat L6 skeletal myoblasts using a retroviral expression vector. At low concentrations of insulin-like growth factor-I (1-10 ng/ml), dose-dependent stimulation of myoblast differentiation was similar in control L6 myoblasts and L6 myoblasts which overexpressed the type-1 insulin-like growth factor receptor. At high concentrations of insulin-like growth factor-I (50-100 ng/ml), L6 myoblasts which overexpressed the receptor did not undergo terminal differentiation and proliferated to form multilayers, while differentiation of control myoblasts was further stimulated. These findings indicate that overexpression of the type-1 IGF receptor can stimulate proliferation and inhibit differentiation in skeletal myoblasts in a ligand-dependent manner.     

Antisense estrogen receptor RNA expression increases epidermal growth factor receptor gene expression in breast cancer cells

In human breast cancer, progression to a more malignant phenotype is often accompanied by decreased expression of estrogen receptor (ER) and increased expression of epidermal growth factor receptor (EGFR). Higher levels of this receptor tyrosine kinase are found in tumors lacking ER, and a quantitative, inverse relationship exists between the level of ER and EGFR mRNA in human breast cell lines. Antisense ER (ASER) RNA was used to evaluate the consequence of decreased ER expression in breast cancer cells, specifically to determine whether ER is involved in the regulation of EGFR gene expression. ER-positive MCF-7 human breast cancer cells were transfected with ASER, and clones constitutively expressing ASER RNA had decreased ER and up to a 3-fold increase in the expression of EGFR mRNA. To confirm that this observation was a direct consequence of ASER expression, a metal-inducible ASER expression construct was transfected into MCF-7 cells, and transfected clones were isolated and characterized. Northern analysis revealed an induction of ASER RNA within 1 h of the addition of zinc, which was followed by a 4-fold increase in EGFR mRNA levels, maximal at 6-12 h. The basal level of expression of the glucocorticoid receptor is also inversely related to that of ER among breast cancer cell lines, but neither constitutive nor inducible expression of ASER affected the expression of glucocorticoid receptor. These data support the hypothesis that the level of expression of ER specifically influences the expression of EGFR in human breast cancer cells and provides a potential link between loss of steroid sensitivity and the acquisition of autonomous growth.     

Increased expression of estrogen receptor beta in chemically transformed human breast epithelial cells

Recent molecular cloning of estrogen receptor beta (ERbeta) suggests alternative pathways of estrogen signaling, but little is known concerning the role of ERbeta in the development of human breast cancer. In the present study, expression of ERalpha and ERbeta mRNA was determined in a series of chemically transformed human breast epithelial cells as well as various normal and malignant breast cancer cell lines. We observed a very low level of ERbeta expression in the mortal S130 and the spontaneously immortalized MCF10-F human breast epithelial cell lines. As MCF-10F cells were treated with environmental chemical carcinogens, an elevated level of ERbeta expression was observed in the resultant transformed BP1, D3 and BP1-ras cells. An even higher level of ERbeta expression was detected in the more transformed BP1-E, D3-1 and D3-1-ras cell lines. Therefore, results from our study indicate that expression of ERbeta can be induced in chemical carcinogen-transformed human breast epithelial cells, and the more transformed cells showed higher levels of ERbeta expression, regardless of which chemical carcinogens were initially used for cell transformation. These results suggest that expression of ERbeta may contribute to the initiation and progression of chemical carcinogen-induced neoplastic transformation.     

Modulation of intestinal estrogen receptor by ovariectomy, estrogen and growth hormone

Ovarian hormone deficiency decreases and estrogen (E2) and growth hormone (GH) administrations increase intestinal absorption of calcium (Ca++). However, the underlying mechanisms are uncertain. To examine whether alterations in the binding characteristics of intestinal estrogen receptors (ERs) are involved, we developed and validated methods for simultaneous measurement of intestinal ERs in cytosolic and nuclear fractions and applied these techniques to four groups of female rats: sham-operated, ovariectomized (Ovx), Ovx + 5 micrograms E2/kg b.wt./day and Ovx + 8 mg GH/kg. b.wt./day. All animals were killed on day 21, and mucosal cells harvested from the duodenum for ER determination. The cytosolic and nuclear ERs were 117.2 +/- 2.7 fmol/mg protein and 64.9 +/- 1.2 fmol/mg DNA, respectively, in sham-operated rats and decreased by 16.1% and 17.0% to 98.4 +/- 1.7 fmol/mg protein and 53.8 +/- 1.3 fmol/mg DNA, respectively in Ovx rats (P < .001). E2 therapy prevented completely the decrease in cytosolic and nuclear ERs that occurred in Ovx rat (126.1 +/- 2.9 fmol/mg protein and 68.0 +/- 3.0 fmol/mg DNA, respectively, in the E2-treated group). Similarly, GH administration prevented the decrease in cytosolic and nuclear ERs that resulted from ovariectomy (119.2 +/- 3.2 fmol/mg protein and 63.4 +/- 1.3 fmol/mg DNA, respectively, in the GH-treated group). The Kd of nuclear ER-ligand complex was 2.0 +/- 0.03 nM in sham-operated rats and was slightly modulated by Ovx, E2 and GH (3.3 +/- 0.02, 2.33 +/- 0.09 and 2.23 +/- 0.04 nM, respectively, P < .001), but the Kd of cytosolic ER-ligand complex was not altered by Ovx, E2 or GH. Our findings indicate that E2 deficiency down-regulates, whereas E2 and GH administrations up-regulate intestinal ERs and prevent ovariectomy-induced decrease in receptor binding affinity. We conclude that E2 deficiency, E2 and GH may modulate intestinal Ca++ absorption, in part, by altering the abundance and binding characteristics of intestinal ERs.     

Possible involvement of calpain in the growth of estrogen receptor positive breast cancer cells

Calpain (Ca2(+)-activated neutral protease, EC 3.4.22.17) has been reported to hydrolyze the estrogen receptor (ER). However, there has been no report available regarding the role of calpain in the growth of breast cancer cells. To investigate the role of calpain in the growth of various breast cancer cell lines, we employed a synthetic peptide, calpeptin, which is a cell permeable specific inhibitor of calpain. Calpeptin inhibited the cell growth of ER positive breast cancer cells, such as MCF-7, T-47D, and ZR-75-1 in a dose dependent manner in the presence of E2. However, the growth of ER negative breast cancer cells, MDA-MB-231, was not inhibited by calpeptin. It is suggested that calpain plays an important role in the growth of ER positive breast cancer cells.     

Insulin-like growth factor binding proteins-2 and -3 stimulate growth hormone receptor binding and mitogenesis in rat osteosarcoma cells

GH exerts its biological actions on osteoblasts through a specific high affinity receptor expressed on these cells. GH receptor binding is positively modulated by a number of factors, including retinoic acid and dexamethasone, whereas fetal calf serum strongly decreases the binding. To identify responsible factors in serum, components of serum, the insulin-like growth factors (IGFs)-I and -II, and IGF binding proteins (IGFBPs)-2 and -3 were tested for a possible negative modulatory role. IGF-I and -II decreased [125I]hGH binding at an optimal concentration of 30 ng/ml for IGF-I and 100 ng/ml IGF-II, reducing the binding to 51% and 55%, respectively, of control values. A stimulation of [125I]hGH binding was observed with IGFBP-2 as well as IGFBP-3, inducing an increase to 148% and 151% of control binding at an optimal concentration of 3000 ng/ml for both peptides. The effects of all peptides were dependent on the incubation time, being significantly increased after 8 h of incubation and reaching the full effect thereafter. The effects were declined at 24 h compared with 16 h for IGFBP-2 and -3 but not for IGF-I and -II. Coincubation of the cells with IGF-I and -II and IGFBP-2 and -3 neutralized the effects of the factors alone. In conclusion, these results show that IGF-I and -II on the one hand and IGFBP-2 and -3 on the other hand exert opposite actions on [125I]hGH binding, IGFBP-2 and -3 exerting probably an IGF-independent effect. Further, IGF-I and -II decreased GH receptor messenger RNA (mRNA) levels, as quantified by a solution hybridization ribonuclease protection assay, from 8.65 +/- 1.78 attomoles (amol)/microgram DNA (control) to 2.4 +/- 0.68 and 2.16 +/- 0.92 amol/microgram DNA, respectively. IGFBP-2 increased GH receptor mRNA levels from 5.26 +/- 1.17 (control) to 13.19 +/- 3.48. Incubation with IGFBP-3 did not result in stimulation of GH receptor mRNA levels (8.59 +/- 2.91 amol/microgram DNA). This shows that the mechanism of regulation of the GH receptor is, except for IGFBP-3, at least in part on the mRNA level. Lastly, IGFBP-2 and IGFBP-3 are mitogenic for UMR-106.01 rat osteosarcoma cells, inducing an increase in cell number to 125% and 142% of control cell counts after 48 h of incubation with 1000 ng/ml IGFBP-2 and -3, whereas IGF-I, IGF-II and Long R3 IGF-I did not stimulate proliferation. IGFBP-2 and -3 potentiate hGH induced mitogenesis at low hGH concentrations of both factors, whereas at higher concentrations no such effect is observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Redundancy of autocrine loops in human osteosarcoma cells

With the aim of identifying innovative therapeutic strategies for osteosarcoma patients who are refractory to conventional chemotherapy, we analyzed the in vitro effects of the blockage of autocrine circuits. Since the insulin-like growth factor-I receptor (IGF-IR)-mediated loop is relevant to the growth of osteosarcoma, we analyzed the activity of the IGF-IR-blocking antibody alphaIR3 in both sensitive and multidrug-resistant osteosarcoma cell lines. Only limited effects, however, were observed, suggesting the simultaneous existence of other autocrine circuits. Indeed, in a representative panel of 12 human osteosarcoma cell lines, in addition to the IGF-IR-mediated circuit, we demonstrated also a loop mediated by epidermal growth factor receptor as well as the presence of nerve growth factor, low-affinity nerve growth factor receptor as well as tyrosine receptor kinase A in the great majority of osteosarcomas. Therapies based on the inhibition of single circuits may have only limited effects in osteosarcoma, whereas the use of suramin, a drug which, besides other activities, non-selectively interferes with the binding of growth factors to their receptors, appears as a promising alternative, in both sensitive and drug-resistant osteosarcoma cells.     

Trans receptor inhibition of human glioblastoma cells by erbB family ectodomains

Our aim has been to understand the features of erbB receptor homo- and heterodimer assembly to develop approaches to disrupt receptor activation. We have developed a general approach to cause erbB receptor-specific trans inhibition of human neoplasia. The clonal progression of human astrocytomas to a more malignant phenotype often involves the amplification and overexpression of the epidermal growth factor receptor (EGFr) gene. We have selectively targeted the EGFr in human glioblastoma cells with kinase-deficient mutants of the erbB family derived from the ectodomain of the Neu oncogene that are able to form heterodimers with EGFr and inhibit EGFr-dependent phenotypes. In EGFr-positive U87MG human glioblastoma cells, expression of the Neu ectodomain inhibits EGF-, but not platelet-derived growth factor-, induced DNA synthesis; inhibits cell proliferation in the presence of EGF, but not platelet-derived growth factor; inhibits the ability of U87MG to form colonies in soft agar; and inhibits transforming efficiency in athymic mice. These studies establish that EGFr-mediated signal transduction is important in the maintenance of malignant glioma, and that trans receptor inhibition is a novel way to abrogate abnormal growth of these tumors. Neu ectodomains will be useful in determining the manner in which the EGFr contributes to glial tumorigenesis and in the design of pharmaceuticals that disable erbB family oncoproteins. In addition, these studies provide a rationale for the application of the Neu ectodomain in gene therapy approaches to human malignant glioma and, potentially, to other systemic epithelial malignancies expressing erbB family receptors.     

Growth hormone receptor activity is stimulated by insulin-like growth factor binding protein 5 in rat osteosarcoma cells

Osteoblast-like UMR-106.01 rat osteosarcoma cells express high affinity growth hormone (GH) receptors (GHRs). Because osteoblasts secrete insulin-like growth factor binding protein-5 (IGFBP-5), we evaluated whether it also modulates GH binding and GHR expression in UMR cells. Human recombinant intact IGFBP-5 stimulated 125I-hGH binding in a dose-dependent manner (dose range 300-3000 ng/ml), inducing an increase to 193.6 +/- 2.1% of control binding at 3000 ng/ml (P < 0.001). Carboxy-truncated IGFBP-5 also stimulated GH binding but with less potency (125 +/- 2.7% of control at 3000 ng/ml, P < 0.01). GHRs identified by chemical crosslinking of 125I-hGH to cell monolayers increased after treatment with IGFBP-5 and decreased in response to insulin-like growth factor-I (IGF-I). GHR mRNA levels, as quantitated by a solution hybridization RNAse protection assay, increased up to 3 to 7-fold in a time-dependent manner by intact IGFBP-5 but not by carboxy-truncated IGFBP-5. An antiserum to IGFBP-5 reduced basal GH binding to 56.7 +/- 4.3% of control value at a concentration of 0.5% (P < 0.001), showing that IGFBP-5 produced by the cells is a strong regulator of GH binding. IGFBP-5 antiserum also decreased GH binding to 85.9 +/- 0.9% of IGFBP-5 stimulated value (P < 0.001), showing the specificity of IGFBP-5 stimulation. To determine whether the GHR upregulation was physiologically significant, cell proliferation was evaluated after coincubation of IGFBP-5 with low, non-stimulatory concentrations of GH. IGFBP-5 (1000 ng/ml) induced cell proliferation to 116.2 +/- 3.2% of control levels, and coincubation with hGH at 10 ng/ml induced an increase to 133.3 +/- 0.1% of control levels. We conclude that exogenous and endogenous IGFBP-5 upregulate GHR mRNA levels and GH binding and this interaction potentiates GH-stimulated mitogenesis in osteoblastic cells.     

Cloning and characterization of human estrogen receptor beta isoforms

BACKGROUND: The purpose of this study was to describe a new anti-cancer drug regimen for endometrial cancer. METHODS: The cytotoxicities of some anti-cancer drugs regimens against human endometrial cancer xenografted into nude mice (TEI, TET, TEU, TEN) have been studied. The activities of ADM, CDDP, CPM, CPT-11, TXL, CDDP + ADM, CDDP + CPM, CDDP + CPT-11, CDDP + TXL, CPT-11 + TXL were evaluated in comparison with a control group using saline. Three mice were used for each group, and when xenografted tumor reached 6 mm of its diameter, 1/5 LD50 of these drugs was administered into the periotoneal cavity mice once a week for three weeks. RESULTS: The effective regimens were CPT-11 + TXL, CDDP + CPT-11, CDDP + CPM, CDDP + TXL for the endometrial cancer. CONCLUSIONS: It is suggested that these new drugs regimens should be tested in clinical studies.     

Enhanced growth inhibition and differentiation of fluorodeoxyuridine-treated human colon carcinoma cells by phenylbutyrate

The effect of phenylbutyrate (PB), a nontoxic differentiation inducer, in human colon carcinoma cell lines treated with 5-fluorodeoxyuridine (FUdR) was evaluated. Two HT-29 human colon carcinoma subclones (U4 well differentiated and U9 poorly differentiated) were equally growth inhibited by 16 h of FUdR (0.2 microM) treatment but recovered cell growth in 3-6 days after the removal of FUdR. PB as a single agent had minimal effect on cell growth, but after FUdR treatment, PB inhibited cell growth for 12 days. The inhibition of cell growth in FUdR-treated cells by PB was more sustained in U4 than U9 cells and was associated with an increased and sustained expression of p21waf1 protein, secretion of transforming growth factor beta1, mediators of p53-dependent or -independent G1 cell cycle arrest, and an increase in the alkaline phosphatase activity as well, considered a marker of differentiation in colon carcinoma cells. These effects of PB were seen only in FUdR-pretreated cells because PB alone had minimal effect on the expression of these genes. The sequential use of FUdR followed by PB in patients with colon carcinoma should be explored because two subclones of HT29, irrespective of their state of differentiation, respond to this clinically achievable regimen.     

Hormone- and DNA-binding mechanisms of the recombinant human estrogen receptor

We have investigated the hormone- and DNA-binding mechanisms of the wild-type human estrogen receptor (hER) overproduced in insect cells using a baculovirus expression system. The recombinant hER was indistinguishable in size (67 kDa) and immunogenically from the native human estrogen receptor in MCF-7 breast carcinoma cells. The recombinant hER was purified to 70-80% homogeneity with a two-step procedure that included ammonium sulfate precipitation and oligonucleotide affinity chromatography using a unique Teflon affinity matrix. The recombinant hER bound estradiol with a positively cooperative mechanism. At hER concentrations in excess of 13 nM the Hill coefficient reached a maximal value of 1.6, whereas, at lower hER concentrations, the Hill coefficient approached 1.0, suggesting that the hER was dissociated to the monomeric species and site-site interactions were diminished. The hER specifically bound an estrogen responsive element (ERE) from chicken vitellogenin II gene as measured by the gel mobility assay, ethylation, and thymine interference footprinting. Specific interference patterns suggest a two-fold symmetry of the hER binding to the ERE with each monomer of the hER bound in the major groove of the DNA. These data indicate that the recombinant hER is valuable to define the biochemical and structural properties of the native estrogen receptor.     

Assay of DNA content and estrogen receptor status in human breast cancer

IVOX was named as an acronym for intravascular oxygenator. The device does not need a blood pomp like an extracorporeal membrane oxygenator (ECMO), and performs intracorporeal gas exchange to be a small elongated, hollow fiber membrane oxygenator designed to lie within the subject's venae cavae so that circulating venous blood can flow freely over and around the external surfaces of the hollow fibers. The amount of gas exchange in IVOX is less than ECMO, however, the equipment is simple and there is no effect to hemodynamics and body temperature. IVOX has been utilized in the management of 165 clinical trials patients in 31 international critical care centers. Currently the gas transfer rate by means of the IVOX device constitutes 1/4 to 1/3 the total metabolic requirement of adult acute respiratory failure patients. Therefore, intentional hypoventilation to limit airway pressures (mild permissive hypercapnia) is recommended to improve CO2 removal with increasing mixed venous CO2 concentrations. In the future, improvements of design, function, and methods of utilization of IVOX device are expected to increase the amount of gas exchange and to enlarge the indications for its use.