Specific cytotoxicity of 16 alpha-[125I]iodoestradiol for estrogen receptor-containing breast cancer cells

We attempted to induce specific killing of estrogen receptor-containing breast cancer cells using estradiol coupled to a high specific activity gamma emitting isotope of iodine. MCF-7 human breast cancer cells were incubated with 16 alpha-[125I]iodoestradiol alone ([125I]E2), or with 16 alpha-[125I]iodoestradiol plus 100-fold excess 17 beta-estradiol (E2), and then viably frozen. After 8 weeks, the [125I]E2 exposed cells and the [125 I]E2 plus E2 exposed cells were 10% and 77% of controls, respectively. When the breast cancer cell line MDA-MB-231 which does not contain estrogen receptors was used, the rate of cell death was similar to the competed MCF-7 cells. When specific cytotoxicity was compared using a cloning technique, nearly a 5 log reduction in surviving cell fraction was seen with [125I]E2, as compared to identical cells treated with [125I]E2 plus competitor. This technique shows promise for selecting a population of cells with defects in their estrogen receptor and in studying subcellular hormone interactions.     

Detection of abscisic-acid-binding proteins in the microsomal protein fraction of Arabidopsis thaliana with abscisic-acid-protein conjugates used as affinity probes

Radioiodinated iododeoxyuridine (IUdR) is a novel, cycle-specific agent that has potential for the treatment of residual malignant glioma after surgery. As only cells in S-phase incorporate IUdR into DNA, a major limitation to this therapy is likely to be proliferative heterogeneity of the tumour cell population. Using a clonogenic end point, we have compared the toxicities of three radioiodoanalogues of IUdR--[123I]IUdR, [125I]IUdR and [131I]IUdR--to the human glioma cell line UVW, cultured as monolayers in the exponential and the plateau phase of growth and as multicellular spheroids. Monolayers treated in the exponential growth phase were most efficiently sterilized by [125I]IUdR (concentration resulting in 37% survival (C37) = 2.36 kBq ml(-1)), while [123I]IUdR and [131I]IUdR were less effective eradicators of clonogens (C37 = 9.75 and 18.9 kBq ml(-1) respectively). Plateau-phase monolayer cultures were marginally more susceptible to treatment with [123I]IUdR and [125I]IUdR (40% clonogenic survival) than [131I]IUdR (60% clonogenic survival). In cells derived from glioma spheroids, both [125I]IUdR and [123I]IUdR were again more effective than [131I]IUdR at concentrations up to and including 20 kBq ml(-1). However, the survival curve for [131I]IUdR crossed the curves for the other agents, resulting in lower survival for [131I]IUdR than [123I]IUdR and [125I]IUdR at concentrations of 40 kBq ml(-1) and higher, the clonogenic survival values at 100 kBq ml(-1) were 13%, 45% and 28% respectively. It was concluded that IUdR incorporating the Auger electron emitters 123I and 125I killed only cells that were in S-phase during the period of incubation with the radiopharmaceutical, whereas the superior toxicity to clonogenic cells in spheroids of [131I]IUdR at higher concentration was due to cross-fire beta-irradiation. These findings suggest that [131I]IUdR or combinations of [131I]IUdR and [123I]IUdR or [125I]IUdR may be more effective than Auger electron emitters alone for the treatment of residual glioma, if proliferative heterogeneity exists.     

Models of estrogen receptor regulation by estrogens and antiestrogens in breast cancer cell lines

The expression and stability of the estrogen receptor (ER) is the result of a complex process that is modulated by estrogens and antiestrogens. Regulation of the steady-state ER mRNA and protein levels in breast cancer cells appears to be the result of either of two distinct regulatory mechanisms. Estrogen exposure causes a rapid down-regulation of the steady-state level of ER mRNA and protein in model I regulation, as exemplified by the MCF-7:WS8 cell line. Conversely, in model II regulation, as observed in the T47D:A18 cell line, estrogen exposure causes an increase in the steady-state ER mRNA level and a maintenance of the ER protein level. In both these cell lines, the nonsteroidal antiestrogen 4-hydroxytamoxifen has little effect on the mRNA level but causes a net accumulation of the ER protein over time. In contrast, the pure antiestrogen ICI 182,780 causes a dramatic reduction of the ER protein in both the MCF-7:WS8 and T47D:A18 cell lines. This loss has little effect upon the ER mRNA level in the MCF-7:WS8 cells but leads to a decline in the ER mRNA in the T47D:Al8 cells. The estrogen-independent MCF-7:2A cell line, which has adapted to growth in estrogen free media, expresses two forms of the ER, a wild-type Mr66,000 ER and a mutant Mr77,000 ER (ER77). ER77 is the product of a genomic rearrangement resulting in a tandem duplication of exons 6 and 7 (J. J. Pink et al, Nucleic Acids Res., 24:962-969,1996). This exon duplication has abolished ligand binding by this protein. Here we demonstrate that the loss of ligand binding has eliminated the effects of 4-OHT and ICI 182,780 on the steady-state ER77 protein level. However, in the MCF-7:2A cells, antiestrogens affect the wild-type ER protein in the same manner as observed in the MCF-7:WS8 and T47D:A18 cells. Estrogen regulates the ER mRNA and wild-type ER and ER77 proteins in the MCF-7:2A cells in the same manner as observed in the MCF-7:WS8 cells. Interestingly, treatment of the MCF-7:2A cells with ICI 182,780 causes a slight increase in ER mRNA, which is reflected in a net increase in the ER77 protein but a dramatic decrease in the wild-type ER. The models presented here describe the response of two human breast cancer cell lines in short-term studies. These distinct regulation pathways are predictive of the response of these cell lines to long-term estrogen deprivation. This study illustrates two alternative regulation pathways that are present in ER-positive, estrogen-dependent breast cancer cells. This variable response highlights the diversity of responses potentially present in the heterogeneous cell populations of clinically observed breast cancer.     

Impaired chemical coupling of cerebral blood flow is compatible with intact neurological outcome in neonates with perinatal risk factors

To develop chelating molecules that provide 99mTc-labeled polypeptides of high in vivo stability and high specific activities under mild reaction conditions, an asymmetrical bis(benzohydroxamamide) compound with an amine group, 4'-aminomethyl-N,N'-trimethylenedibenzohydroxamamide [NH2-C3(BHam)2], was designed and synthesized. The amine residue of NH2-C3(BHam)2 was converted to a maleimide group by reaction with N-succinimidyl-6-maleimidohexanoate, and the conjugation product was coupled to thiol groups of a monoclonal antibody against osteogenic sarcoma (OST7, IgG1) pretreated with 2-iminothiolane to prepare C3(BHam)2-OST7. 99mTc radiolabeling of C3(BHam)2-OST7 was performed by the exchange reaction with [99mTc]glucoheptonate. [99mTc]C3(BHam)2-OST7 was further characterized using directly radioiodinated OST7 ([125I]OST7) and [111In]labeled OST7 with 1-[4-[(5-maleimidopentyl)amidobenzyl]ethylenediamine-N,N, N'N'-tetraacetic acid (EMCS-Bz-EDTA) as references. [99mTc]C3(BHam)2-OST7 was obtained with radiochemical yields of over 94% at protein concentrations as low as 0.2 mg/mL at room temperature for 1 h. [99mTc]C3(BHam)2-OST7 remained stable after incubation in freshly prepared murine plasma and in the presence of cysteine. Similar binding affinities to tumor cells were observed between [99mTc]C3(BHam)2-OST7 and [125I]OST7. When injected into normal mice, [99mTc]C3(BHam)2-OST7 exhibited radioactivity levels in the blood similar to [111In]-EMCS-Bz-EDTA-OST7 up to 24 h postinjection with significantly faster elimination rate of the radioactivity from the liver. In nude mice bearing osteogenic sarcoma, no significant differences were observed in the radioactivity levels in the blood and the tumor between [99mTc]C3(BHam)2-OST7 and [125I]OST7 at 24 h postinjection. These findings indicated that C3(BHam)2 provided 99mTc chelate of high stability at low concentrations even when conjugated to an intact antibody. Such characteristics render bis(hydroxamamide) compounds useful as chelating molecules for preparation of 99mTc-labeled polypeptides.     

A comparison of mass balance, pharmacokinetics and disposition of [14C(U)]- and [125I]recombinant human interleukin-2 in cynomolgus monkeys

Recombinant human interleukin-2 (rHuIL-2) has been metabolically labeled with 14C amino acids in Escherichia coli and affinity purified on a rHuIL-2 receptor affinity column. The radiolabeled molecule had a specific radioactivity of 238 dpm/unit and the identical amino acid sequence and biological activity as unlabeled rHuIL-2. In this study, we used this labeled [14C(U)]rHuIL-2 and commercially available [125I]rHuIL-2 (identical in sequence to the [14C(U)]rHuIL-2) to compare the mass balance, pharmacokinetics, and disposition in cynomolgus monkeys. After a single intravenous bolus dose of 4 x 10(5) units/kg, serum samples were collected for 7 days and examined for biological activity, total radioactivity, and by molecular size exclusion chromatography. Urine and feces were analyzed for total radioactivity. When analyzed for biological activity, both [14C(U)]- and [125I]rHuIL-2 exhibited the following pharmacokinetic parameters: terminal elimination half-life of 1-2 hr, AUC0-infinity ranged from 2005 to 4659 units x hr/ml, clearance was 90-200 ml/hr/kg, and volume of distribution ranged from 103 to 163 ml/kg. Comparison of the pharmacokinetic profiles of the two radiolabels were very different from bioactivity, in that the elimination half-lives for radioactivity were approximately 8 days and 10 hr for [14C(U)]- and [125I]rHuIL-2, respectively. We conclude that the [14C(U)]rHuIL-2 was metabolized to constituent amino acids and recycled into newly synthesized proteins from our size exclusion chromatography studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid sulfation of 3,3',5'-triiodothyronine in native Xenopus laevis oocytes

Sulfation is an important metabolic pathway facilitating the degradation of thyroid hormone by the type I iodothyronine deiodinase. Different human and rat tissues contain cytoplasmic sulfotransferases that show a substrate preference for 3,3'-diiodothyronine (3,3'-T2) > T3 > rT3 > T4. During investigation of the expression of plasma membrane transporters for thyroid hormone by injection of rat liver RNA in Xenopus laevis oocytes, we found uptake and metabolism of iodothyronines by native oocytes. Groups of 10 oocytes were incubated for 20 h at 18 C in 0.1 ml medium containing 500,000 cpm (1-5 nM) [125I]T4, [125I]T3, [125I]rT3, or [125I]3,3'-T2. In addition, cytosol prepared from oocytes was tested for iodothyronine sulfotransferase activity by incubation of 1 mg cytosolic protein/ml for 30 min at 21 C with 1 microM [125I]T4, [125I]T3, [125I]rT3, or [125I]3,3'-T2 and 50 microM 3'-phosphoadenosine-5'-phosphosulfate. Incubation media, oocyte extracts, and assay mixtures were analyzed by Sephadex LH-20 chromatography for production of conjugates and iodide. After 20-h incubation, the percentage of added radioactivity present as conjugates in the media and oocytes amounted to 0.9 +/- 0.2 and 1.0 +/- 0.1 for T4, less than 0.1 and less than 0.1 for T3, 32.5 +/- 0.4 and 29.3 +/- 0.2 for rT3, and 3.8 +/- 0.3 and 2.3 +/- 0.2 for 3,3'-T2, respectively (mean +/- SEM; n = 3). The conjugate produced from rT3 was identified as rT3 sulfate, as it was hydrolyzed by acid treatment. After injection of oocytes with copy RNA coding for rat type I iodothyronine deiodinase, we found an increase in iodide production from rT3 from 2.3% (water-injected oocytes) to 46.2% accompanied by a reciprocal decrease in rT3 sulfate accumulation from 53.7% to 7.1%. After 30-min incubation with cytosol and 3'-phosphoadenosine-5'-phosphosulfate, sulfate formation amounted to 1.8% for T4, less than 0.1% for T3, 77.9% for rT3, and 2.9% for 3,3'-T2. These results show that rT3 is rapidly metabolized in native oocytes by sulfation. The substrate preference of the sulfotransferase activity in oocytes is rT3 > 3,3'-T2 > T4 > T3. The physiological significance of the high activity for rT3 sulfation in X. laevis oocytes remains to be established.     

Targeted radiotherapy of multicell neuroblastoma spheroids with high specific activity [125I]meta-iodobenzylguanidine

PURPOSE: Iodine-125 induces cell death by a mechanism similar to that of high linear energy transfer (high-LET) radiation. This study investigates the cytotoxicity of high-specific-activity [125I]meta-iodobenzylguanidine (125I-mIBG) in human SK-N-MC neuroblastoma cells grown as three-dimensional multicellular spheroids. MATERIALS AND METHODS: Spheroids were incubated with high-specific-activity 125I-mIBG (6 mCi/microg, 1000 times that of the conventional specific activity used for autoradiography). Cytotoxicity was assessed by fluorescence viability markers and confocal microscopy for intact spheroids, fluorescence-activated cell sorting and clonogenic assay, and clonogenic assays for dispersed whole spheroids. Distribution of radioactive mIBG was determined by quantitative light-microscope autoradiography of spheroid cryostat sections. Dose estimation was based on temporal knowledge of the retained radioactivity inside spheroids, and of the radiolabel's emission characteristics. Findings were compared with those of spheroids treated under the same conditions with 131I-mIBG, cold mIBG, and free iodine-125. RESULTS: 125I-mIBG exerted significant cell killing. Complete spheroids were eradicated when they were treated with 500 microCi of 125I-mIBG, while those treated with 500 microCi or 1000 microCi of 131I-mIBG were not. The observed difference in cytotoxicity between treatments with 125I- and 131I-mIBG could not be accounted for by the absorbed dose of spheroid alone. The peripheral, proliferating cell layer of the spheroids remained viable at the moderate radioactivity of 100 microCi for both isotopes. Cytotoxicity induced by 125I-mIBG was quantitatively comparable by the peripheral rim thickness to that of 131I-mIBG at the dose of 100 microCi. The peripheral rim thickness decreased most significantly in the first 17 hours after initial treatment. There was no statistical decrease in the rim thickness identified afterwards for the second, third, and fourth days of incubation. CONCLUSION: The cytotoxic effect of high-specific-activity 125I-mIBG appears to be comparable to, if not more efficient than that of conventionally used 131I-mIBG at the same level of total radioactivity. 125I-mIBG may improve the therapeutic index over that of 131I-mIBG in the clinical management of metastatic neuroblastoma due to the short range of Auger electrons.     

Overexpression of HER2 modulates bcl-2, bcl-XL, and tamoxifen-induced apoptosis in human MCF-7 breast cancer cells

Overexpression of HER2 in estrogen receptor (ER)-positive human breast tumors has been associated with resistance to endocrine therapy. Here we investigated the effects of HER2 on expression of apoptotic pathways and modulation of tamoxifen-induced apoptosis in ER-positive MCF-7 breast cancer cells. We report that HER2 overexpression in MCF-7 cells is accompanied by up-regulation of antiapoptotic Bcl-2 and Bcl-XL proteins and suppression of tamoxifen-induced apoptosis. In addition, human tumor cell lines that are both ER positive and overexpress HER2 also express enhanced levels of Bcl-2 compared to cells that are either ER positive or overexpress HER2 alone. Our findings suggest that possible deregulation of antiapoptotic Bcl-2 and Bcl-XL may be associated with the enhanced survival of HER2-overexpressing and ER-positive breast cancer cells treated with antiestrogens.     

A model to describe how a point mutation of the estrogen receptor alters the structure-function relationship of antiestrogens

The antiestrogen tamoxifen [(Z)-1(p-beta-dimethylamino-ethoxyphenyl)-1,2- diphenylbut-1-ene] is an effective anticancer agent for the treatment of hormone responsive breast cancer. Previous studies have demonstrated that a point mutation in the estrogen receptor (ER) resulted in an alteration of the pharmacology of 4-hydroxytamoxifen, the active metabolite of tamoxifen (Jiang et al, Mol Endocrinol 6:2167-2174, 1992). We have extended our studies to evaluate the effect of a point mutation, a Val substitution for Gly at amino acid 400 in the ligand binding domain of ER, on the pharmacology of other antiestrogens in ER stable transfectants derived from the ER-negative breast cancer cell line MDA-MB-231 CL10A. The compounds were tested with or without estradiol-17 beta (E2) for their effects on cell growth in cells expressing the wild type ER (S30) or the mutant ER (ML alpha 2H) or in control antisense ER transfectant AS23 which does not express ER protein. MCF-7 cells, which express the wild type ER, were also used as a control. The growth of AS23 cells was not affected by any of the compounds at a concentration of 1 microM. E2 stimulated the growth of MCF-7 cells but inhibited the growth of ER transfectants S30 and ML alpha 2H. The ML alpha 2H cells were about 10 to 100-fold less sensitive to E2 and antiestrogens than S30 and MCF-7 cells. Keoxifene, an antiestrogen with a high affinity for the ER, maintained antiestrogenic activities in both ER transfectants and MCF-7 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in radiation sensitivity and steroid receptor content induced by hormonal agents and ionizing radiation in breast cancer cells in vitro

Possible influences of tamoxifen and estradiol on in vitro radiation sensitivity and cellular receptor content after irradiation and/or tamoxifen treatment were studied in breast cancer cell lines; estrogen receptor (ER) and progesterone receptor (PgR) positive cell lines MCF-7 and MCF-7/TAM(R)-1 and the ER and PgR negative cell line MDA-MB-231. The tamoxifen resistant MCF-7/TAM(R)-1 cells were more resistant to ionizing radiation than the MCF-7 and MDA-MB-231 cells. Exposure to tamoxifen made the MCF-7 cells more radiation resistant, while estradiol made the MDA-MB-231 cells more radiation sensitive. A radiation dose of 6 Gy reduced the ER content in cytosol in both MCF-7 and MCF-7/TAM(R)-1 cells, but brought no alterations to the PgR content. In MCF-7/TAM(R)-1 cells tamoxifen exposure significantly increased the ER and reduced the PgR content, an effect not observed in the MCF-7 cells. To conclude, the present study indicates that irradiation and tamoxifen may modify the ER and PgR content in cytosol in breast cancer cells. Hormonal treatment may alter the radiation sensitivity, even in ER negative cells, suggesting that hormonal agents may act both via receptor and non-receptor binding mechanisms.     

Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium

Estradiol-17beta (E2) acts through the estrogen receptor (ER) to regulate uterine growth and functional differentiation. To determine whether E2 elicits epithelial mitogenesis through epithelial ER versus indirectly via ER-positive stromal cells, uteri from adult ER-deficient ER knockout (ko) mice and neonatal ER-positive wild-type (wt) BALB/c mice were used to produce the following tissue recombinants containing ER in epithelium (E) and/or stroma (S), or lacking ER altogether: wt-S + wt-E, wt-S + ko-E, ko-S + ko-E, and ko-S + wt-E. Tissue recombinants were grown for 4 weeks as subrenal capsule grafts in intact female nude mice, then the hosts were treated with either E2 or oil a week after ovariectomy. Epithelial labeling index and ER expression were determined by [3H]thymidine autoradiography and immunohistochemistry, respectively. In tissue recombinants containing wt-S (wt-S + wt-E, wt-S + ko-E), E2 induced a similar large increase in epithelial labeling index compared with oil-treated controls in both types of tissue recombinants despite the absence of epithelial ER in wt-S + ko-E tissue recombinants. This proliferative effect was blocked by an ER antagonist, indicating it was mediated through ER. In contrast, in tissue recombinants prepared with ko-S (ko-S + ko-E and ko-S + wt-E), epithelial labeling index was low and not stimulated by E2 despite epithelial ER expression in ko-S + wt-E grafts. In conclusion, these data demonstrate that epithelial ER is neither necessary nor sufficient for E2-induced uterine epithelial proliferation. Instead, E2 induction of epithelial proliferation appears to be a paracrine event mediated by ER-positive stroma. These data in the uterus and similar studies in the prostate suggest that epithelial mitogenesis in both estrogen and androgen target organs are stromally mediated events.     

Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: no apparent synergism

The estrogenic activity of dieldrin, toxaphene, and an equimolar mixture of both compounds (dieldrin/toxaphene) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 human breast cancer cells, and in yeast-based reporter gene assays. Treatment of the animals with 17beta-estradiol (E2) (0.0053 kg/day x3) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and progesterone receptor binding, respectively. In contrast, treatment with 2.5, 15 and 60 micromol/kg (x3) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dose-dependent increase in any of the E2-induced responses. The organochlorine pesticides alone and the binary mixture did not bind to the mouse uterine estrogen receptor (ER) in a competitive binding assay using [3H]E2 as the radioligand. In parallel studies, estrogenic activities were determined in MCF-7 cells by using a cell proliferation assay and by determining induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with plasmids containing estrogen-responsive 5'-promoter regions from the rat creatine kinase B and human cathepsin D genes. E2 caused a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with creatine kinase B and a 3.8-fold increase in cells transiently transfected with the human cathepsin D construct. Treatment of MCF-7 cells with dieldrin, toxaphene, or an equimolar mixture of dieldrin plus toxaphene (10(-8)-10(-5) M) did not significantly induce cell proliferation or CAT activity in the transient transfection experiment with both plasmids. The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with 10(-9) M [3H]E2 in the presence or absence of 2 x 10(-7) M unlabeled E2 (to determine nonspecific binding), toxaphene (10(-5) M), dieldrin (10(-5) M), and equimolar concentrations of the dieldrin plus toxaphene mixture (10(-5) M). The binding observed for [3H]E2 in the whole cell extracts was displaced by unlabeled E2, whereas the organochlorine pesticides and binary mixture exhibited minimal to nondetectable competitive binding activity. E2 caused a 5000-fold induction of beta-galactosidase (beta-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene. Treatment with 10(-6)-10(-4) M chlordane, dieldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 10(-4) M endosulfan caused a 2000-fold increase in beta-gal activity. Diethylstilbestrol caused a 20-fold increase in activity in yeast transformed with the mouse ER and a single estrogen responsive element upstream of the beta-gal reporter gene. Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in activity at a concentration of 2.5 x 10(-5) M. Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 x 10(-5) M or 2.5 x 10(-4) M. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based reporter gene assays, the activities of both dieldrin and toxaphene were minimal, and no synergistic interactions were observed with a binary mixture of the two compounds.     

Estimation of unit risk for coke oven emissions

We are investigating the hypothesis that biotin multimers can be used with streptavidin and monoclonal antibody conjugates in cancer pretargeting protocols to provide a method of increasing the amount of radioactivity bound on cancer cells in patients. As part of that investigation, a series of biotinylated Starburst dendrimers (BSBDs) have been prepared and evaluated in vitro and in vivo. In this study, a new biotinidase-stabilized, water-solubilizing biotinylation reagent was prepared and reacted with Starburst (PAMAM) dendrimers, generations 0, 1, 2, 3, and 4. The reaction conditions employed resulted in perbiotinylation of generation 0 (four biotin moieties conjugated), generation 1 (eight biotin moieties conjugated), generation 2 (16 biotin moieties conjugated), and generation 3 (32 biotin moieties conjugated). With generation 4, incomplete biotinylation was achieved resulting in the largest portion of that BSBD having 51 biotin moieties (of 64 possible) conjugated. The ability of each BSBD to cross-link streptavidin (SAv) was examined in an in vitro assay. In that assay, an assessment was made of the quantity of [125I]SAv bound with polystyrene-bound SAv after treatment with the synthesized BSBDs. All BSBDs cross-linked the polystyrene-bound SAv with [125I]SAv; however, the amount of [125I]SAv bound varied with the different BSBDs. Roughly 1 equiv of [125I]SAv was bound when Starburst dendrimers containing three or four biotin moieties (generation 0) were used. Two equivalents were bound with BSBD generation 1, and 4 equiv were bound with BSBDs generations 2, 3, and 4. To assess the distribution of BSBDs generations 0, 1, and 2 in mice (at 4 h postinjection), a method was developed for radioiodinating them using the NHS ester of p-[125I]iodobenzoate ([125I]PIB). It was found that the radioiodinated BSBDs had low blood concentrations (i.e., 0.13-0.20% ID/g) at the 4 h time point. In fact, most tissues examined had low concentrations of biotinylated dendrimers, except kidney and liver. Kidney had the highest concentration of [125I]-labeled BSBDs, and its concentration increased with increasing size and charge of dendrimer (e.g., 8-48% ID/g). On the basis of the increased radioactivity observed in the in vitro assay and the rapid clearance from blood in mice, additional in vivo studies with perbiotinylated Starburst dendrimer, generation 2, are planned.     

Interplay between sex steroids and melatonin in regulation of human benign prostate epithelial cell growth

Human benign prostatic epithelial cells contain functional melatonin receptors that can suppress cell growth and viability. The development of benign prostatic hyperplasia in men is assumed to result from androgen-estrogen imbalance. The impact of sex steroids on melatonin receptors in human benign prostate epithelial cells was investigated. The suppression by melatonin of [3H]thymidine incorporation and cGMP, and the enhancement of cAMP levels in the cells were used as markers of melatonin responses. Dihydrotestosterone (DHT) and 17 beta-estradiol (E2) separately increased [3H]thymidine incorporation into the cells, but suppressed it when combined. In cells grown with DHT, melatonin responses were extenuated. E2 greatly reduced the apparent affinity of [125I]melatonin binding in these cells without affecting binding site density. In parallel, the ability of melatonin to suppress [3H]thymidine incorporation into the cells was ablated within 1 h after the addition of E2. The melatonin-mediated increase in cAMP and decrease in cGMP concentrations were also ablated by E2. Preincubation of the cells with bis-indolylmaleimide (GF 102903X), a specific inhibitor of protein kinase C, prevented the E2-mediated inactivation of melatonin binding and the inhibitory action on [3H]thymidine incorporation. Prolonged (18-h) incubation of the cells with phorbol 12-myristate 13-acetate to down regulate protein kinase activity, partially restored [125I]melatonin binding and responsiveness in the E2-treated cells. These data indicate that 1) DHT and E2 enhance prostate epithelial cells growth, but reduce cell growth when combined; 2) DHT extenuates the inhibitory effects of melatonin on epithelial cell growth; and 3) E2 acts to inactivate melatonin receptors and consequently responses in human epithelial benign prostatic hyperplasia cells. This process is probably mediated by protein kinase C. Together, these results show an interplay between melatonin and sex steroids in the regulation of benign prostatic epithelial cell growth.     

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.     

Decrease of hormone binding capacity of estrogen receptor by calcium

We have addressed the question as to whether calcium may modify the [3H]estradiol ([3H]E2) binding properties of the estrogen receptor (ER). A human recombinant full length ER (yER) expressed in yeast was used to limit the potential interference of ER-associated proteins and proteases present in the target tissues. Ca++ (0.1-10 mM) always produced an important loss of [3H]E2 binding capacity without any effect on the hormone binding affinity of residual receptors. This loss was reflected in a decrease of immunoreactivity for monoclonal antibodies raised against the hormone binding domain. An ER recombinant expressing solely this domain confirmed that the ion operated at this level. Binding of [125I]Z-17 alpha-(2-iodovinyl)-11 beta-chloromethyl estradiol-17 beta (an compound with very high selectivity for ER) as well as [125I]tamoxifen aziridine were similarly affected. Size-exclusion chromatography failed to reveal the emergence of any ER isoforms of low molecular weight rejecting the hypothesis of a Ca(++)-induced proteolysis. In agreement with this conclusion, EDTA reversed the loss of [3H]E2 binding capacity. Phosphoamino acids (PY, PT and PS) partly antagonized the effect of Ca++ suggesting its interaction with phosphoamino acid residues. Worthy of note, the effect of Ca++ appeared more marked when assessed by DCC than HAP assay. The phosphocalcic nature of the HAP matrix may explain this phenomenon which was observed with cytosolic ER from various origins.     

Efflux of intracellular versus plasma membrane cholesterol in HepG2 cells: different availability and regulation by apolipoprotein A-I

We have compared the efflux of cholesterol from different cellular pools of human hepatoma cells HepG2 using intact cells or isolated membrane fractions. To label different pools, cells were incubated with either unesterified [14C]cholesterol that had been incorporated into high density lipoproteins ([14C]FC-HDL), low density lipoproteins ([14C]FC-LDL), or phosphatidylcholine liposomes ([14C]FC-PC), or with [14C]acetate. Cell fractionation revealed that labeling of cells with [14C]FC-PC resulted in the incorporation of [14C]cholesterol almost exclusively into the plasma membrane (PM), while incubation with [14C]FC-HDL resulted in the majority of [14C]cholesterol incorporation into the PM, but with a smaller component associated with lysosomes. Labeling with [14C]FC-LDL or [14C]acetate led to an accumulation of [14C]cholesterol predominantly in lysosomes or the endoplasmic reticulum (ER), respectively. When the kinetics of [14C]cholesterol efflux was analyzed after pulse-labeling of different cellular pools, half-times of cholesterol efflux from lysosomes and ER were significantly longer than that from PM. In another set of experiments, when both labeling and efflux times varied, efflux of [14C]cholesterol from the PM to human serum after 1.5 h pulse and chase incubations was double that from lysosomes and 8-fold that from ER. Extension of the incubation times from 1.5 to 3 h diminished the difference in cholesterol efflux from different membranes. Further incubation to 6 h almost abolished the different responses. Cell-free preparations of membranes, obtained from cells labeled with [14C]cholesterol, showed no differences in cholesterol efflux. No differences in the distribution of [14C]cholesterol released into serum among lipoprotein subfractions was observed. Pretreatment of the serum with Fab fragments of polyclonal rabbit anti-human apolipoprotein A-I antibodies reduced its ability to promote efflux of cholesterol from the ER by 77%, but had no effect on cholesterol efflux from the PM. Fab fragments of non-immune IgG had no effect on the efflux of both ER and PM cholesterol. We conclude that the availability of cellular cholesterol for efflux from HepG2 cells is strongly influenced by its subcellular location, and is regulated by apolipoprotein A-I.     

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)