Identification of an amino acid residue in multidrug resistance protein 1 critical for conferring resistance to anthracyclines.
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Murine multidrug resistance protein 1 (mrp1), unlike human MRP1, does not confer resistance to anthracyclines. Previously, we have shown that a human/murine hybrid protein containing amino acids 959-1187 of MRP1 can confer resistance to these drugs. We have now examined the functional characteristics of mutant proteins in which we have converted individual amino acids in the comparable region of mrp1 to those present at the respective locations in MRP1. These mutations had no effect on the drug resistance profile conferred by mrp1 with the exception of converting glutamine 1086 to glutamate, as it is in the corresponding position (1089) in MRP1. This mutation created a protein that conferred resistance to doxorubicin without affecting vincristine resistance, or the ability of mrp1 to transport leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG). Furthermore, mutation Q1086D conferred the same phenotype as mutation Q1086E while the mutation Q1086N did not detectably alter the drug resistance profile of mrp1, suggesting that an anionic side chain was required for anthracycline resistance. To confirm the importance of MRP1 E1089 for conferring resistance to anthracyclines, we mutated this residue to Gln, Asp, Ala, Leu, and Lys in the human protein. The mutation E1089D showed the same phenotype as MRP1, while the E1089Q substitution markedly decreased resistance to anthracyclines without affecting LTC(4) and E(2)17betaG transport. Conversion of Glu-1089 to Asn, Ala, or Leu had a similar effect on resistance to anthracyclines, while conversion to a positive amino acid, Lys, completely eliminated resistance to anthracyclines and vincristine without affecting transport of LTC(4), E(2)17betaG, and the GSH-dependent substrate, estrone-3-sulfate. These results demonstrate that an acidic amino acid residue at position 1089 in predicted TM14 of MRP1 is critical for the ability of the protein to confer drug resistance particularly to the anthracyclines, but is not essential for its ability to transport conjugated organic anions such as LTC(4) and E(2)17betaG. (+info)
Up-regulation of steroid sulphatase activity in HL60 promyelocytic cells by retinoids and 1alpha,25-dihydroxyvitamin D3.
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HL60 promyeloid cells express both classes of oestrogen receptor (ERalpha and ERbeta). We show that hydrolysis of oestrone sulphate by steroid sulphatase is a major source of oestrone in HL60 cells, and that most of the released oestrone is not metabolized further to 17beta-oestradiol. Treatment of HL60 cells with retinoids or 1alpha,25-dihydroxyvitamin D3 increased steroid sulphatase mRNA and activity in parallel with the induction of CD11b, an early marker of myeloid differentiation that is expressed before the differentiating cells stop proliferating. Use of agonists and antagonists against retinoid receptor-alpha and retinoid receptor-X revealed that both classes of retinoid receptor can drive steroid sulphatase up-regulation. Steroid sulphatase activity fluctuates during the cell cycle, being highest around the transition from G1 to S phase. During the differentiation of HL60 cells induced by all-trans-retinoic acid or 1alpha,25-dihydroxyvitamin D3, there is increased conversion of 17beta-oestradiol into oestrone by an oxidative 17beta-hydroxysteroid dehydrogenase. Treatment of Caco-2 colon adenocarcinoma cells with all-trans-retinoic acid or 1alpha,25-dihydroxyvitamin D3 also increases 17beta-oestradiol oxidation to oestrone. An increase in local oestrone production therefore occurs in multiple cell types following treatment with retinoids and 1alpha,25-dihydroxyvitamin D3. The possible involvement of locally produced oestrogenic steroids in regulating the proliferation and differentiation of myeloid cells is discussed. (+info)
Pharmacologic, but not dietary, genistein supports endometriosis in a rat model.
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Endometriosis is a disease in which uterine tissue proliferates in extrauterine sites. Using a surgical model to simulate endometriosis, we explored the potential for the phytoestrogen genistein, by injection and diet, to sustain endometriosis in rats. Uterine tissue was attached to intestinal mesentery of 8-week-old Sprague Dawley rats. After 3 weeks, the rats were ovariectomized and the implants measured. Following 3 weeks of daily injections or exposure to dietary genistein, animals were necropsied and implants located and measured. Injections of genistein (50 and 16.6 microg/g BW) or estrone (1 microg/rat) sustained the implants; injection of sesame oil (vehicle for estrone), dimethylsulfoxide (DMSO; vehicle for genistein), or genistein at 5.0 microg/g BW did not sustain implants. Dietary genistein (250 or 1000 mg genistein/kg AIN-76A diet) did not support the implants. In ovary-intact rats exposed to 250 mg genistein/kg AIN-76A diet, implant size was not altered, compared to control-fed animals. To assess estrogenic actions of genistein, we measured uterine estrogen receptor alpha (ER-alpha) and progesterone receptor (PR) isoforms A and B by Western blot analyses. Injections of estrone or genistein (50 or 16.6 microg/g BW) significantly reduced uterine ER-alpha compared to vehicle-treated animals. PR (B) was significantly increased by all injected doses of genistein or estrone and by the higher dietary dose (1000 mg genistein/kg AIN-76A). PR (A) was significantly increased by injected doses of genistein (16.6 and 5.0 microg/g BW). We conclude that pharmacologic injections, but not dietary physiological concentrations of genistein, support surgically induced endometriosis in rats. Our results suggest a critical role for ER modulation and genistein bioavailability in the maintenance of the implants. (+info)
Phospholipid-dependence of oestrone UDP-glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase.
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Hepatic UDP-glucuronyltransferase activity was resolved into two fractions, one exhibiting oestrone glucuronyltransferase activity and the other exhibiting p-nitrophenol glucuronyltransferase activity. Hydroxyapatite-column chromatography removed greater than 95% of the phospholipids from both preparations. The partially purified delipidated enzymes were essentially devoid of catalytic activity, but activities were restored by the addition of phospholipids or phosphatidylcholine mixtures containing various saturated and unsaturated fatty acids. Both oestrone and p-nitrophenol glucuronyl-transferase activities were reconstituted to similar degrees with the phosphatidylcholine mixtures. When purified phospholipids were tested, phosphatidylcholine and lysophosphatidylcholine were most effective in restoring activity, whereas phosphatidylethanolamine was the least effective. These results further suggest that oestrone and p-nitrophenol UDP-glucuronyltransferases are dependent on phospholipids for their activity. (+info)
Do urinary estrogen metabolites reflect the differences in breast cancer risk between Singapore Chinese and United States African-American and white women?
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Breast cancer risk is substantially lower in Singapore than in women from the United STATES: Part of the risk discrepancy is probably explained by differences in the production of endogenous estrogens, but differences in the pathway by which estrogen is metabolized may also play a role. We undertook a study to determine whether the ratio of urinary 2-hydroxyestrone (2OHE(1)):16alpha-hydroxyestrone (16alpha-OHE(1)) was higher in Singapore Chinese than in a group of United States (predominantly African-American) women living in Los ANGELES: We also wanted to determine whether any difference in estrogen metabolite ratio between these two groups of women was greater than that in estrone (E(1)), estradiol (E(2)) and estriol (E(3)). The participants in this study were randomly selected healthy, non-estrogen using women participating in the Singapore Chinese Health Study (n = 67) or the Hawaii/Los Angeles Multiethnic Cohort Study (n = 58). After adjusting for age and age at menopause, mean urinary 2-OHE(1) was only 23% (P = 0.03) higher in Singapore Chinese than in United States women, and there were no statistically significant differences in 16alpha-OHE(1) levels or in the ratio of 2-OHE(1):16alpha-OHE(1) between the two groups. The adjusted mean 2-OHE(1):16alpha-OHE(1) ratio was 1.63 in Singapore Chinese and 1.48 in United States women (P = 0.41). In contrast, the adjusted mean values of E1, E2, and E3 were 162% (P < 0.0001), 152% (P < 0.0001), and 92% (P = 0.0009) higher, respectively, in United States women than in Singapore Chinese women. Our study suggests that urinary E1, E2, and E3 reflect the differences in breast cancer risk between Singapore Chinese and United States women to a stronger degree than the estrogen metabolites 2OHE(1) and 16alpha-OHE(1) or the ratio of 2OHE(1):16alpha-OHE(1.) (+info)
Dexamethasone blocks antioestrogen- and oxidant-induced death of pituitary tumour cells.
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The oestrogen receptor is fundamental to the growth and survival of the rat pituitary tumour cell line, GH(3). Our previous studies have shown that antioestrogens such as RU 58668 and ZM 182780 will reduce the rate of cell division and also induce cell death. Death of these cells in response to antioestrogen treatment appears to be due to a heightened sensitivity to reactive oxygen species (ROS). As part of a study to determine the cross-talk between steroid receptor systems in these cells, we have observed that the glucocorticoid, dexamethasone (Dex), inhibits antioestrogen-induced cell death. Cell death induced by H(2)O(2) is enhanced by ZM 182780 and this effect is also blocked by Dex. As apoptotic cell death in a number of systems involves an early loss of mitochondrial membrane potential (DeltaPsi(m)), we have performed detailed studies on the time-course of DeltaPsi(m) loss in relation to the loss in cell membrane function. These studies have indicated that a loss of DeltaPsi(m) parallels a loss of cell membrane function - this is more characteristic of necrosis than of apoptosis. From microscopic observations of these cells in response to H(2)O(2), it has been noted that early cell membrane blebbing, induced by H(2)O(2), is blocked in the presence of ZM 182780. Cell membrane blebbing can precede necrosis as well as apoptosis and it is thought to involve cytoskeletal changes, for which localised glycolytic reactions provide ATP. These observations, together with those showing that removal of glucose, but not inhibition of mitochondrial function, enhances ROS-induced cell death, prompted studies on the glycolytic pathway. As a strong candidate mechanism, it would appear that, via an effect on one of the rate-limiting glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase, Dex is able to overcome the antioestrogen-enhanced loss of glycolytic function following exposure of cells to ROS. This report contributes to the growing body of evidence showing that glucocorticoids provide a survival advantage to both normal and tumour cell types. (+info)
Effects of 2-[3-estrone-N-ethyl-piperazine-methyl]tetracycline (XW630) on osteoporosis in ovariectomized rats.
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AIM: To study the effects of 2-[3-estrone-N-ethyl-piperazine-methyl]tetracycline (XW630) on experimental osteoporosis in ovariectomized (OVX) rats. METHODS: Serum estradiol (E2) content and bone 1-carboxyglutamic acid-containing protein (BGP) content were measured by radioimmunoassay. With undecalcified bone section and tetracycline intraperitoneal labeling, the bone static and dynamic data were studied in right femur samples. RESULTS: After treatment with XW630 2.5 mg.kg-1, serum BGP content increased by 75.7% but there was no change in serum E2 content and uterus weight compared with OVX rats. Compared with OVX rats, the static data of trabecular bone volume/total tissue volume, trabecular bone volume/sponge bone volume, and mean trabecular plate density were enhanced after treatment with XW630 for 13 wk. The dynamic data of single-labeled surface, double-labeled surface, trabecular osteoid surface, and bone formation rate in tissue level in XW630 group were increased and osteoid maturation period was shortened. CONCLUSION: XW630 enhanced bone activation frequency and increased trabecular connectivity, stability, and strength. XW630 stimulated bone formation and inhibited bone resorption with no effect on reproductive system. (+info)
Effects of XW630 on cell proliferation, iNOS activity, and cGMP content in human osteoblast-like cell line TE85.
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AIM: To study the effects of 2-[3-estrone-N-ethyl-piperazine-methyl] tetracycline (XW630) in human osteoblast-like cell line TE85. METHODS: [3H]Thymidine incorporation and cell count for cell proliferation, radioimmunoassay for cyclic GMP (cGMP) content, and monitoring the conversion of [3H]arginine for inducible nitric-oxide synthase (iNOS) activity assay. RESULTS: After treatment with XW630 for 48 h, [3H]thymidine incorporation and cell numbers increased by 62.7% and 96.9%, respectively. NG-monomethyl-L-arginine (L-NMMA, an NOS inhibitor) induced a concentration-dependent inhibitory effect on the proliferation after treatment for 48 h. The inhibitory effect was prevented partially by XW630 (1.0 nmol.L-1). After treatment with XW630 for 12-48 h, iNOS activity and cGMP concentration increased in time-dependent manners. CONCLUSION: XW630 stimulated cell proliferation, enhanced iNOS activity and cGMP content in human osteoblast-like cell line TE85. (+info)