GFP-reporter for a high throughput assay to monitor estrogenic compounds

In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor α (ERα) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing β-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years.     

Induction of estrogen-regulated genes differs in immortal and tumorigenic human mammary epithelial cells expressing a recombinant estrogen receptor.

Studies on estrogen receptor (ER)-positive human breast cancer cell lines have shown that estrogen treatment positively modulates the expression of the genes encoding transforming growth factor-alpha (TGF alpha), 52-kDa cathepsin-D, and pS2. To determine whether these genes would be similarly regulated by estrogens in normal human mammary epithelial cells, we stably transfected immortal nontumorigenic human mammary epithelial cells with an ER-encoding expression vector. ER-negative tumor cells were also transfected for comparison. Levels of TGF alpha and 52-kDa cathepsin-D mRNA were enhanced by estrogen treatment of both ER-transfected immortal and tumorigenic cells, demonstrating that the ER by itself is sufficient to elicit estrogenic regulation of the expression of these genes. In contrast, expression of the pS2 gene was detected only in the ER-transfected tumor cells. The ER in both cell lines is capable of recognizing the pS2 promoter, however, since estrogen enhanced the activity of an introduced pS2-CAT reporter plasmid in transient expression analyses. These and other experiments with somatic cell hybrids between the immortal cells and ER+/pS2+ MCF-7 tumor cells, where pS2 gene expression is extinguished, support the conclusion that the immortal nontumorigenic cells encode gene products that block endogenous pS2 expression. These results also imply that such repressors are not active in the tumor cells.     

A novel estrogen sensor based on recombinant Arxula adeninivorans cells

A novel yeast cell-based assay was developed for the detection of estrogenic activity in wastewater. Recombinant Arxula adeninivorans strains were engineered to co-express the human estrogen receptor alpha (hERalpha) and a Klebsiella-derived phytase (phyK) reporter gene under the control of an A. adeninivorans-derived glucoamylase (GAA) promoter which had been modified by the insertion of estrogen-responsive elements (EREs). In the presence of estrogenic compounds, hERalpha dimerizes and binds to the estrogen. Reporter gene expression is induced by subsequent binding of the hERalpha-dimer/estrogen complex to estrogen responsive elements (ERE) in the promoter. The insertion of different numbers of EREs in three alternative promoter positions and its effect on reporter gene expression were assessed. In one of the constructs, a detection limit of 5 ng l(-1) and a determination limit of 10 ng l(-1) for 17beta-estradiol-like activity was achieved. The photometric assay used enabled estrogen determination in sewage samples within 30 h.     

Effects of environmental estrogenic chemicals on AP1 mediated transcription with estrogen receptors alpha and beta

There has been much discussion concerning endocrine disrupting chemicals suspected of exerting adverse effects in both wildlife and humans. Since the majority of these compounds are estrogenic, a large number of in vitro tests for estrogenic characteristics have been developed for screening purpose. One reliable and widely used method is the reporter gene assay employing estrogen receptors (ERs) and a reporter gene with a cis-acting estrogen responsive element (ERE). Other elements such as AP1 also mediate estrogenic signals and the manner of response could be quite different from that of ERE. Since this has yet to be explored, the ER mediated AP1 activity in response to a series of environmental estrogens was investigated in comparison with ERE findings. All the compounds exhibited estrogenic properties with ERE-luc and their AP1 responses were quite similar. These was one exception, however, p,p'-DDT (1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane) did not exert any AP1-luc activity, while it appeared to be estrogenic at 10(-7) to 10(-5)M with the ERE action. None of the compounds demonstrated ER beta:AP1 activity. These data suggest that significant differences can occur in responses through the two estrogen pathways depending on environmental chemicals.     

Construction of high sensitive detection system for endocrine disruptors with yeast n-alkane-assimilating Yarrowia lipolytica

To construct a highly sensitive detection system for endocrine disruptors, we have compared the activity of promoters with the ALK1, ICL1, RPS7 and TEF1 for heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of lacZ or hERalpha reporter gene, respectively, and the activity was evaluated by beta-galactosidase assay by lacZ or western blot analysis by hERalpha. The expression analysis revealed that the ALK1 and ICL1 promoter were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly high level of expression in the presence of glucose and glycerol, respectively. Particularly, the TEF1 promoter showed the highest beta-galactosidase activity and a significant signal by western blotting with the anti-estrogen receptor compared with the other promoters. Moreover, the detection system was constructed with promoters were linked to the upstream of expression vector for hERalpha gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hERalpha and CXAU1-2XERE was the most effective system for the E2-dependent induction of the beta-galactosidase activity. This system showed the highest beta-galactosidase activity at 10-6 M E2 and the activity could be detected at even the concentration of 10-10 M E2. As the result, we constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity and reproducibility of the system for characterizing environmental estrogens.     

高通量筛选雌激素类化合物重组绿色荧光蛋白酵母细胞测评体系

以载体双表达的方式构建重组酵母环境雌激素的评价体系, 用于快速筛选雌激素类化合物。在表达载体中, 用3-磷酸甘油醛脱氢酶(GPD)启动子驱动a人雌激素受体基因(hERa)的表达; 在报告载体中, 用雌激素效应元件(ERE)调控的绿色荧光蛋白(yEGFP)作为报告基因。将两者转化于酵母细胞(W303-1A)中, 构建成重组绿色荧光蛋白酵母细胞。该酵母细胞经不同浓度的雌激素类化合物作用后, 发现GFP的表达量与此类受试物具有明显的剂量效应关系。与其他环境雌激素酵母评价体系相比, 该重组酵母评价细胞, 在应用时不需要破坏细胞壁, 也不需要底物和相关试剂, 可直接在96孔板中操作完成, 具有快速、高通量、敏感性高、重现性好及廉价等特点。     

Construction of a bacterial assay for estrogen detection based on an estrogen-sensitive intein

Escherichia coli strain DIER was constructed for estrogen detection by inserting an estrogen-sensitive intein (VMA(ER) intein) into the specific site of the constitutively expressed chromosomal lacZ gene. This VMA(ER) intein was generated by replacing the endonuclease region of the Saccharomyces cerevisiae VMA intein with the estrogen binding region of the human estrogen receptor α (hERα). When there were estrogens or analogs, the splicing of the VMA(ER) intein was induced to produce the mature LacZ protein, which was detected through a β-galactosidase colorimetric assay. Eight typical chemicals (17-β-estradiol, bisphenol A, chrysene, 6-OH-chrysene, benz[a]anthracene, pyrene, progesterone, and testosterone) were detected using this DIER strain, and the whole detection procedure was accomplished in 2 h. Their 50% effective concentrations (EC(50)), relative estrogenic activities, and estradiol equivalency factors were calculated and were quite consistent with those detected with the yeast estrogen screening (YES) system. Furthermore, the estrogenic activities of the synthetic musk samples extracted from the wastewater and waste sludge of a sewage treatment plant of Shanghai (China) were detected, and their results were comparable to those obtained from the YES system and gas chromatography-mass spectrometry (GC-MS). In conclusion, the DIER bioassay could fill a niche for the efficient, rapid, high-throughput screening of estrogenic compounds and has potential for the remote, near-real-time monitoring of environmental estrogens.     

Regulation of pS2 gene expression by affinity labeling and reversibly binding estrogens and antiestrogens: comparison of effects on the native gene and on pS2-chloramphenicol acetyltransferase fusion genes transfected into MCF-7 human breast cancer cells

We have examined the effects of reversibly and irreversibly binding estrogenic and antiestrogenic ligands for the estrogen receptor on pS2 RNA accumulation in MCF-7 human breast cancer cells and on pS2-chloramphenicol acetyl transferase (CAT) fusion gene expression in transfected MCF-7 cells. In MCF-7 cells grown in the absence of estrogens, the reversibly binding estrogen, estradiol, and the affinity labeling estrogen, ketononestrol aziridine, KNA, evoked a 13-fold increase in pS2 RNA level. The reversibly binding antiestrogen trans-hydroxytamoxifen and the affinity labeling antiestrogens tamoxifen aziridine or desmethylnafoxidine aziridine behaved as partial agonists/antagonists. In thymidine kinase-chloramphenicol acetyltransferase (tk-CAT) fusion genes containing a 1000 base pair fragment of the pS2 5'-flanking region encompassing the estrogen responsive element of the gene [pS2 (-1100/-90) tk-CAT], estradiol and ketononestrol aziridine evoked a marked stimulation of CAT activity and, in transfected cells grown in both the presence or absence of the weak estrogen phenol red, the antiestrogens behaved as partial agonists/antagonists. This pS2 5'-flanking region displayed both estrogen-dependent and estrogen-independent enhancer activity as monitored by stimulation of CAT activity. Hormonal regulation of the transfected pS2 fusion gene was similar to that observed in the native pS2 gene of MCF-7 cells; however, antiestrogens, while still partial agonists-antagonists, were relatively more agonistic on the transfected fusion gene than on the native gene. One antiestrogen (ICI 164,384) that behaved as a pure estrogen antagonist on the native gene was a partial agonist-antagonist of pS2 gene expression in the plasmid. This study illustrates that the hormonal regulation of the pS2 gene, as characterized by the agonist-antagonist balance of estrogens and antiestrogens, is influenced by the DNA context of the pS2 estrogen responsive element. Also, the fact that estrogens and antiestrogens that form covalent bonds with the estrogen receptor modulate activity of the native pS2 gene and the pS2-tk-CAT fusion gene in a manner similar to that of their reversibly binding counterparts suggests that it may be possible to use these irreversibly binding ligands to follow the interaction of hormone-receptor complexes with regions regulating estrogenic stimulation of the pS2 gene.     

Regulation of estrogen activity by sulfation in human Ishikawa endometrial adenocarcinoma cells.

Sulfation is an important conjugation reaction in the metabolism of steroids. Steroids sulfates do not interact with the appropriate hormone receptors; additionally, the presence of the charged sulfate moiety increases the aqueous solubility and excretion of most steroids. Estrogen sulfotransferase (EST) is the major form of human cytosolic ST involved in the conjugation of estrogens. EST is important in the inactivation of beta-estradiol (E2) during the luteal phase of the menstrual cycle. EST has a significantly higher affinity for the sulfation of E2 and 17alpha-ethinylestradiol (EE2) than for other potent estrogens such as diethylstilbestrol (DES) and equine estrogens. The ability of EST to sulfate these estrogenic compounds at physiologic concentrations is important in regulating their activation of the ER in estrogen responsive cells. Human Ishikawa endometrial adenocarcinoma (ISH) cells possess an estrogen receptor (ER)-regulated alkaline phosphatase (AlkPhos) which is used to assay ER activation. To study the effects of EST activity on the ER activation of different estrogenic compounds, ISH cells were stably transformed with an EST expression vector. Dose-response curves for the induction of AlkPhos activity by the different estrogenic compounds were generated with EST/ISH and control pcDNA/ISH cells. EST/ISH cells were 200-fold less sensitive to E2 and EE2 than were control cells. No differences were observed in the dose response curves for DES between EST/ISH and pcDNA/ISH cells. EST/ISH cells were approximately 3-10-fold less sensitive to the equine estrogens equilin and 17-equilin as compared to control cells. The ability of EST to decrease the ER activation of an estrogen correlates with the sulfation of these compounds at nanomolar concentrations by EST/ISH and pcDNA/ISH ISH cells. These results indicate that EST is capable of efficiently inactivating E2 and EE2 but is significantly less effective in inhibiting the ER binding of other potent estrogenic compounds.     

PC-1分子转录激活功能研究

PC-1基因是在人前列腺癌细胞中克隆的新基因,表达水平随前列腺癌恶性程度增加而升高,其表达产物具有转录因子的一些特征.为研究PC-1分子的转录激活功能,首先应用酵母双杂交系统将PC-1全长以及不同区段的cDNA克隆到表达载体pAS2-1中,然后分别转化酵母细胞株CG-1945. lacZ和His3报告基因激活的检测结果表明,该分子具有转录激活活性并将该活性定位于N端的46个氨基酸区域.此外,将PC-1分子不同区段的cDNA分别克隆至表达载体pZHO1中,将它们与报告基因质粒pTRE-luc共转染哺乳动物细胞COS7和C4-2,Firefly荧光素酶相对活性的检测结果表明,该分子N端的46个氨基酸区域具有转录激活活性.     

Effects of analogs of indole-3-carbinol cyclic trimerization product in human breast cancer cells

5,6,11,12,17,18-Hexahydrocyclonona[1,2-b:4,5-b*:7,8-b**]triindole (CTr) is a major digestive product of indole-3-carbinol (I3C) from Brassica vegetables and exhibits strong estrogenic activities. CTr increases proliferation of estrogen-dependent breast tumor cells, binds with strong affinity for the estrogen receptor-alpha (ERalpha), and activates expression of estrogen (E(2))-dependent genes. To begin to examine the structural features that determine the biological activity of CTr, we prepared and studied the effects of two analogs, 9,18-dihydro-12H-[1,2,5]trithionino[3,4-b:6,7-b*:9,8-b**]triindole (S(3)CTr) and 5,6,11,12,17,18-hexahydro-5,11,17-trimethylcyclonona[1,2-b:4,5-b*:7,8-b**]triindole (Me(3)CTr). N-Methylation of CTr completely ablated the estrogenic activities of CTr. In the dose range in which CTr was clearly estrogenic, Me(3)CTr exhibited no detectable effect on cell growth, ERalpha binding to E(2), or ERalpha-responsive gene expression. S(3)CTr showed mixed ERalpha agonist activities. It bound to the ERalpha and activated receptor binding with DNA, weakly activated expression of transfected E(2)-responsive reporter gene constructs, and strongly inhibited the E(2)-induced activation of these reporter constructs. S(3)CTr activated aryl hydrocarbon receptor (AhR)-mediated pathways, consistent with the moderately strong binding affinity of S(3)CTr for the AhR. Comparisons of the conformational characteristics among CTr and its two analogs indicated that the estrogenic effects of CTr are highly sensitive to apparently minor structural modifications, and further supported the hypothesis for a central role of hydrogen bonding around the nitrogen atom in CTr binding to the ligand binding site of ERalpha.     

Estrogen and prostate cancer: an eclipsed truth in an androgen-dominated scenario

Prostate cancer is the commonest non-skin cancer in men. Incidence and mortality rates of this tumor vary strikingly throughout the world. Although several factors have been implicated to explain this remarkable variation, lifestyle and dietary factors may play a dominant role, with sex hormones behaving as intermediaries between exogenous factors and molecular targets in development and progression of prostate cancer. Human prostate cancer is generally considered a paradigm of androgen-dependent tumor; however, estrogen role in both normal and malignant prostate appears to be equally important. The association between plasma androgens and prostate cancer remains contradictory and mostly not compatible with the androgen hypothesis. Similar evidence apply to estrogens, although the ratio of androgen to estrogen in plasma declines with age. Apart from methodological problems, a major issue is to what extent circulating hormones can be considered representative of their intraprostatic levels. Both nontumoral and malignant human prostate tissues and cells are endowed with key enzymes of steroid metabolism, including 17betahydroxysteroid dehydrogenase (17betaHSD), 5beta-reductase, 3alpha/3betaHSD, and aromatase. A divergent expression and/or activity of these enzymes may eventually lead to a differential prostate accumulation of steroid derivatives having distinct biological activities, as it occurs for hydroxylated estrogens in the human breast. Locally produced or metabolically transformed estrogens may differently affect proliferative activity of prostate cancer cells. Aberrant aromatase expression and activity has been reported in prostate tumor tissues and cells, implying that androgen aromatization to estrogens may play a role in prostate carcinogenesis or tumor progression. Interestingly, many genes encoding for steroid enzymes are polymorphic, although only a few studies have supported their relation with risk of prostate cancer. In animal model systems estrogens, combined with androgens, appear to be required for the malignant transformation of prostate epithelial cells. Although the mechanisms underlying the hormonal induction of prostate cancer in experimental animals remain uncertain, there is however evidence to support the assumption that long term administration of androgens and estrogens results in an estrogenic milieu in rat prostates and in the ensuing development of dysplasia and cancer. Both androgen and estrogen have been reported to stimulate proliferation of cultured prostate cancer cells, primarily through receptor-mediated effects. As for estrogens, the two major receptor types, ERalpha and ERbeta, are expressed in both normal and diseased human prostate, though with a different cellular localization. Since these two receptors are different in terms of ligand binding, heterodimerization, transactivation, and estrogen response element activity, it is likely that an imbalance of their expression may be critical to determine the ultimate estrogen effects on prostate cancer cells. In prostate cancer, ERbeta activation appears to limit cell proliferation directly or through ERalpha inhibition, and loss of ERbeta has been consistently associated with tumor progression. Several splicing variants of both ERalpha and ERbeta exist. Little is known about their expression and function in the human prostate, although reciprocal regulation and interaction with gene promoter both warrant further investigation. In summary, although multiple consistent evidence suggests that estrogens are critical players in human prostate cancer, their role has been only recently reconsidered, being eclipsed for years by an androgen-dominated interest.