Estrogen receptor α (ERα) is a ligand-inducible transcription factor that mediates the biological effects of estrogens and antiestrogens. Many point mutations in the human ERα gene have been reported to be associated with breast cancer, endometrial cancer, and psychiatric diseases. However, functional analyses for most mutants with amino acid changes are still lacking. In the present study, to investigate the effects of point mutations on the function, gel-shift assays and luciferase assays were performed for eight kinds of mutated ERα proteins, including a single nucleotide change of C207G (N69K), G478T (G160C), T887C (L296P), A908G (K303R), C926T (S309F), A1058T (E353V), A1186G (M396V) and G1231deletion (411fsX7). The mutated ERα expression plasmids were constructed by sitedirected mutagenesis. With gel-shift assays using in vitro translated ERα proteins, binding to the consensus estrogen response element (ERE) was observed for the mutated ERα proteins except ERα (G160C) and ERα (411fsX7), the binding of which was comparable with that of the wild type. Western blot analyses showed that ERα (G160C) could not be efficiently translated with the in vitro transcription/translation system and that ERα (411fsX7) produced a truncated protein. To investigate the transactivation potency, wild-type or mutated ERα expression plasmids were cotransfected with pGL3-3EREc38 reporter plasmid into human breast adenocarcinoma MDA-MB-435 cells. The concentration-response curves (10 pM -100 nM E2) of the mutant ERα proteins except ERα (E353V) and ERα (411fsX7) were similar to that of wild-type ERα. However, at a low level of E2 (100 pM), the mutants ERα (N69K), ERα (L296P), ERα (S309F), and ERα (M396V) showed a significant decrease of transactivation compared with that of the wild-type ERα. The mutants ERα (E353V) and ERα (411fsX7) did not show responsiveness to E2 and antiestrogens, 4-hydroxytamoxifen (4OHT) and ICI 182,780. The mutant ERα (S309F) showed decreased responsiveness for the antiestrogenicity of 4OHT. In conclusion, we found that some of the naturally occurring human ERα mutants with amino acid changes may have an altered responsiveness to estrogen and antiestrogens.

ABSTRACT:UDP-glucuronosyltransferases (UGTs) are major phase II drug metabolism enzymes that catalyze the glucuronidation of numerous endogenous and exogenous compounds. UGTs are divided into two families, UGT1 and UGT2, based on evolutionary divergence and homology. Nine UGT1A and seven UGT2B functional isoforms have been identified in humans. Glucuronidation occurs mainly in liver but also in various extrahepatic tissues, possibly affecting the pharmacokinetics. In the present study, we comprehensively determined the expression of all functional UGT1A and UGT2B isoforms in normal human tissues including liver, lung, stomach, small intestine, colon, kidney, bladder, adrenal gland, breast, ovary, uterus, and testis by semiquantitative reverse transcription-polymerase chain reaction. In addition, the expressions of these UGTs mRNA in 15 kinds of human tissue-derived cell lines were also analyzed. Many UGT isoforms were abundantly expressed in the liver, gastrointestinal tract, and kidney, supporting previous studies. Interestingly, we found that all UGTs except UGT2B17 were expressed in bladder. In steroidrelated tissues, UGTs were expressed in tissue-and isoform-specific manners. Expression profiles in human tissue-derived cell lines were not necessarily consistent with those in corresponding normal tissues. Different expression profiles were observed in distinct cell lines derived from the same organ. The information presented here will be helpful for understanding the glucuronidation in various tissues and for choosing appropriate cell lines for in vitro studies.

ABSTRACT:UDP-glucuronosyltransferases (UGTs) catalyze glucuronidation of a variety of xenobiotics and endobiotics. UGTs are divided into two families, UGT1 and UGT2. The purpose of this study was to estimate the absolute expression levels of each UGT isoform in human liver and to evaluate the interindividual variability. Real-time reverse transcriptase-polymerase chain reaction analysis was performed to determine the copy numbers of nine functional UGT1A isoforms and seven UGT2B isoforms. We noticed that not only primers but also templates as a standard for quantification should prudently be selected. Once we established appropriate conditions, the mRNA levels of each UGT isoform in 25 individual human livers were determined. UGT1A1 (0.9-138. ity. Abundant isoforms were UGT2B4 and UGT2B10, followed by UGT1A1, UGT2B15, and UGT1A6. The sum of the UGT2B mRNA levels was higher than that of UGT1A mRNA levels. It is interesting to note that the mRNA levels normalized with glyceraldehyde-3-phosphate dehydrogenase mRNA for almost UGT isoforms that are substantially expressed in liver showed significant correlations to each other. Western blot analysis was performed using antibodies specific for UGT1A1, UGT1A4, UGT1A6, or UGT2B7. Correlation between the protein and mRNA levels was observed in only UGT1A1 (r ‫؍‬ 0.488; p < 0.01). In conclusion, this study comprehensively determined the absolute values of mRNA expression of each UGT isoform in human livers and found considerable interindividual variability.