Protective roles of quinone reductase and tamoxifen against estrogen-induced mammary tumorigenesis.

We previously reported that antiestrogen-liganded estrogen receptor beta (ERbeta) transcriptionally activates the major detoxifying enzyme quinone reductase (QR) (NAD(P)H:quinone oxidoreductase). Further studies on the functional role of ERbeta-mediated upregulation of antioxidative enzymes indicated protective effects against estrogen-induced oxidative DNA damage (ODD). We now report on in vivo and in vitro studies that show that ERbeta-mediated upregulation of QR are involved in the protection against estrogen-induced mammary tumorigenesis. Using the August Copenhagen Irish (ACI) model of estrogen-induced carcinogenesis, we observed that increased ODD and decreased QR expression occur early in the process of estrogen-induced mammary tumorigenesis. Prevention of ACI mammary gland tumorigenesis by tamoxifen was accompanied by decreased ODD and increased QR levels. These correlative findings were supported by our findings that downregulation of QR levels led to increased levels of estrogen quinone metabolites and enhanced transformation potential of 17beta-estradiol treated MCF10A non-tumorigenic breast epithelial cells. Concurrent expression of ERbeta and treatment with 4-hydroxytamoxifen decreased tumorigenic potential of these MCF10A cells. We conclude that upregulation of QR, through induction by tamoxifen, can inhibit estrogen-induced ODD and mammary cell tumorigenesis, representing a possible novel mechanism of tamoxifen prevention against breast cancer.

Activation of transcriptionally inactive human estrogen receptors by cyclic adenosine 3',5'-monophosphate and ligands including antiestrogens.

We show that some transcriptionally inactive human estrogen receptor (ER) mutants can be activated by 17 beta-estradiol (E2), and sometimes by antiestrogens, in the presence of elevated levels of intracellular cAMP. ER-deficient Chinese hamster ovary or 3T3 mouse fibroblast cells were transfected with mutant ERs (the point mutant L540Q, the frameshift mutant S554fs, or the carboxy-terminal truncated receptor ER1-530) and various estrogen response element-containing reporter genes. Individual treatments with E2, the antiestrogens trans-hydroxytamoxifen and ICI 164,384, or with 3-isobutyl-1-methyl-xanthine plus cholera toxin (IBMX plus CT) which raise intracellular cAMP, generally do not activate the mutant receptors. However, cotreatment with IBMX/CT and one of the three ligands (E2, trans-hydroxytamoxifen, or ICI164,384) results in the unexpected recovery of strong activation of the L540Q or S554fs receptors, the magnitude of which is dependent upon promoter- and cell-contexts. Unlike L540Q and S554fs, the transcriptionally inactive ER1-530 is not activated by any combination of ligands and IBMX/CT. These data demonstrate that some ER mutants that form transcriptionally nonproductive ER-E2 complexes can be successfully activated by the combination of an agonist or antagonist ligand and an agent thought to act via phosphorylation pathways. Also highlighted is the promoter- and cell-specific nature of the transcriptional response to different ligand-ER complexes. Lastly, the enhanced transcriptional activity of wild type ER and some ER mutants in the presence of antiestrogens and elevated intracellular cAMP may provide a partial explanation of the ability of some estrogen-dependent human breast tumors to resist antiestrogen therapies currently employed.

Identification of multiple, widely spaced estrogen-responsive regions in the rat progesterone receptor gene.

Progesterone receptors (PRs) mediate the actions of progestin hormones and play important roles during the reproductive cycle and pregnancy. Since PR expression is known to be regulated by estrogen, we have undertaken studies to examine the mechanisms underlying this regulation. We have identified multiple distinct regions of the rat PR gene, widely spaced and spread throughout the 5'-flanking region, the 5'-untranslated region, and the first exon (between -2264 and +2241), that can form a strong estrogen-responsive enhancer when linked together. Estrogen-responsive activities for two of the regions in isolation (+461/+636 and +2176/+2241) were demonstrated in one or more homologous or heterologous promoter contexts. The contributions of the other regions (-2264/-1970, -1167/-957 and +2088/+2110) to the overall activity of the assembled enhancer were cryptic in that they were only observed in the context of the other PR gene fragments, not in isolation. We identified four weak, but functional, imperfect estrogen response elements (EREs) in these regions of the PR gene, each differing from the consensus by 2 base pairs. In addition, we identified four ERE half-sites in the PR gene, three of which are paired (i.e. < 150 base pairs away) with the EREs in the estrogen-responsive regions. Competitive gel shift assays demonstrated weak, but detectable, binding of estrogen receptor to the EREs. Of note, the estrogen-responsive enhancer assembled from the five regions of the PR gene exhibited promoter specificity; it conferred estrogen responsiveness of the distal PR gene promoter, but it failed to enhance the endogenous estrogen responsiveness of the proximal PR gene promoter. The positioning of response elements in the rat PR gene, which we show to be unique among steroid hormone-regulated genes, may have functional consequences for the regulation of the magnitude and timing of PR gene expression by estrogen.

Cloning of the rat progesterone receptor gene 5'-region and identification of two functionally distinct promoters.

To examine some of the molecular mechanisms controlling transcription of the rat progesterone receptor (PR) gene, we have cloned and sequenced the 5'-region of the gene. Northern blot analyses with a series of probes identified two regions where distinct subsets of the multiple PR gene transcripts initiated, suggesting the presence of two promoters in the gene. Promoter activities for two gene fragments encompassing these regions, -131/+65 (P; distal) and +461/+675 (P'; proximal), were demonstrated in transient transfection experiments using reporter constructs containing the gene fragments linked individually upstream of the chloramphenicol acetyltransferase (CAT) gene. Cotransfection of P-CAT or P'-CAT constructs containing two upstream GAL4 binding sites into primary cultures of rat uterine cells with a vector expressing a GAL4 DNA binding domain-VP16 activating region fusion protein resulted in a 10-fold increase in CAT activity relative to cells transfected with either reporter and a vector expressing only the GAL4 DNA binding domain. The estrogen inducibility of the promoter-CAT constructs was assessed by transfection into MCF-7 breast cancer cells, which contain high levels of estrogen receptor (ER). P'-CAT, but not P-CAT, was induced by estradiol (E2; 8-fold). In primary rat uterine cells, which contain lower levels of ER, P'-CAT required the addition of one upstream consensus estrogen response element (ERE) to be estrogen inducible, whereas P-CAT required the addition of two EREs. Point and deletion mutants of the proximal promoter region in the P'-CAT reporter, screened in MCF-7 cells, were used to identify a 20-base pair fragment (+617/+636) that retained the promoter activity and 50% of the estrogen inducibility of P'. This fragment contained an ERE-like sequence conserved in 8 of 10 positions relative to the consensus ERE. Two copies of this sequence conferred estrogen inducibility (4-fold) when placed upstream of the distal promoter in P-CAT. To examine ER-dependent stimulation of the two PR gene promoters by cAMP, P-CAT and P'-CAT reporter constructs containing two upstream consensus EREs were cotransfected into ER-negative 3T3 cells with an ER expression vector. Induction by E2 was greater than 50-fold for both constructs. Treatment of the cells with agents that increase intracellular cAMP levels, namely cholera toxin plus isobutyl methylxanthine, resulted in CAT activity that was 8% and 51% of the E2-stimulated activity for the P and P' constructs, respectively.

The carboxy-terminal F domain of the human estrogen receptor: role in the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists.

Of the steroid hormone receptor family members, the estrogen receptor (ER) is notable in containing a sizable (42-amino acid) C-terminal region, denoted domain F. This F region differs from its adjacent hormone-binding domain, domain E, in that it is not well conserved among different vertebrate ER species, and its role in the biological activity of the ER is not well defined. We report an important role for the F domain of the ER in modulating the magnitude of gene transcription by estrogen and antiestrogen, and in determining the effectiveness of antiestrogens in suppressing estrogen-stimulated gene transcription. Using transient transfections, we have examined, in several cell types, the transcriptional activity of the full-length wild type human ER and ER lacking the carboxy-terminal F domain (delta F ER, containing amino acids 1-554) or ER altered in the F domain by point mutations. In some cells, namely Chinese hamster ovary (CHO) cells and MDA-MB-231 human breast cancer cells expressing wild type ER or delta F ER, estradiol (E2) stimulates equally transcription of several estrogen-responsive promoter-reporter gene constructs [estrogen ca-18119 element, (ERE)2-TATA-CAT, (ERE)2-pS2-CAT, (ERE)2-progesterone receptor(distal)-CAT]; however, the antiestrogens trans-hydroxytamoxifen and ICI 164,384, which stimulate transcription of some of these reporter constructs with the wild type ER, were unable to stimulate transcription with delta F ER. In addition, these antiestrogens were more effective antagonists of E2-stimulated transcription by delta F ER than by wild type ER. By contrast, in HeLa human cervical cancer cells and 3T3 mouse fibroblast cells, the delta F ER exposed to E2 is much less effective than wild type ER in stimulating transcription, and antiestrogens were less potent in suppressing E2-stimulated transcription by the delta F ER. These differences in response of the delta F and wild type ER to estrogen or antiestrogen do not appear to be due to a change in receptor expression level, binding affinity for ligands, or binding to estrogen response element DNA. Our data support the supposition that the conformation of the receptor-ligand complex is different with estrogen vs. antiestrogen and with wild type vs. delta F ER, such that its potential for interaction with protein cofactors or transcription factors is different and is markedly influenced by cell context. Thus, the F domain of the ER has a specific modulatory function that affects the agonist/antagonist effectiveness of antiestrogens and the transcriptional activity of the liganded ER in cells.

Identification of a novel transferable cis element in the promoter of an estrogen-responsive gene that modulates sensitivity to hormone and antihormone.

The estrogen receptor (ER) is a ligand-regulated transcription factor that acts at the promoters of estrogen-regulated genes to modulate their expression. In the present study, we examined three estrogen-regulated promoters, namely the rat progesterone receptor gene distal (PRD) and proximal (PRP) promoters and the human pS2 gene promoter, and observed marked differences in their sensitivity to stimulation by estrogen and repression of estrogen-stimulated transcription by antiestrogen (AE)-occupied ER. ER-containing MCF-7 human breast cancer cells were transfected with reporter gene constructs containing estrogen response elements upstream of the three gene promoters. In this system, PRP and PRD showed similar dose-response curves for stimulation by estradiol whereas pS2 was activated by even lower concentrations of estradiol. By contrast, PRD was much less sensitive to repression of estrogen-stimulated activity by all AEs studied, relative to the PRP and the pS2 promoters. Using deletion and mutational analysis, we have identified a transferable cis element at -131 to -94 bp in PRD that is involved in modulating the sensitivity of this promoter to both estrogens and AEs. The element reduced the magnitude of estrogen-stimulated activity, enhanced the ability of AEs to repress estrogen-stimulated activity, and elicited similiar effects when transferred to the promoter of another estrogen-responsive gene. Thus, removal of this region from PRD further accentuated the insensitivity of this promoter to AE while enhancing its sensitivity (both EC50 and fold induction) to estrogen. Gel mobility shift assays showed that proteins from nuclear extracts of MCF-7 cells interact with this element and that the binding of these proteins is inversely correlated with the transcriptional effectiveness of the ER. The findings demonstrate that a specific cis element from the promoter of an estrogen-responsive gene can alter the transcriptional activity of hormone and antihormone-occupied receptor bound at its response element near the promoter. Such ligand response modulatory elements, and changes in the levels and activity of factors that bind to such elements, may underlie the different sensitivities of steroid hormone-regulated genes to both hormones and antihormones.

Human estrogen receptor ligand activity inversion mutants: receptors that interpret antiestrogens as estrogens and estrogens as antiestrogens and discriminate among different antiestrogens.

The estrogen receptor (ER) is a transcription factor whose activity is normally activated by the hormone estradiol and inhibited by antiestrogen. It has been found that certain mutational changes in the activation function-2 region in the hormone-binding domain of the human ER result in ligand activity inversion mutants, i.e. receptors that are now activated by antiestrogen and inhibited by estrogen. The ER point mutant L540Q is activated by several antiestrogens (the more pure antiestrogens ICI 164,384 and RU 54,876 or the partial antiestrogen trans-hydroxytamoxifen) but not by estradiol. The presence of the F domain and an intact activation function-i in the A/B domain are required for this activity, as is the DNA-binding ability of the receptor. This inverted ligand activity is observed with several estrogen-responsive promoters, both simple and complex; however, the activating ability of antiestrogens is observed only in some cells, highlighting the important role of cell-specific factors in ligand interpretation. The introduction of two additional amino acid changes close to 540 results in receptors that are still not activated by estradiol but are now able to distinguish between partial antiestrogens (which remain agonistic) and pure antiestrogens (which show a greatly reduced stimulatory activity). These ligand activity inversion mutants remain stable in cells in the presence of the antiestrogen ICI 164,384, as does a related ER mutant receptor that shows the normal, wild type ER ligand activity profile in which ICI 164,384 is transcriptionally inactive. Thus, the presence of adequate levels of mutant ER may be necessary but not sufficient for ICI 164,384 to elicit transcriptional activity. These findings highlight the means by which the carboxyl-terminal region in domain E functions to interpret the activity of a ligand, and they demonstrate that rather minimal changes in the ER can result in receptors with inverted response to antiestrogen and estrogen. Such point mutations, if present in estrogen target cells, would result in antiestrogens being seen as growth stimulators, rather than suppressors, with potentially detrimental consequences in terms of breast cancer treatment with antiestrogens.

Antiestrogens: mechanisms and actions in target cells.

Antiestrogens, acting via the estrogen receptor (ER) evoke conformational changes in the ER and inhibit the effects of estrogens as well as exerting anti-growth factor activities. Although the binding of estrogens and antiestrogens is mutually competitive, studies with ER mutants indicate that some of the contact sites of estrogens and antiestrogens are likely different. Some mutations in the hormone-binding domain of the ER and deletions of C-terminal regions result in ligand discrimination mutants, i.e. receptors that are differentially altered in their ability to bind and/or mediate the actions of estrogens vs antiestrogens. Studies in a variety of cell lines and with different promoters indicate marked cell context- and promoter-dependence in the actions of antiestrogens and variant ERs. In several cell systems, estrogens and protein kinase activators such as cAMP synergize to enhance the transcriptional activity of the ER in a promoter-specific manner. In addition, cAMP changes the agonist/antagonist balance of tamoxifen-like antiestrogens, increasing their agonistic activity and reducing their efficacy in reversing estrogen actions. Estrogens, and antiestrogens to a lesser extent, as well as protein kinase activators and growth factors increase phosphorylation of the ER and/or proteins involved in the ER-specific response pathway. These changes in phosphorylation alter the biological effectiveness of the ER. Multiple interactions among different cellular signal transduction systems are involved in the regulation of cell proliferation and gene expression by estrogens and antiestrogens.

Effects of social stress and intrauterine position on sexual phenotype in wild-type house mice (Mus musculus).

Wild-type house mice were used to test the effect of intrauterine position on anogenital distance (AGD) and to verify whether crowding stress would masculinize female pups, developing at all intrauterine positions, as has been demonstrated in CF-1 mice stressed by restraint, heat, and light. Stress of crowding was documented by comparing aggressive behavior, litter birth weights, and plasma corticosterone levels among females in different densities. AGDs were recorded from pups born to females housed from day 12 to 19 of gestation either individually with their mate (nonstressed) or in one of two group-housed densities. Female pups from nonstressed dams positioned between two males in utero (2M females) had longer AGDs than females positioned between two females (0M females). AGDs of males from nonstressed dams did not differ on the basis of intrauterine position. Group-housed pregnant females in the higher of two densities had female pups with longer AGDs than female pups of other dams. However, variance in female pup AGD was no different among dams in different densities. These results extend to the wild house mouse previous findings in albino mice that intrauterine position influences sexual phenotype. In addition, social stress can induce masculinization of female pups in wild mice as physical stress has been shown to do in albinos. This suggests that intrauterine position effects and their modification by crowding stress can potentially influence the dynamics of wild house mouse populations.

Serum corticosterone in fetal mice: sex differences, circadian changes, and effect of maternal stress.

The serum concentration of corticosterone was examined in control and stressed pregnant female mice (Mus domesticus) as well as male and female fetuses due to our interest in the behavioral effects of material stress on offspring in mice. Pregnant females were restrained under flood lights (2 sessions/day, 45 min/session) from Day 13-17 of pregnancy. On Day 17 of pregnancy a significant increase in maternal serum corticosterone was exhibited 1 h after the onset of a stress session, and serum corticosterone did not return to baseline until 16 h later. We also observed a significant increase in serum corticosterone in male fetuses during the first 4 h after maternal stress, while no significant change in serum concentration of corticosterone was observed in female fetuses throughout 24 h after maternal stress. Daily variation in serum concentration of corticosterone was also determined at 4-h intervals in pregnant mice and their fetuses from Day 16-18 of pregnancy. Pregnant females maintained on a 12 L:12 D cycle exhibited peak serum corticosterone concentrations at and just before the onset of the darkness. Daily fluctuations in serum concentrations of corticosterone in male and female fetuses reflected the pattern observed in the mothers. A sex difference in serum corticosterone in fetuses was observed at some, but not all times of the day, with the difference being greatest during the dark phase of the mother's light:dark cycle.

Inhibition of metastasis by HEXIM1 through effects on cell invasion and angiogenesis.

We report on the role of hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) as an inhibitor of metastasis. HEXIM1 expression is decreased in human metastatic breast cancers when compared with matched primary breast tumors. Similarly we observed decreased expression of HEXIM1 in lung metastasis when compared with primary mammary tumors in a mouse model of metastatic breast cancer, the polyoma middle T antigen (PyMT) transgenic mouse. Re-expression of HEXIM1 (through transgene expression or localized delivery of a small molecule inducer of HEXIM1 expression, hexamethylene-bis-acetamide) in PyMT mice resulted in inhibition of metastasis to the lung. Our present studies indicate that HEXIM1 downregulation of HIF(-)1α protein allows not only for inhibition of vascular endothelial growth factor-regulated angiogenesis, but also for inhibition of compensatory pro-angiogenic pathways and recruitment of bone marrow-derived cells (BMDCs). Another novel finding is that HEXIM1 inhibits cell migration and invasion that can be partly attributed to decreased membrane localization of the 67 kDa laminin receptor, 67LR, and inhibition of the functional interaction of 67LR with laminin. Thus, HEXIM1 re-expression in breast cancer has therapeutic advantages by simultaneously targeting more than one pathway involved in angiogenesis and metastasis. Our results also support the potential for HEXIM1 to indirectly act on multiple cell types to suppress metastatic cancer.

The exonuclease activity of hPMC2 is required for transcriptional regulation of the QR gene and repair of estrogen-induced abasic sites.

We have previously reported that the expression of antioxidative stress enzymes is upregulated by trans-hydroxytamoxifen (TOT) in breast epithelial cell lines providing protection against estrogen-induced DNA damage. This regulation involves Estrogen Receptor ß (ERß) recruitment to the Electrophile Response Element (EpRE) and a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2). We have also demonstrated that ERß and hPMC2 are required for TOT-dependent recruitment of poly (ADP-ribose) polymerase 1 (PARP-1) and Topoisomerase IIß (Topo IIß) to the EpRE. Sequence analysis reveals that the C-terminus of hPMC2 encodes a putative exonuclease domain. Using in vitro kinetic assays, we found that hPMC2 is a 3'-5' non-processive exonuclease that degrades both single-stranded and double-stranded substrates. Mutation of two conserved carboxylate residues drastically reduced the exonuclease activity of hPMC2, indicating the relative importance of the catalytic residues. Western blot analysis of breast cancer cell lines for Quinone Reductase (QR) levels revealed that the intrinsic exonuclease activity of hPMC2 was required for TOT-induced QR upregulation. Chromatin immunoprecipitation (ChIP) assays also indicated that hPMC2 was involved in the formation of strand breaks observed with TOT treatment and is specific for the EpRE-containing region of the QR gene. We also determined that the transcription factor NF-E2-related factor-2 (Nrf2) is involved in the specificity of hPMC2 for the EpRE. In addition, we determined that the catalytic activity of hPMC2 is required for repair of abasic sites that result from estrogen-induced DNA damage. Thus, our study provides a mechanistic basis for transcriptional regulation by hPMC2 and provides novel insights into its role in cancer prevention.

HEXIM1 is a critical determinant of the response to tamoxifen.

Tamoxifen resistance is a major problem in the treatment of estrogen receptor (ER)-positive patients. We have previously reported that hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) inhibits ERα activity by competing with ERα for binding to cyclin T1, a subunit of positive transcription elongation b (P-TEFb). This results in the inhibition of the phosphorylation of RNA polymerase II (RNAPII) at serine 2 and the inhibition of transcription elongation of ERα target genes. As HEXIM1 can inhibit ER activity, we examined whether it has a critical role in the inhibitory effects of tamoxifen on ER. We observed that tamoxifen-induced HEXIM1 recruitment to the promoter region of ER target genes and decreased the recruitment of cyclin T1 and serine 2 phosphorylated RNAPII to the coding regions of these genes. Conversely, in cells wherein HEXIM1 expression has been downregulated we observed attenuation of the inhibitory effects of tamoxifen on estrogen-induced cyclin T1 recruitment to coding regions of ER target genes. As a consequence, downregulation of HEXIM1 resulted in the attenuation of the repressive effects of tamoxifen on estrogen-induced gene expression and proliferation. Conferring clinical relevance to our studies is our analysis of human breast cancer tissue samples that indicated association of lower expression of HEXIM1 with tumor recurrence in patients who received tamoxifen. Our studies provide a better understanding of the mechanistic basis for the inhibitory effect of tamoxifen on ER activity and may suggest new therapeutic targets for the treatment of tamoxifen-resistant breast cancer.

HEXIM1 modulates vascular endothelial growth factor expression and function in breast epithelial cells and mammary gland.

Recently, we found that mutation of the C-terminus of transcription factor hexamethylene bisacetamide-inducible protein 1 (HEXIM1) in mice leads to abnormalities in cardiovascular development because of aberrant vascular endothelial growth factor (VEGF) expression. HEXIM1 regulation of some genes has also been shown to be positive transcription elongation factor b (P-TEFb) dependent. However, it is not known whether HEXIM1 regulates VEGF in the mammary gland. We demonstrate that HEXIM1 regulates estrogen-induced VEGF transcription through inhibition of estrogen receptor-alpha recruitment to the VEGF promoter in a P-TEFb-independent manner in MCF-7 cells. Under hypoxic conditions, HEXIM1 inhibits estrogen-induced hypoxia-inducible factor-1 alpha (HIF-1alpha) protein expression and recruitment of HIF-1alpha to the hypoxia-response element in the VEGF promoter. In the mouse mammary gland, increased HEXIM1 expression decreased estrogen-driven VEGF and HIF-1alpha expression. Conversely, a mutation in the C-terminus of HEXIM1 (HEXIM1(1-312)) led to increased VEGF and HIF-1alpha expression and vascularization in mammary glands of heterozygous HEXIM1(1-312) mice when compared with their wild-type littermates. In addition, HEXIM1(1-312) mice have a higher incidence of carcinogen-induced mammary tumors with increased vascularization, suggesting an inhibitory role for HEXIM1 during angiogenesis. Taken together, our data provide evidence to suggest a novel role for HEXIM1 in cancer progression.

hPMC2 is required for recruiting an ERbeta coactivator complex to mediate transcriptional upregulation of NQO1 and protection against oxidative DNA damage by tamoxifen.

In the presence of ERbeta, trans-hydroxytamoxifen (TOT) protects cells against 17beta-estradiol (E(2))-induced oxidative DNA damage (ODD) and this correlates with increased expression of the antioxidative enzyme quinone reductase (QR). Here, we investigate the molecular mechanism responsible for ERbeta-mediated protection against ODD. We observe constitutive interaction between ERbeta and the novel protein hPMC2. Using a combination of breast epithelial cell lines that are either positive or negative for ERalpha, we demonstrate TOT-dependent recruitment of both ERbeta and hPMC2 to the EpRE (electrophile response element)-regulated antioxidative enzyme QR. We further demonstrate TOT-dependent corecruitment of the coactivators Nrf2, PARP-1 (poly (ADP-ribose) polymerase 1) and topoisomerase IIbeta, both in the presence and absence of ERalpha. However, absence of either ERbeta or hPMC2 results in nonrecruitment of PARP-1 and topoisomerase IIbeta, loss of antioxidative enzyme induction and attenuated protection against ODD by TOT even in the presence of Nrf2 and ERalpha. These findings indicate minor role for Nrf2 and ERalpha in TOT-dependent antioxidative gene regulation. However, downregulation of PARP-1 attenuates TOT-dependent antioxidative gene induction. We conclude that ERbeta and hPMC2 are required for TOT-dependent recruitment of coactivators such as PARP-1 to the EpRE resulting in the induction of antioxidative enzymes and subsequent protection against ODD.

Glucocorticoid effects on the skeletal muscle differentiation program: analysis of clonal proliferation, morphological differentiation and the expression of muscle-specific and regulatory genes.

We examined the effect of glucocorticoids on the proliferation and differentiation of skeletal muscle cells using the C2C12 cell line. We found that treatment with glucocorticoids enhanced muscle cell differentiation but had only minor effects on the clonal growth rate of C2C12 cells. The stimulatory effect of glucocorticoids on myogenic differentiation was reflected in the increased expression of muscle-specific genes, creatine kinase (CK) and acetylcholine receptor gamma subunit (AChR). Dexamethasone had no effect on CK and AChR mRNA stability and enhanced transcription from a CAT reporter genes containing the 3.3kb 5' flanking region of the murine CK gene (-3300MCK-CAT). Since dexamethasone did not affect the expression levels of the myogenic regulatory genes such as myoD and myogenin, the enhancement of muscle-specific transcription might reflect an increase in the functional activity of the regulatory proteins. Other possible mechanisms involved in the differentiation-enhancing effect of glucocorticoids are discussed.

The quinone reductase gene: a unique estrogen receptor-regulated gene that is activated by antiestrogens.

Antiestrogens are thought to exert most of their beneficial effects in breast cancer by antagonizing the actions of estrogen. We report here that antiestrogens also stimulate the expression of quinone reductase (QR) [NAD(P)H:quinone oxidoreductase, EC 1.6.99.2], which may provide protective effects against the toxicity and mutagenicity caused by quinones. QR is up-regulated by low concentrations of antiestrogens (trans-hydroxytamoxifen, tamoxifen, and ICI182,780) in estrogen receptor (ER)-containing breast cancer cells, and this increase is suppressed by estrogen via an ER-dependent mechanism. Since regulation of the QR gene, as well as other genes involved in detoxification such as the glutathione S-transferase Ya subunit (GST Ya) gene, is known to be mediated by an electrophile/antioxidant response element (EpRE/ARE), we examined the effects of antiestrogens on a 41-bp electrophile responsive region derived from the GST Ya gene. Transfection of this EpRE-containing region into ER-negative breast cancer cells in the presence or absence of an expression vector for the human ER, as well as mutagenesis studies, revealed that the EpRE-containing construct was activated by antiestrogen to the same extent as by tert-butylhydroquinone (TBHQ), a known activator of EpREs; however, only the stimulation by antiestrogen, and not TBHQ, required ER and was repressed by estradiol, although activation by both inducers mapped to the same 10-bp EpRE consensus sequence. Thus, there appear to be two pathways for QR induction, one that is activated by electrophile inducers such as TBHQ and is ER independent, and a second that is antiestrogen regulated and ER dependent; both pathways act through the EpRE. The anticancer action of antiestrogens may thus derive not only from the already well-known repression of estrogen-stimulated activities but also from the activation of detoxifying enzymes, such as QR, that may contribute to the beneficial antioxidant activity of antiestrogens.

Identification and characterization of a novel factor that regulates quinone reductase gene transcriptional activity.

The regulation of the quinone reductase (QR) gene as well as other genes involved in detoxification is known to be mediated by an electrophile/antioxidant response element (EpRE/ARE). We have previously observed that QR is up-regulated by the antiestrogen trans-hydroxytamoxifen in breast cancer cells. QR gene regulation by the antiestrogen-occupied estrogen receptor (ER) is mediated by the EpRE-containing region of the human QR gene, and the ER is one of the complex of proteins that binds to the EpRE. In an effort to further understand the mechanism for ER regulation of QR gene we identified other protein factors that regulate QR gene transcriptional activity in breast cancer cells. One of these protein factors, hPMC2 (human homolog of Xenopus gene which prevents mitotic catastrophe), directly binds to the EpRE and interacts with the ER in yeast genetic screening and in vitro assays. Interestingly hPMC2 interacts more strongly to ER beta when compared with ER alpha. In transient transfection assays using reporter constructs containing the EpRE, hPMC2 alone can slightly activate reporter in ER-negative MDA-MB-231 breast cancer cells. The activation of QR gene activity by hPMC2 is enhanced in the presence of ER beta.