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Biomedical subjects

Stephen Safe

Publications and source records attributed to Stephen Safe.

At least 55 records · Page 3Linked to original sources
PubMed2004

Peroxisome proliferator-activated receptor gamma-dependent activation of p21 in Panc-28 pancreatic cancer cells involves Sp1 and Sp4 proteins.

1,1-Bis(3'-indolyl)-1-(p-trifluoromethylphenyl)methane (DIM-C-pPhCF3) and troglitazone activate peroxisome proliferator-activated receptor gamma (PPARgamma) in Panc-28 pancreatic cancer cells and also inhibit cell proliferation. DIM-C-pPhCF3 was more active than troglitazone and was used as a model to investigate the mechanism of PPARgamma-dependent inhibition of Panc-28 cell growth. DIM-C-pPhCF3 significantly inhibited G0/G1-->S phase progression, as determined by FACS analysis, and this was associated with decreased retinoblastoma protein phosphorylation and increased p21 protein and mRNA expression, but no change in p27 or cyclin D1. PPARgamma antagonists blocked DIM-C-pPhCF3-induced growth inhibition and induction of p21 protein, and similar inhibitory effects were observed in Panc-28 cells transfected with a construct (pWWP) containing a -2325 to +8 p21 promoter insert. Deletion analysis of the p21 promoter indicated that PPARgamma-dependent activation of p21 promoter constructs by DIM-C-pPhCF3 required GC-rich sites 3 and 4 in the proximal region (-124 to -60) of the p21 promoter. The results of RNA interference and protein expression/DNA binding assays suggest that DIM-C-pPhCF3 induced p21 expression through a novel mechanism that involves PPARgamma interactions with both Sp1 and Sp4 proteins bound to the proximal GC-rich region of the p21 promoter.

Anticarcinogenic Agents↗
PubMed2004

1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes induce peroxisome proliferator-activated receptor gamma-mediated growth inhibition, transactivation, and differentiation markers in colon cancer cells.

1,1-Bis(3'indolyl)-1-(p-substitutedphenyl)methanes containing p-trifluoromethyl (DIM-C-pPhCF)), p-t-butyl (DIM-C-pPhtBu), and p-phenyl (DIM-C-pPhC6H5) groups induce peroxisome proliferator-activated receptor gamma (PPARgamma)-mediated transactivation in HT-29, HCT-15, RKO, and SW480 colon cancer cell lines. Rosiglitazone also induces transactivation in these cell lines and inhibited growth of HT-29 cells, which express wild-type PPARgamma but not HCT-15 cells, which express mutant (K422Q) PPARgamma. In contrast, DIM-C-pPhCF3, DIM-C-pPhtBu, and DIM-C-pPhC6H5 inhibited growth of both HT-29 and HCT-15 cells with IC50 values ranging from 1 to 10 micromol/L. Rosiglitazone and diindolylmethane (DIM) analogues did not affect expression of cyclin D1, p21, or p27 protein levels or apoptosis in HCT-15 or HT-29 cells but induced keratin 18 in both cell lines. However, rosiglitazone induced caveolins 1 and 2 in HT-29 but not HCT-15 cells, whereas these differentiation markers were induced by DIM-C-pPhCF3 and DIM-C-pPhC6H5 in both cell lines. Because overexpression of caveolin 1 is known to suppress colon cancer cell and tumor growth, the growth inhibitory effects of rosiglitazone and the DIM compounds are associated with PPARgamma-dependent induction of caveolins.

Apoptosis↗
PubMed2004

Hormonal regulation of lactate dehydrogenase-A through activation of protein kinase C pathways in MCF-7 breast cancer cells.

Lactate dehydrogenase A (LDH-A) is hormonally regulated in rodents, and increased expression of LDH-A is observed during mammary gland tumorigenesis. The mechanisms of hormonal regulation of LDH-A were investigated using a series of deletion and mutant constructs derived from the rat LDH-A gene promoter. Results of these studies show that constructs containing the -92 to -37 region of the LDH-A promoter are important for basal and E2-induced transactivation, and mutation of the consensus CRE motif within this region results in significant loss of basal activity and hormone-responsiveness. Gel mobility shift assays using nuclear extracts from MCF-7 cells show that both CREB and ATF-1 interact with the CRE. Studies with kinase inhibitors show that E2-induced activation of this CRE is dependent on protein kinase C, and these data indicate that LDH-A is induced through a non-genomic pathway of estrogen action.

Animals↗
PubMed2004

Interaction of the aryl hydrocarbon receptor ligand 6-methyl-1,3,8-trichlorodibenzofuran with estrogen receptor alpha.

The polycyclic aromatic hydrocarbon 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) is related to the industrial byproduct dioxin and is a weak agonist and partial antagonist at the aryl hydrocarbon receptor (AhR). Tamoxifen is used for the treatment and prevention of breast cancer and interferes with the interaction of estrogen with estrogen receptor alpha (ER). The combination of MCDF and tamoxifen lowered the effective dose of both drugs required to inhibit 7,12-dimethylbenz(a)anthracene-induced mammary tumor growth in rats and protected against the estrogenic effects of tamoxifen on the uterus in rats (A. McDougal et al., Cancer Res 2001;61:3902-7), pointing to the potential use of MCDF in breast cancer treatment. Potential AhR-ER cross-talk is evidenced by the antiestrogenic activity of MCDF and the degradative effect of MCDF on ER protein levels. Our studies confirmed that MCDF degraded the ER. MCDF displayed antiestrogenic activity at higher concentrations in MCF-7 human breast cancer cells, but MCDF alone (10(-6) M) stimulated the growth of MCF-7 cells. MCDF also activated an estrogen response element (ERE)-luciferase reporter and increased mRNA levels of the estrogen-responsive gene transforming growth factor (TGF)-alpha. The estrogenic effects of MCDF are ER dependent because they were blocked by the pure antiestrogen ICI 182,780. MCDF induced ER-coactivator interaction in glutathione S-transferase pull-down assays and the formation of an ER.ERE complex in gel mobility shift assays, further indicating that the estrogenic actions of MCDF are mediated by the ER. In addition, knockdown of the AhR with small interfering RNA did not affect MCDF-induced ERE-luciferase activity. Overall, these data support the conclusion that MCDF is a partial agonist at the ER. This study provides the first evidence for the direct interaction of the ER with MCDF and challenges the view that MCDF is simply an AhR-specific ligand.

Basic Helix-Loop-Helix Proteins↗
PubMed2004

Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor alpha and SP proteins.

Vascular endothelial growth factor (VEGF) is expressed in multiple hormone-dependent cancer cells/tumors. Treatment of ZR-75 breast cancer cells with 17beta-estradiol (E2) induced a greater than fourfold increase of VEGF mRNA levels. ZR-75 breast cancer cells were transfected with pVEGF1, a construct containing a -2018 to +50 VEGF promoter insert, and E2 induced reporter gene (luciferase) activity. Deletion and mutation analysis of the VEGF gene promoter identified a GC-rich region (-66 to -47) which was required for E2-induced transactivation of pVEGF5, a construct containing the minimal promoter (-66 to +54) that exhibited E2-responsiveness. Interactions of nuclear proteins from ZR-75 cells with the proximal GC-rich region of the VEGF gene promoter were investigated by electrophoretic mobility shift and chromatin immunoprecipitation assays. The results demonstrate that both Sp1 and Sp3 proteins bound the GC-rich motif (-66 to -47), and estrogen receptor alpha (ERalpha) interactions were confirmed by chromatin immunoprecipitation. Moreover, E2-dependent activation of constructs containing proximal and distal GC/GT-rich regions of the VEGF promoter was inhibited in ZR-75 cells transfected with small inhibitory RNAs for Sp1 and Sp3. These results were consistent with a mechanism of hormone activation of VEGF through ERalpha/Sp1 and ERalpha/Sp3 interactions with GC-rich motifs.

Breast Neoplasms↗
PubMed2004

Estrogen-dependent regulation of ornithine decarboxylase in breast cancer cells through activation of nongenomic cAMP-dependent pathways.

17beta-estradiol (E2) induces ornithine decarboxylase (ODC) activity in several E2-responsive tissues/cells, and this study investigated the mechanism of hormone-induced transactivation in MCF-7 human breast cancer cells. E2-induced reporter gene (luciferase) activity in MCF-7 cells transfected with a construct (pODC1) containing the -164 to +29 region of the human ODC gene promoter linked to bacterial luciferase. This promoter sequence contains GC-rich Sp1 binding sites, CAAT, LSF, cAMP response element (CRE), and TATA motifs. Deletion and mutational analysis of the ODC promoter showed that both CAAT and LSF sites were required for hormone-induced transactivation. Gel mobility shift and DNA footprinting assays indicated that NFYA and LSF bound the CAAT and LSF motifs, respectively, and GAL4-NFYA/GAL4-LSF chimeras were also activated by E2, 8-bromo-cAMP, and protein kinase A (PKA) expression plasmid. However, E2-induced transactivation of GAL4-NFYA and GAL4-LSF was blocked by the PKA inhibitor SQ22356 indicating that the mechanism of ODC induction by E2 involves upregulation of cAMP/PKA through nongenomic pathways of estrogen action.

Breast Neoplasms↗
PubMed2004

Aryl hydrocarbon receptor-mediated inhibition of LNCaP prostate cancer cell growth and hormone-induced transactivation.

LNCaP prostate cancer cells express the aryl hydrocarbon receptor (AhR), and treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and an Ah-responsive reporter gene. Similar results were obtained with the selective AhR modulator 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF); however, TCDD but not 6-MCDF induced degradation of the AhR protein. TCDD and 6-MCDF inhibited growth of LNCaP cells, and inhibitory AhR-androgen receptor (AR) crosstalk was investigated in cells transfected with constructs containing the androgen-responsive probasin promoter (-288 to +28) (pPB) or three copies of the -244 to -96 region of this promoter (pARR(3)). Ten nanomolar dihydrotestosterone (DHT) and 17 beta-estradiol (E2) induced transactivation in LNCaP cells transfected with pPB or pARR(3); however, inhibitory AhR-AR crosstalk was observed only with the latter construct. 6-MCDF and TCDD did not inhibit DHT- or E2-induced transactivation in ZR-75 human breast cancer cells, indicating that these interactions were promoter and cell context-dependent. Both E2 and DHT stabilized AR protein in LNCaP cells; however, cotreatment with TCDD or 6-MCDF decreased AR protein levels. These results indicate that inhibitory AhR-AR crosstalk in prostate cancer cells is complex and for some responses, AR protein stability may play a role.

Benzofurans↗
PubMed2004

A new class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists that inhibit growth of breast cancer cells: 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes.

1,1-Bis(3'-indolyl)-1-(p-trifluoromethylphenyl)methane (DIM-C-pPhCF(3)) and several p-substituted phenyl analogues have been investigated as a new class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. Structure-activity studies in PPARgamma-dependent transactivation assays in MCF-7 breast cancer cells show that 5-20 micro M concentrations of compounds containing p-trifluoromethyl, t-butyl, cyano, dimethylamino, and phenyl groups were active, whereas p-methyl, hydrogen, methoxy, hydroxyl, or halogen groups were inactive as PPARgamma agonists. Induction of PPARgamma-dependent transactivation by 15-deoxy-Delta12,14-prostaglandin J2 (PGJ2) and DIM-C-pPhCF(3) was inhibited in MCF-7 cells cotreated with the PPARgamma-specific antagonist N-(4'-aminopyridyl)-2-chloro-5-nitrobenzamide. In mammalian two-hybrid assays, DIM-C-pPhCF(3) and PGJ2 (5-20 micro M) induced interactions of PPARgamma with steroid receptor coactivator (SRC) 1, SRC2 (TIFII), and thyroid hormone receptor-associated protein 220 but not with SRC3 (AIB1). In contrast, DIM-C-pPhCF(3), but not PGJ2, induced interactions of PPARgamma with PPARgamma coactivator-1. C-substituted diindolylmethanes inhibit carcinogen-induced rat mammary tumor growth, induce differentiation in 3T3-L1 preadipocytes, inhibit MCF-7 cell growth and G(0)/G(1)-S phase progression, induce apoptosis, and down-regulate cyclin D1 protein and estrogen receptor alpha in breast cancer cells. These compounds are a novel class of synthetic PPARgamma agonists that induce responses in MCF-7 cells similar to those observed for PGJ2.

3T3-L1 Cells↗
PubMed2003

Peroxisome proliferator-activated receptor gamma agonists induce proteasome-dependent degradation of cyclin D1 and estrogen receptor alpha in MCF-7 breast cancer cells.

Treatment of MCF-7 cells with the peroxisome proliferator-activated receptor (PPAR) gamma agonists ciglitazone or 15-deoxy-Delta 12,14-prostaglandin J2 resulted in a concentration- and time-dependent decrease of cyclin D1 and estrogen receptor (ER) alpha proteins, and this was accompanied by decreased cell proliferation and G(1)-G(0)-->S-phase progression. Down-regulation of cyclin D1 and ER alpha by PPARgamma agonists was inhibited in cells cotreated with the proteasome inhibitors MG132 and PSII, but not in cells cotreated with the protease inhibitors calpain II and calpeptin. Moreover, after treatment of MCF-7 cells with 15-deoxy-Delta 12,14-prostaglandin J2 and immunoprecipitation with cyclin D1 or ER alpha antibodies, there was enhanced formation of ubiquitinated cyclin D1 and ER alpha bands. Thus, PPARgamma-induced inhibition of breast cancer cell growth is due, in part, to proteasome-dependent degradation of cyclin D1 (and ER alpha), and this pathway may be important for other cancer cell lines.

Breast Neoplasms↗
PubMed2003

Domains of estrogen receptor alpha (ERalpha) required for ERalpha/Sp1-mediated activation of GC-rich promoters by estrogens and antiestrogens in breast cancer cells.

Estrogen receptor alpha (ERalpha)/Sp1 activation of GC-rich gene promoters in breast cancer cells is dependent, in part, on activation function 1 (AF1) of ERalpha, and this study investigates contributions of the DNA binding domain (C) and AF2 (DEF) regions of ERalpha on activation of ERalpha/Sp1. 17Beta-estradiol (E2) and the antiestrogens 4-hydroxytamoxifen and ICI 182,780 induced reporter gene activity in MCF-7 and MDA-MB-231 cells cotransfected with human or mouse ERalpha (hERalpha or MOR), but not ERbeta and GC-rich constructs containing three tandem Sp1 binding sites (pSp13) or other E2-responsive GC-rich promoters. Estrogen and antiestrogen activation of hERalpha/Sp1 was dependent on overlapping and different regions of the C, D, E, and F domains of ERalpha. Antiestrogen-induced activation of hERalpha/Sp1 was lost using hERalpha mutants deleted in zinc finger 1 [amino acids (aa) 185-205], zinc finger 2 (aa 218-245), and the hinge/helix 1 (aa 265-330) domains. In contrast with antiestrogens, E2-dependent activation of hERalpha/Sp1 required the C-terminal F domain (aa 579-595), which contains a beta-strand structural motif. Moreover, in peptide competition experiments overexpression of a C-terminal (aa 575-595) F domain peptide specifically blocked E2-dependent activation of hERalpha/Sp1, suggesting that F domain interactions with nuclear cofactors are required for ERalpha/Sp1 action.

Base Composition↗
PubMed2003

17 beta-estradiol- and 4-hydroxytamoxifen-induced transactivation in breast, endometrial and liver cancer cells is dependent on ER-subtype, cell and promoter context.

The pattern of transcriptional activation by 17beta-estradiol (E2) and 4-hydroxytamoxifen (4-OHT) was determined in ZR-75 and MDA-MB-231 breast, ECC1 and HEC1A endometrial and HepG2 liver cancer cell lines cotransfected with E2-responsive constructs and wild-type estrogen receptor alpha (ER alpha) or ER beta (ER beta) or variant forms of ER alpha expressing activation function 1, AF1 (ER alpha-AF1) or activation function 2, AF2 (ER alpha-AF2). The E2-responsive constructs contained promoter inserts from the human complement C3 (pC3), human cathepsin D (pCD) and rat creatine kinase B (pCKB) genes. Minimal ER beta-dependent transactivation (<2.5-fold induction) was observed for E2 only in ECC1 and MDA-MB-231 cells transfected with pCKB or pC3, whereas 4-OHT was inactive as an ER beta agonist for all promoters in the four cell lines. The ER alpha agonist and/or antagonist activities for E2 and 4-OHT were highly variable and the transactivation was dependent on ER subtype, ER alpha variant expressed, gene promoter, and cell context. For example, E2 did not activate pCD in HepG2 cells transfected with wild-type or variant ER alpha, whereas E2 activated reporter gene activity in the four endometrial and breast cancer cell lines transfected with ER alpha and pCD, pCKB or pC3. Hormone activation of these constructs by ER alpha-AF1 or ER alpha-AF2 was highly variable among the different cell lines and even in the same cell line transfected with the three E2-responsive constructs. Similar variability was observed for 4-OHT. For example, 4-OHT activates pC3 in HepG2 cells transfected with ER alpha or ER alpha-AF1, and pCKB in HEC1A cells. However, AF1-dependent activation by 4-OHT is not observed for pCKB in ECC1 cells or for pC3 and pCD in HEC1A or ECC1 endometrial cancer cells. The results of this study suggest that transcriptional activation by E2 and 4-OHT induces recruitment of different transcription factor complexes that are dependent on the cell type and also the gene promoter.

Animals↗
PubMed2003

Aryl hydrocarbon receptor gene silencing with small inhibitory RNA differentially modulates Ah-responsiveness in MCF-7 and HepG2 cancer cells.

Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNAs for the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) mRNAs in MCF-7 breast cancer cells resulted in a 60 to 80% decrease in levels of AhR and ARNT proteins in whole-cell extracts and decreased binding of nuclear extracts to 32P-labeled dioxin-responsive element. siRNA for the AhR also decreased 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 protein, CYP1A1-dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17beta-estradiol (E2) induces proliferation of MCF-7 cells through enhanced G0/G1 --> S phase progression, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell-cycle progress were partially blocked in MCF-7 cells transfected with siRNA for AhR. The results also indicated that siRNA-dependent decreases in AhR protein in MCF-7 cells were accompanied by increased G0/G1 --> S phase progression, suggesting a growth-inhibitory role for the "endogenous" AhR. Surprisingly, TCDD alone induced G0/G1 --> S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 --> S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cyclin-dependent kinase 2 (cdk2), and cdk4. In the absence of ligand, the AhR exhibits growth-inhibitory (MCF-7) and growth-promoting (HepG2) activity that is cell context-dependent.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed2003

The aryl hydrocarbon receptor mediates degradation of estrogen receptor alpha through activation of proteasomes.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands suppress 17beta-estradiol (E)-induced responses in the rodent uterus and mammary tumors and in human breast cancer cells. Treatment of ZR-75, T47D, and MCF-7 human breast cancer cells with TCDD induces proteasome-dependent degradation of endogenous estrogen receptor alpha (ERalpha). The proteasome inhibitors MG132, PSI, and PSII inhibit the proteasome-dependent effects induced by TCDD, whereas the protease inhibitors EST, calpain inhibitor II, and chloroquine do not affect this response. ERalpha levels in the mouse uterus and breast cancer cells were significantly lower after cotreatment with E plus TCDD than after treatment with E or TCDD alone, and our results indicate that AhR-mediated inhibition of E-induced transactivation is mainly due to limiting levels of ERalpha in cells cotreated with E plus TCDD. TCDD alone or in combination with E increases formation of ubiquitinated forms of ERalpha, and both coimmunoprecipitation and mammalian two-hybrid assays demonstrate that TCDD induces interaction of the AhR with ERalpha in the presence or absence of E. In contrast, E does not induce AhR-ERalpha interactions. Thus, inhibitory AhR-ERalpha cross talk is linked to a novel pathway for degradation of ERalpha in which TCDD initially induces formation of a nuclear AhR complex which coordinately recruits ERalpha and the proteasome complex, resulting in degradation of both receptors.

Animals↗
PubMed2003

Cell context-dependent differences in the induction of E2F-1 gene expression by 17 beta-estradiol in MCF-7 and ZR-75 cells.

17 beta-Estradiol (E2) induces E2F-1 gene expression in ZR-75 and MCF-7 human breast cancer cells. Analysis of the E2F-1 gene promoter in MCF-7 cells previously showed that hormone-induced transactivation required interactions between estrogen receptor alpha (ER alpha)/Sp1 bound to upstream GC-rich sites and NFYA bound to downstream CCAAT sites within the -169 to -54 region of the promoter. This same region of the E2F-1 promoter was also E2 responsive in ER alpha-positive ZR-75 cells; however, further analysis of the promoter showed that cooperative ER alpha/Sp1/NFY interactions were not necessary for hormone-induced transactivation in ZR-75 cells. The upstream GC-rich motifs (-169 to -111) are activated independently by ER alpha/Sp1 in ZR-75 but not MCF-7 cells, and a construct (pE2F-1j(m1)) containing the -122 to -54 downstream CCAAT site that bound NFYA was also E2 responsive. E2 also induced reporter gene activity in ZR-75 cells transfected with an expression plasmid for a chimeric protein containing the DNA-binding domain of the yeast GAL4 protein fused to NFYA (pM-NFYA) and a construct containing five tandem GAL4 response elements. Subsequent studies showed that hormonal activation of pE2F-1j(m1) and pM-NFYA are dependent on nongenomic pathways in which E2 activates cAMP/protein kinase A. Hormone-dependent regulation of E2F-1 gene expression in ZR-75 and MCF-7 involves the same cis elements and interacting transcription factors but different mechanisms, demonstrating the importance of cell context on transactivation pathways, even among ER-positive breast cancer cell lines.

CCAAT-Binding Factor↗
PubMed2003

Estrogen receptor/Sp1 complexes are required for induction of cad gene expression by 17beta-estradiol in breast cancer cells.

The cad gene is trifunctional and expresses carbamoylphosphate synthetase/aspartate carbamyltransferase/dihydroorotase, which are required for pyrimidine biosynthesis. Cad gene activities are induced in MCF-7 human breast cancer cells, and treatment of MCF-7 or ZR-75 cells with 10 nM 17beta-estradiol (E2) resulted in a 3- to 5-fold increase in cad mRNA levels in both cell lines. The mechanism of hormone-induced cad gene expression was further investigated using constructs containing the growth-responsive -90 to +115 (pCAD1) region of the cad gene promoter. E2 induced reporter gene (luciferase) activity in MCF-7 and ZR-75 cells transfected with pCAD1, which contains three upstream GC-rich and two downstream E-box motifs. Deletion and mutation analysis of the cad gene promoter demonstrated that only the GC boxes that bind Sp1 protein were required for E2 responsiveness. Results of electrophoretic mobility shift and chromatin immunoprecipitation assays show that both Sp1 and estrogen receptor alpha interact with the GC-rich region of the cad gene promoter. Moreover, in transactivation assays with pCAD1, hormone-induced transactivation was inhibited by cotransfection with dominant-negative Sp1 expression plasmid and small inhibitory RNA for Sp1, which silences Sp1 expression in the cells. These results demonstrate that, in common with many other genes involved in E2-induced cell proliferation, the cad gene is also regulated by a nonclassical ERalpha/Sp1-mediated pathway.

Aspartate Carbamoyltransferase↗