Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Lactate dehydrogenase A (LDHA) is hormonally regulated in rodents, and increased expression of LDHA is observed during mammary gland tumorigenesis. The mechanisms of hormonal regulation of LDHA were investigated in breast cancer cells using a series of deletion and mutant reporter constructs derived from the rat LDHA gene promoter. Results of transient transfection studies showed that the -92 to -37 region of the LDHA promoter was important for basal and estrogen-induced transactivation, and mutation of the consensus CRE motif (-48/-41) within this region resulted in significant loss of basal activity and hormone-responsiveness. Gel mobility shift assays using nuclear extracts from MCF-7 cells indicated that CREB family proteins interacted with the CRE. Studies with kinase inhibitors showed that estrogen-induced activation of this CRE was dependent on protein kinase C, and these data show that LDHA is induced through a nongenomic (extranuclear) pathway of estrogen action. Estrogen activates several nongenomic pathways in MCF-7 cells, and this study investigated the effects of structurally diverse estrogenic compounds on activation of mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), protein kinase A (PKA), and calcium/calmodulin-dependent protein kinase IV (CaMKIV). Activation of kinases was determined by specific substrate phosphorylation and transactivation assays that were diagnostic for individual kinases. The compounds investigated in this study include E2, diethylstilbestrol (DES), the phytoestrogen resveratrol, and the following synthetic xenoestrogens: bisphenol-A (BPA), nonylphenol, octylphenol, endosulfan, kepone, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and 2',3',4',5'-tetrachloro-4-biphenylol (HO-PCB-Cl4). With theexception of resveratrol, all the compounds activated PI3K and MAPK whereas activation of PKC by the xenoestrogens was structure-dependent and resveratrol, kepone and HO-PCB-Cl4 were inactive. Only minimal estrogen/xenoestrogen-dependent activation of PKA was observed. CaMKIV was activated only by E2 and DES, and HO-PCB-Cl4 was a potent inhibitor of CaMKIV-dependent activity. These results demonstrate that activation of nongenomic pathways by estrogenic compounds in MCF-7 cells is structure-dependent.

The estrogen receptor (ER) is a ligand-activated transcription factor that regulates gene expression. The classical mechanisms of nuclear ER action include ligand-induced dimerization of ER which binds estrogen responsive elements (EREs) in promoters of target genes. In addition, non-genomic pathways of ER action have also been identified in breast cancer cells. Cdc25A is a tyrosine phosphatase that catalyzes dephosphorylation of cyclin/cyclin-dependent kinase complexes to regulate G1- to S-phase cell cycle progression. Cdc25A mRNA levels are induced by 17β-estradiol (E2) in ZR-75 breast cancer cells, and deletion analysis of the Cdc25A promoter identified the -151 to -12 region as the minimal E2-responsive sequence. Subsequent mutation/deletion analysis showed that at least three different cis-elements were involved in activation of Cdc25A by E2, namely, GC-rich Sp1 binding sites, CCAAT motifs, and E2F sites. Studies with inhibitors and dominant negative expression plasmids show that E2 activates Cdc25A expression through activation of genomic ERα/Sp1 and E2F1 and cAMP-dependent activation of NF-YA. Thus, both genomic and non-genomic pathways of estrogen action are involved in induction of Cdc25A in breast cancer cells. The PIAS family was initially identified as cytokine-induced inhibitors of STATs which contain several conserved domains involved in binding to other nuclear coactivators. In this study we have investigated coactivation of ERα by PIAS3 in breast cancer cell lines transiently cotransfected with the pERE3 constructs which contain three tandem EREs linked to a luciferase reporter gene. PIAS3 coactivated ERα-mediated transactivation in cells cotransfected with pERE3 and wild-type ERα. In contrast to many other coactivators, PIAS3 also enhanced transactivation of ERα when cells were cotransfected with the TAF1 ERα mutant. In addition, PIAS3 does not interact with activation function 2 (AF2) domain of ERα in a mammalian two-hybrid assay. These data indicate that coactivation of ERα by PIAS3 was AF2-domain independent. Analysis of several PIAS3 deletion mutants showed that the region containing amino acids 274 to 416 of PIAS3 are required for coactivation suggesting that the RING finger domain and acidic region of PIAS3 are important for interactions with wild-type ERα. These results demonstrate that PIAS3 coactivated ERα and this represents a non-classical LXXLL-independent coactivation pathway.

Vitamin D interacting protein 205 (DRIP205) is a mediator complex protein that anchors the complex to the estrogen receptor (ER) and other nuclear receptors (NRs). In ZR-75 breast cancer cells treated with 17?-estradiol (E2) and transfected with a construct containing three tandem estrogen responsive elements (pERE3), DRIP205 coactivates ER?-mediated transactivation. DRIP205?587-636 is a DRIP205 mutant in which both NR boxes within amino acids 587-636 have been deleted and, in parallel transfection studies, DRIP205?587-636 also coactivates ER?. Moreover, both wild-type and variant DRIP205 also colocalize with ER? in the nuclei of transfected cells. AF1 and AF2 of ER? are both required for DRIP205 coactivation. Extensive deletion analysis of DRIP205 shows that multiple domains of this protein play a role in coactivation of ER? and in interactions with ER?. On the other hand, both DRIP205 and DRIP205?587-636 coactivate E2-induced transactivation of ER?/Sp1 in cells transfected with a construct containing three GC-rich sites (pSp13). Coactivation of ER?/Sp1 by DRIP205 is dependent on AF1 of ER?. Enhancement of ER? and ER?/Sp1 by DRIP205 does not require NR boxes of DRIP205, and deletion mutants DRIP205 (1-714) and DRIP205 (516-1566) significantly coactivate ER? and ER?/Sp1. RNA interference study showed that DRIP205 coactivation of ER?/Sp was abolished in cells transfected with iSp3 and iSp4, suggesting that Sp3 and Sp4 are required for coactivation of ER?/Sp by DRIP205 in ZR-75 cells.

Estrogen Receptor ? (ER?)/Sp1 activation of GC-rich gene promoters in breast cancer cells is dependent, in part, on the activation function 1 (AF1) of ER?. This study investigates contributions of the DNA binding domain (C) and AF2 (DEF) regions of ER? on activation of ER?/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 ER? (hER? or MOR), but not ER? and GC-rich constructs containing three tandem Sp1 binding sites (pSp13) or other E2-responsive GC-rich promoters. Estrogen and antiestrogen activation of hER?/Sp1 was dependent on overlapping and different regions of the C, D, E, and F domains of ER?. Antiestrogen-induced activation of hER?/Sp1 was lost using hER? 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 hER?/Sp1 required the C-terminal F domain (aa 579-595), which contains a ?-strand structural motif. Moreover, in peptide competition experiments overexpression of NR-box peptides inhibits E2-induced luciferase activity of pERE3, which contains three tandem repeats of consensus ERE sites, whereas E2-induced hER?/Sp1 action was not inhibited by NR-box peptide expression. In contrast, overexpression of a C-terminal (aa 575-595) F domain peptide specifically blocked E2-dependent activation of hER?/Sp1, but not on activation of pERE3, suggesting that F domain interactions with nuclear cofactors are specifically required for ER?/Sp1 action. Furthermore, direct physical interactions between hER? and Sp1 protein in vivo have been investigated by using Fluorescence Resonance Energy Transfer (FRET) microscopy and image analysis. Consistent with results from transient transfection assay, E2, 4OHT, and ICI enhanced hER?/Sp1 interactions in living cells and these interactions were also confirmed by coimmunoprecipitation. In addition, endogenous hER?/Sp1 action was evaluated by using si RNA for Sp1 and a significant decrease in ligand-induced hER?/Sp1 action was observed after decreased Sp1 expression.

Lactate dehydrogenase A (LDHA) is hormonally regulated in rodents, and increased expression of LDHA is observed during mammary gland tumorigenesis. The mechanisms of hormonal regulation of LDHA were investigated in breast cancer cells using a series of deletion and mutant reporter constructs derived from the rat LDHA gene promoter. Results of transient transfection studies showed that the -92 to -37 region of the LDHA promoter was important for basal and estrogen-induced transactivation, and mutation of the consensus CRE motif (-48/-41) within this region resulted in significant loss of basal activity and hormone-responsiveness. Gel mobility shift assays using nuclear extracts from MCF-7 cells indicated that CREB family proteins interacted with the CRE. Studies with kinase inhibitors showed that estrogen-induced activation of this CRE was dependent on protein kinase C, and these data show that LDHA is induced through a nongenomic (extranuclear) pathway of estrogen action. Estrogen activates several nongenomic pathways in MCF-7 cells, and this study investigated the effects of structurally diverse estrogenic compounds on activation of mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), protein kinase A (PKA), and calcium/calmodulin-dependent protein kinase IV (CaMKIV). Activation of kinases was determined by specific substrate phosphorylation and transactivation assays that were diagnostic for individual kinases. The compounds investigated in this study include E2, diethylstilbestrol (DES), the phytoestrogen resveratrol, and the following synthetic xenoestrogens: bisphenol-A (BPA), nonylphenol, octylphenol, endosulfan, kepone, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and 2',3',4',5'-tetrachloro-4-biphenylol (HO-PCB-Cl4). With theexception of resveratrol, all the compounds activated PI3K and MAPK whereas activation of PKC by the xenoestrogens was structure-dependent and resveratrol, kepone and HO-PCB-Cl4 were inactive. Only minimal estrogen/xenoestrogen-dependent activation of PKA was observed. CaMKIV was activated only by E2 and DES, and HO-PCB-Cl4 was a potent inhibitor of CaMKIV-dependent activity. These results demonstrate that activation of nongenomic pathways by estrogenic compounds in MCF-7 cells is structure-dependent.

Thesis (Ph.D.)--Bowling Green State University, 2009. / Document formatted into pages; contains xx, 271 p. : ill. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 127-153).

Thesis (Ph. D.) -- Lincoln University, 2009. / Also available via the World Wide Web. Some images in print version are not in digital version.

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.