ORCID Profile
0000-0002-0420-6888
Current Organisation
The University of Manitoba
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Publisher: Bioscientifica
Date: 04-2005
DOI: 10.1677/JME.1.01688
Abstract: To investigate the effect of altered oestrogen receptor (ER)α and ERβ expression on oestrogen and anti-oestrogen action in breast cancer, we have stably expressed an inducible ERβ1 in MCF7 breast cancer cells. Stably expressing clones were isolated and over-expression of ERβ1 correlated with increased levels of specific radiolabelled oestradiol (E2) binding. Increased ERβ1 did not affect endogenous levels of ERα but increased progesterone receptor (PR) levels. Over-expression of ERβ1 reduced growth responses to E2 in contrast to little if any effect of over-expression of ERα. In oestrogen-replete conditions, over-expression of ERβ1 but not ERα reduced proliferation. Over-expression of ERβ1 did not result in anti-oestrogen resistance but was associated with increased sensitivity to 4-hydroxytamoxifen. Our results suggested that over-expression of ERβ1 in the presence of an endogenously expressed ERα was associated with tamoxifen sensitivity but may negatively modulate ERα-mediated growth. However, not all ERα activities were inhibited since endogenous PR expression was increased by both ERα and ERβ1 over-expression. These data paralleled those seen in some in vivo studies showing a relationship between PR and ERβ expression as well as ERβ expression and tamoxifen sensitivity of ER-positive breast cancer patients. These models are relevant and will be useful for dissecting the role of ERβ1 expression in ER-positive breast cancer.
Publisher: Elsevier BV
Date: 11-1998
Publisher: Wiley
Date: 11-1996
DOI: 10.1002/(SICI)1097-4644(19961101)63:2<174::AID-JCB5>3.0.CO;2-V
Abstract: The tissue matrix consists of linkages and interactions of the nuclear matrix, cytoskeleton, and extracellular matrix. This system is a dynamic structural component of the cell that organizes and processes structural and functional information to maintain and coordinate cell function and gene expression. We have studied estrogen regulation of nuclear matrix associated proteins, including the intimately connected cytoskeletal intermediate filaments, in T-47D5 human breast cancer cells. Three proteins (identified as cytokeratins 8, 18, and 19) present in the nuclear matrix-intermediate filament fraction (NM-IF) of cells grown in estrogen-replete conditions were dramatically reduced when the cells were grown in acute (1 week) estrogen-depleted conditions. Replacing estrogen in the medium of acute estrogen-depleted cells restored expression of these proteins. T-47D5 cells that are chronically depleted of estrogen (T5-PRF) are estrogen-nonresponsive in culture. These cells overexpressed these three proteins, compared to parent cells grown in the presence of estrogen. Treatment of the T5-PRF cells with estrogen did not lead to further up-regulation of these proteins. Treating T-47D5 cells in estrogen-replete conditions with the antiestrogens 4-hydroxytamoxifen and ICI 164 384 (100 nM, 3 days) resulted in a significant reduction in these proteins, while no effect was seen in long-term chronic estrogen-depleted T-47D5 cells. In conclusion, we have identified NM-IF proteins (cytokeratins 8, 18, and 19) in human breast cancer cells that are estrogen regulated and may play a role in estrogen action in human breast cancer cells.
Publisher: Wiley
Date: 15-03-1996
DOI: 10.1002/(SICI)1097-4644(19960315)60:4<560::AID-JCB12>3.0.CO;2-L
Abstract: The expression of the c-myc gene is essential for the proliferation of both hormone-dependent and -independent human breast cancer cells. The regulation of c-myc gene expression in MCF-7 (hormone-dependent, estrogen-receptor (ER)-positive) and MDA MB 231 (hormone-independent, ER-negative) human breast cancer cells differs, with the c-myc gene of MCF-7 but not MDA MB 231 cells being regulated at the transcriptional level by estrogen. We have shown previously that the DNAase I hypersensitive (DH) sites in the c-myc chromatin of hormone-dependent and -independent human breast cancer cells were similar, with the exception of DH site II2. DH site II2, which maps near the P0 promoter, was less sensitive in hormone-dependent than in hormone-independent cells. As DH sites generally indicate the presence of sequence-specific DNA-binding proteins, we undertook a study to identify the nuclear proteins isolated from MCF-7 and MDA MB 231 cells that bound to the P0 and P2 promoter regions of the c-myc gene in vitro. The studies presented here provide evidence that Sp1 and/or Sp1-like proteins bind to the P0 and P2 promoter regions of the c-myc gene of MCF-7 and MDA MB 231 cells. Furthermore, evidence is presented for the presence of several previously unidentified sequence-specific DNA-binding proteins binding to these promoters. The DNA-binding activities of these latter proteins differed in the nuclear extracts of the MCF-7 and MDA MB 231 human breast cancer cells.
Publisher: Canadian Science Publishing
Date: 06-2007
DOI: 10.1139/O07-009
Abstract: Dihydropyrimidine dehydrogenase (DPD) is one of the factors that determine the efficacy and toxicity of 5-fluorouracil. Variations in DPD activity may result from alterations at the transcriptional level of the DPYD gene. Heterogeneity in DPYD expression has been reported, but the molecular mechanisms responsible for this remain unclear. We investigated methylation of the DPYD promoter as a mechanism for transcriptional regulation of DPYD in the RKO colorectal cancer cell line. We demonstrate that the active transcription machinery for DPYD is present in RKO cells, but promoter binding of Sp1, a transactivator of DPYD, was inhibited, which on subsequent examination was shown to be associated with dense promoter methylation. Treatment with 5-aza-2′-deoxycytidine alone or the combination of 5-aza-2′-deoxycytidine and trichostatin A induced demethylation of the promoter and markedly increased the DPYD mRNA level in RKO cells but not in unmethylated WiDr cells. Furthermore, in vitro methylation of the DPYD promoter decreased promoter activity. These data suggest an important role for methylation in DPYD suppression. The transcriptional suppression of DPYD by methylation may be responsible for the increased 5-fluorouracil sensitivity observed in some patients. This may also provide insight into the mechanism underlying the downregulation of DPYD in some colorectal cancers.
Publisher: Elsevier BV
Date: 08-1994
Abstract: The effect of inhibiting transcription and/or replication on the steady state levels of the ubiquitinated histone isoforms was investigated. We show that treatment of chinese hamster ovary (CHO), monkey kidney (COS), human endometrial carcinoma (Hec-50 and Ishikawa) cells with actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, inhibitors of heterogeneous nuclear RNA synthesis, selectively reduced the levels of ubiquitinated (u) H2B, but not uH2A, uH2A.Z, polyubiquitinated H2A or a novel ubiquitinated histone species, in the chromatin of these cells. The level of the ubiquitinated histones was not affected when synthesis of DNA was arrested. These results show that, in general, maintenance of the levels of uH2B in chromatin is dependent upon ongoing transcription.
Publisher: American Association for Cancer Research (AACR)
Date: 15-10-2006
DOI: 10.1158/0008-5472.CAN-05-4111
Abstract: Detection of estrogen receptor (ER)-α phosphorylated at Ser118 (P-Ser118-ER-α) may be an indicator of an intact ligand-dependent ER-α in breast tumors in vivo and may predict responsiveness to endocrine therapy. The current study addresses whether P-Ser118-ER-α is functionally involved in ER target gene transcription and if this is modulated by HER-2 overexpression. Using chromatin immunoprecipitation analysis, P-Ser118-ER-α was found associated with the promoters of several estrogen-regulated genes in MCF-7 breast cancer cells 30 minutes following estrogen treatment. Coactivators AIB1 and p300 were coimmunoprecipitated with P-Ser118-ER-α following estrogen treatment. The overexpression of HER-2 protein in MCF-7 cells did not affect estrogen induction of phosphorylation of Ser118 or its presence at the promoters of several estrogen-regulated genes. U0126, an inhibitor of mitogen-activated protein kinase (MAPK) pathway, had no effect on P-Ser118-ER-α. The lack of effect of HER-2 overexpression on P-Ser118-ER-α expression in cell models is supported by similar levels of expression of P-Ser118-ER-α in ER+/HER-2-overexpressing and ER+/HER-2− breast tumors in vivo. Using inhibitors of cyclin-dependent kinase 7 (Cdk7), [(5,6-dichloro-1-β-d-ribofuranosylbenzimidazole and 2-(R)-1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine], and IκB kinase-α (IKK-α BAY-11-7082), we show that IKK-α, but not Cdk7, is at least in part involved in estrogen-mediated phosphorylation at Ser118 in MCF-7 cells. Our data provide direct evidence for a functional role of P-Ser118-ER-α in estrogen-regulated signaling and do not support the hypothesis that resistance of breast tumors to tamoxifen therapy involves ligand independent activation of ER-α due to constitutive phosphorylation of Ser118 by constitutive activation of MAPK pathway. (Cancer Res 2006 66(20): 10162-70)
Publisher: Elsevier BV
Date: 02-1993
DOI: 10.1016/0303-7207(93)90258-L
Abstract: Expression of the c-myc protooncogene is estrogen regulated in estrogen receptor (ER) positive, hormone-dependent human breast cancer cells, but it is constitutively active in ER negative, hormone-independent breast cancer cells. To determine whether these differences are reflected in c-myc chromatin, DNase I hypersensitive sites (DHS) were mapped. Six DHS were detected in all cell lines studied, with DHS 3(2) being more prominent than DHS 3(1). The accessibility of DHS 2 was markedly greater in ER negative cells than in ER positive cells, and this relative accessibility remained unchanged when cells were grown in estrogen free medium. DHS 2, 3(1) and 3(2) map near the P0, P1 and P2 promoters, respectively. An analysis of promoter usage demonstrated that P2 was the preferred promoter. Thus, the differences in the accessibility of DHS 2 in c-myc chromatin of ER positive and negative cells likely reflects alterations in DNA-protein interactions in this region.
Publisher: Elsevier BV
Date: 12-2006
DOI: 10.1016/J.JSBMB.2006.09.021
Abstract: Post-translational modifications of proteins are known to be important in protein activity and ERalpha is known to be phosphorylated at multiple sites within the protein. The exact function of site-specific phosphorylation in ERalpha is unknown, although several hypotheses have been developed using site-directed mutagenesis and cell culture models. Targeting the ERalpha at the level of such post-translational modification pathways would be a new and exciting approach to endocrine therapy in breast cancer, but adequate knowledge is lacking with regard to the relevance of site-specific phosphorylation in ERalpha in human breast cancer in vivo. Recently, antibodies to P-Serine(118)-ERalpha and P-Serine(167)-ERalpha, two major sites of phosphorylation in ERalpha, have become available and some in vivo data are now available to complement studies in cells in culture. However, the in vivo data are somewhat contradictory and limited by the small cohorts used and the lack of standard well-characterized reagents and protocols.
Publisher: Springer Science and Business Media LLC
Date: 29-09-2006
DOI: 10.1007/S10549-006-9390-X
Abstract: We have previously observed a paradoxical relationship of the psoriasin/S100A7 gene with estrogen response in-vitro in ERalpha positive cells but its association with ERalpha negative status in-vivo raising the possibility that S100A7 might be regulated by ERbeta in breast cancer. Using doxycycline-inducible ERbeta and ERalpha expressing MCF-7 cells the hypothesis that psoriasin/S100A7 is ERbeta regulated was investigated To explore the relationship between psoriasin/S100A7 and ERbeta expression in-vivo, we also assessed a cohort of 233 ERalpha negative breast tumors using tissue microarrays and immunohistochemistry. Psoriasin/S100A7 was increased by 17beta-estradiol (E2) following ERbeta induction, in several clones of ERbeta over-expressing but not in the original MCF-7 cells, nor clones over-expressing ERalpha. The effect of E2 on psoriasin/S100A7 was inhibited by 4-hydroxytamoxifen and ICI 182780 but not with a selective ERalpha antagonist. An ERbeta selective-agonist but not an ERalpha selective-agonist, induced psoriasin/S100A7. This induction still occurred after stable down-regulation of ERalpha using siRNA in ERbeta inducible cells. E2 increased psoriasin/S100A7 mRNA but cycloheximide treatment inhibited this effect. A relationship between ERbeta and psoriasin/S100A7 was observed in the p53 immunohistochemically negative subset of invasive breast tumors in-vivo (r = 0.225, p = 0.046, n = 79). In conclusion we demonstrate that E2 induction of psoriasin/S100A7 can be specifically regulated through ERbeta in-vitro and associated with ERbeta in-vivo. These data support the hypothesis that psoriasin/S100A7 is specifically regulated by ERbeta activity and could be useful to guide future therapies targeting ERbeta in certain phenotypic subsets of breast cancers in-vivo.
No related grants have been discovered for James Davie.