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.

Human tumor susceptibility gene 101, TSG101, exhibits a variety of functions including protein sorting, vesicular trafficking, and regulation of transcription, epithelial growth and differentiation. The upstream sequence of TSG101 gene shows a typical housekeeping TATA-less and Sp1 containing promoter. Our previous data indicated the essential role of TSG101 in skin keratinocyte differentiation that is under the regulation of PKC-Sp1 signaling. In this report, we investigated the correlation of TSG101 and Sp1 expression in the specimens of cervical intraepithelial neoplasia. Cervical intraepithelial neoplasia specimens used in this study were 129 paraffin blocks from 41 normal, 35 CIN I, 28 CIN II and 25 CIN III/CIS patients collected in Cancer Prevention and Screening Center at Kaohsiung from January 2005 to July 2007. The expression of TSG101 and Sp1 were analyzed by immunohistochemistry and digitally quantified by Image Pro-plus 6.1 Microimage software according to the method described by Eliane Pedra Dias et al. The quantified data were statistically analyzed using Spearman's rho coefficient and SPSS for Win, v.14 statistical software (SPSS Inc., Chicago, USA). Values were considered significantly different when the P value < 0.05. We found that TSG101 and Sp1 are expressed in cells of parabasal and intermediate layers in normal cervical epithelium, whereas their expressions in basal and superficial layers were either absence or reduced. Interestingly, the expressions of these two markers are significantly increased in more advanced progression stages (CIN II and CIN III/CIS) of cervical intraepithelial neoplastic specimens (P < 0.05). Congruous expression pattern of TSG 101 and Sp1 in normal cervical epithelium confirms the important of cellular Sp1 signaling in regulating TSG101 expression, which is essential during epithelial cell growth and differentiation. Our results also indicate upregulation of these two markers might be important for the progression of cervical intraepithelial neoplasia. Further analysis using more specimens should reveal the prognostic value of these two markers.

Maintenance of genomic stability is paramount for survival of an organism; failure to repair DNA damage ultimately leads to the accumulation of genetically unstable cells and the onset of different human diseases including cancer. DNA single strand breaks and base oxidation/alkylation are among the most frequent types of DNA damage occurring spontaneously in cells. Base excision repair (BER), which copes with the majority of these lesions, is therefore a fundamental DNA repair system. Accordingly, it is important to understand how BER is regulated, and particularly, how and if BER is affected by the cellular load of DNA damage. Although functions of key BER proteins are well-defined, regulation of their expression is poorly understood. During BER, the protein XRCC1 is particularly important. It functions as a scaffold, stabilising repair complexes at sites of DNA damage thereby promoting efficient DNA repair. As a central coordinator in BER, it is therefore of great interest to understand how expression of XRCC1 is controlled. In this thesis I demonstrate that modulation of XRCC1 expression is mediated by transcription factor Sp1. Importantly, Sp1 is also affected during the DNA damage response, suggesting an indirect mechanism promoting BER modulation in response to the cellular DNA damage load. In fact, I show that, in response to persistent DNA strand breaks, the key DNA damage signalling factor ATM phosphorylates Sp1. This initiates Sp1 degradation, negatively affecting BER. Therefore, this thesis identifies a mechanism involving signalling from ATM that regulates BER in response to persistent DNA damage, which I link to susceptibility to apoptosis and cell elimination. I hypothesise that regulation of DNA repair in response to persistent DNA damage constitutes a mechanism to promote the elimination of potentially pre-cancerous cells that accumulate unrepairable levels of DNA lesions.

The metastatic spread of tumor cells is the major lethal aspect of cancer, and lymphatic metastasis is one of the most important routes. Recent studies indicated that cyclooxygenase-2 (COX-2) expression is frequently associated with lymph node metastasis and over-expression of COX-2 can enhance lymphatic invasion of cancer cells. The interaction of chemokines and their cognate receptors also plays a critical role in cancer metastasis. Previous results of our laboratory demonstrated that CCR7 is a downstream target for COX-2 and COX-2 up-regulated CCR7 expression via the EP2 and EP4 receptor. We also found that protein kinase A (PKA) and AKT kinase are involved in COX-2-induced CCR7. In this study, we provided further evidences that COX-2 directly stimulates CCR7 expression via promoter activation. Promoter deletion and mutation assay indicated that COX-2 stimulated CCR7 promoter via the Sp1 binding site located at the -61/-52 bp region upstream of the transcription start site. Increase of Sp1 binding to CCR7 promoter by COX-2 was confirmed by chromatin immunoprecipitation (ChIP) assay. Furthermore, knockdown of Sp1 expression resulted in inhibition of PGE2-induced CCR7, and over-expression of Sp1 potently up-regulated CCR7 in MCF-7 cells. In vitro kinase assay indicated that AKT could directly phosphorylate Sp1 at S42, T679 and S698 sites. And the phosphorylation of Sp1 by AKT led to enhanced protein stability and DNA binding affinity of Sp1. The results of immunohistochemistry indicated that CCR7 expression was significantly associated with Sp1 and phosphor-AKT. Taken together, COX-2 may act via the EP receptor/PKA/AKT/Sp1 signaling pathway to stimulate CCR7 expression in breast cancer cells to promote lymphatic spread.

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Phosphatidylcholine (PC) is made in all nucleated mammalian cells via the CDP-choline pathway. Another major pathway for PC biosynthesis in liver is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). We have identified 3T3-L1 adipocytes as a cell culture model that expresses PEMT endogenously. Analysis of the proximal PEMT promoter in 3T3-L1 adipocytes revealed an important regulatory region. Sp1 binds to a GC-rich site within this section of the promoter and inhibits PEMT transcriptional activity. Tamoxifen is an anti-estrogen drug widely used for the treatment of hormone-responsive breast cancer but has a frequent side-effect of increasing accumulation of lipid in the liver (hepatic steatosis). Tamoxifen represses PEMT gene expression by promoting Sp1 binding to the promoter. However, decreased catalytic activity of PEMT was not a major initial contributor to tamoxifen-mediated hepatic steatosis. We found that increased de novo fatty acid synthesis is the primary event which leads to tamoxifen-induced steatosis in mouse liver. Tamoxifen did not significantly alter hepatic fatty acid uptake, triacylglycerol secretion or fatty acid oxidation. Finally, we provide evidence that deletion of the PEMT gene in a well-established mouse model of atherosclerosis (apolipoprotein E deficient) reduces the formation of aortic lesions and prevents the associated development of dilated cardiomyopathy. This beneficial effect is likely due a reduction of atherogenic lipoproteins. These results indicate that treatment strategies aimed at the inhibition of PEMT could prevent the development of atherosclerosis that predisposes individuals to heart failure.

TSG101 is a tumor susceptibility gene exhibits multiple biological functions, including the regulation of vesicular trafficking, transcription, cellular growth and differentiation. The intracellular steady-state level of TSG101 was shown to under stringent control in a narrow range. Either deprivation or overexpression of mouse tsg101 in NIH3T3 cells leads to neoplastic transformation and subsequent tumorigenic potential of the transformed cells. However, the detail mechanism for regulation of TSG101 gene promoter activity is not clear. Our results indicated TSG101 is a housekeeping gene and contains a TATA-less and Sp1 binding site promoter. Here, we demonstrate in vivo binding of Sp1 transcription factor on TSG101 promoter region by chromatin immunoprecipitation(ChIP). In addition, Sp1-associated transcription regulators were purified using DNA affinity precipitation assay (DAPA) method and subjected to two-dimensional gel electrophoresis and the subsequent MALDI-TOF analysis. Our results verify the biding of Sp1 transcription on the DAPA probe containing wildtype but not the mutant Sp1 biding sequence by subsequent western blotting. Our MALDI-TOF analysis of protein spots from two-dimensional gel did not reveal the binding of Sp1 protein, instead the identified a number of cellular proteins, such as U5 small nuclear RNP¡BATP-dependent DNA helicase 2 and actin of unknown significance.

Thioredoxin-1 (Trx-1) is a redox protein that is overexpressed in many tumors where it is associated with tumor growth, inhibited apoptosis and decreased patient survival. Through redox reactions, Trx-1 is able to reduce a number of proteins including transcription factors. Sp1 activation has been implicated in the regulation of many genes involved in cellular growth and survival and its overexpression in certain cancer correlates with decreased patient survival. We demonstrate that Trx-1 is able to activate Sp1 in a redox dependent manner. Trx-1 overexpression increases Sp1 transactivation and DNA binding whereas a redox inactive Trx-1 has no effect on Sp1 DNA binding.Sp1 has been implicated in vascular endothelial growth factor regulation and we have shown that Trx-1 expression results in increased hypoxic VEGF expression and increased tumor permeability in vivo. Trx-1 overexpression results in an increase in VEGF expression that is dependent upon Sp1, as inhibition of Sp1 expression with siRNA prevented the induction of VEGF expression by Trx-1. These results suggest that Trx-1 increases VEGF expression under normoxic conditions through a redox dependent increase in the DNA binding of the Sp1 transcription factor. VEGF regulation by Sp1 could increase angiogenesis in relatively perfused areas contributing to the stimulation of tumor growth by Trx-1.We hypothesized that Trx-1 regulation of Sp1 may be part of the mechanism of Trx-1 induction of cellular growth. Sp1 regulates many genes involved in cellular growth including epidermal growth factor receptor (EGFR) and insulin-like growth factor I receptor (IGF-IR). These two growth factor receptors are important for cellular growth and have been shown to be important therapeutic targets for cancer treatment. We report that treatment with the Trx-1 inhibitor PX-12 results in decreased Sp1 DNA binding as well as decreased Sp1 activation and transactivation of VEGF, EGFR, and IGF-IR. These results indicate that Trx-1 promotes cellular growth and survival, in part, through the redox regulation of Sp1 responsive growth genes EGFR and IGF-IR. Inhibition of Trx-1, via PX-12, results in a decrease in EGFR and IGF-IR expression and suggests a new mechanism by which Trx-1 inhibition is clinically effective for treating cancer.

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