Expression Levels Of Sp1 Research Articles

Concordant over‐expression of transcription factor Sp1 and vascular endothelial growth factor in extramammary Paget's disease

Specificity protein 1 (Sp1) is a transcription factor and shown to be a sequence-specific DNA-binding protein that activate a broad and diverse spectrum of mammalian gene, such as vascular endothelial growth factor (VEGF) gene. But the expression of Sp1 and VEGF has not previously been investigated in extramammary Paget's disease (EMPD). To investigate the expression of Sp1 and VEGF proteins in EMPD and to assess their relationships and potential contribution to malignant transduction of EMPD, paraffin-embedded EMPD specimens (35 tissue samples from 33 patients with primary EMPD, including two samples of metastatic lymph nodes from two patients) were subjected to immunohistochemical staining for Sp1 and VEGF. All of the 35 EMPD specimens, including all of six invasive EMPD and two metastatic lymph node specimens, showed strong nuclear positive staining for Sp1 and strong cytoplasmic positive staining for VEGF. The expression levels (% positive cells) of Sp1 and VEGF in EMPD were significantly higher than those of normal skin (NS). There was a significantly high correlation between expression levels of Sp1 and VEGF in EMPD. The present study reveals that the concordant over-expression of Sp1 and VEGF may play a pivotal role in the tumorigenesis and further malignant transduction of EMPD.

Therapeutic inhibition of Sp1 expression in growing tumors by mithramycin a correlates directly with potent antiangiogenic effects on human pancreatic cancer

Human pancreatic cancer over expresses the transcription factor Sp1. However, the role of Sp1 in pancreatic cancer angiogenesis and its use as target for antiangiogenic therapy remain unexplored. Archived human pancreatic cancer specimens were used to assess gene expression and microvessel density (MVD) status by immunohistochemistry: Small-interfering RNA (siRNA) was used to determine the impact of altered Sp1 expression on tumor growth and angiogenesis, and mithramycin A (MIT) was used to evaluate Sp1-targeted antiangiogenic treatment of human pancreatic cancer in animal models. The expression level of Sp1 was correlated directly with the MVD status (P < .001) and the expression level of vascular endothelial growth factor (VEGF) (P < .05). Knockdown of Sp1 expression did not affect the growth of pancreatic cancer cells in vitro but inhibited their growth and metastasis in mouse models. This antitumor activity was consistent with the in vitro and in vivo antiangiogenic activity resulting from Sp1 knockdown. Subcutaneous and intraperitoneal injection of MIT significantly suppressed the growth of human pancreatic cancer in mouse models. This tumor suppression was correlated with the suppression of Sp1 expression in growing tumors but not in normal tissues. Moreover, treatment with MIT reduced tumor MVD, which was consistent with the down-regulation of VEGF, platelet-derived growth factor, and epidermal growth factor receptor. Both clinical and experimental evidence indicated that Sp1 is a critical regulator of human pancreatic cancer angiogenesis and the antitumor activity of MIT is a result, at least in part, of the suppression of Sp1 expression and consequent down-regulation the downstream targets of Sp1 that are key to angiogenesis.

The interaction with Sp1 and reduction in the activity of histone deacetylase 1 are critical for the constitutive gene expression of IL-1α in human melanoma cells

A375-6 human melanoma cells are sensitive to the antiproliferative effect of IL-1. After a long period of culturing, we have obtained cells resistant to IL-1. The resistant clone A375-R8 constitutively produced IL-1 alpha. In this study, we identified a sequence, CGCC, located at -48 to -45 upstream of the transcription start site, to be essential for the constitutive IL-1 alpha gene activation. Specificity protein 1 (Sp1) and Sp3 bound to the nucleotide containing the sequence. Although the binding level to the nucleotide and expression level of Sp1 and Sp3 are comparable in A375-R8 and A375-6 cells, transactivation activity of Sp1 is higher in A375-R8 cells as compared with A375-6 cells. Sp3 could not transactivate the IL-1 alpha promoter. These results suggest that Sp1 but not Sp3 is important for IL-1 alpha gene activation. Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), greatly augmented the IL-1 alpha promoter activity in A375-6 cells to the level comparable with that in A375-R8 cells. TSA also induced IL-1 alpha mRNA expression in A375-6 cells. Sp1 and Sp3 bound to HDAC1 in A375-R8 and A375-6 cells. The chromatin immunoprecipitation assay revealed the binding of Sp1 and HDAC1 to the promoter region of the IL-1 alpha gene. The activities of HDAC bound to Sp1 and Sp3, and that of HDAC1 was lower in A375-R8 cells as compared with A375-6 cells. These results indicate that the reduction in the activity and interaction of HDAC1 with Sp1 are critical for the constitutive IL-1 alpha gene expression.

Elevated expression of vascular endothelial growth factor correlates with increased angiogenesis and decreased progression‐free survival among patients with low‐grade neuroendocrine tumors

Vascular endothelial growth factor (VEGF) is a critical proangiogenic factor in solid tumors. However, its expression and role in human neuroendocrine tumor development and progression remains unclear. Using immunohistochemistry, VEGF and Sp1 expression patterns were investigated in 50 cases of human gastrointestinal neuroendocrine tumor having various clinicopathologic characteristics. It was found that strong VEGF expression was detected in tumor cells, whereas no or very weak VEGF expression was detected in stromal cells surrounding or within the tumors. The levels of VEGF expression directly correlated with the expression levels of Sp1 and microvessel density. Strong, weak, and negative VEGF expression was observed in 32%, 54%, and 14% of cases, respectively. Compared with the group with negative VEGF expression, VEGF (weak/strong) expression was associated with metastasis (14% versus 58%; P = .03). The median progression-free survival (PFS) durations of patients with strong and weak VEGF expression were 29 months and 81 months, respectively. With a median follow-up duration of 50 months, the median PFS duration for the group with negative VEGF expression has not been reached. Compared with the log-rank test, VEGF expression was associated with poor PFS (P = .02). Using in vitro and in vivo models, human carcinoid cell lines were treated with bevacizumab, a monoclonal antibody targeting VEGF. Bevacizumab did not inhibit the growth of carcinoid cells in vitro but significantly reduced tumor angiogenesis and impaired tumor growth in animals. The data suggest that overexpression of VEGF promotes the growth of human neuroendocrine tumors in part through up-regulation of angiogenesis.

Synergistic Induction of Folate Receptor β by All-Trans Retinoic Acid and Histone Deacetylase Inhibitors in Acute Myelogenous Leukemia Cells: Mechanism and Utility in Enhancing Selective Growth Inhibition by Antifolates

The folate receptor (FR) type beta is a promising target for therapeutic intervention in acute myelogenous leukemia (AML), owing particularly to its selective up-regulation in the leukemic cells by all-trans retinoic acid (ATRA). Here we show, using KG-1 and MV4-11 AML cells and recombinant 293 cells, that the histone deacetylase (HDAC) inhibitors trichostatin A (TSA), valproic acid (VPA), and FK228 potentiated ATRA induction of FR-beta gene transcription and FR-beta mRNA/protein expression. ATRA and/or TSA did not induce de novo FR synthesis in any of a variety of FR-negative cell lines tested. TSA did not alter the effect of ATRA on the expression of retinoic acid receptor (RAR) alpha, beta, or gamma. Chromatin immunoprecipitation assays indicate that HDAC inhibitors act on the FR-beta gene by enhancing RAR-associated histone acetylation to increase the association of Sp1 with the basal FR-beta promoter. Under these conditions, the expression level of Sp1 is unaltered. A decreased availability of putative repressor AP-1 proteins may also indirectly contribute to the effect of HDAC inhibitors. Finally, FR-beta selectively mediated growth inhibition by (6S) dideazatetrahydrofolate in a manner that was greatly potentiated in AML cells by ATRA and HDAC inhibition. Therefore, the combination of ATRA and innocuous HDAC inhibitors may be expected to facilitate selective FR-beta-targeted therapies in AML.

Regulation of KiSS-1 Metastasis Suppressor Gene Expression in Breast Cancer Cells by Direct Interaction of Transcription Factors Activator Protein-2α and Specificity Protein-1

KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2alpha (AP-2alpha) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2alpha binding elements, AP-2alpha-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2alpha into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2alpha wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2alpha regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2alpha directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2alpha and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2alpha in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis.

Abnormal Sp1 transcription factor expression in Alzheimer disease and tauopathies

Sp1 transcription factor expression was examined by immunohistochemistry, immunofluorescence and confocal microscopy in Alzheimer disease (AD), Pick disease (PiD), progressive supranuclear palsy (PSP), Parkinson disease (PD) and Dementia with Lewy bodies (DLB). Sp1 partly co-localizes with hyper-phosphorylated tau deposits in neurofibrillary tangles, dystrophic neurites of senile plaques and neuropil threads in AD, and in neurons, astrocytes and oligodendrocytes bearing hyper-phosphorylated tau in PiD and PSP. Sp1 is not found in α-synuclein inclusions in PD and DLB. These modifications are not associated with changes in the total expression levels of Sp1, as revealed with gel electrophoresis and Western blotting of brain homogenates. Furthermore, no co-immunoprecipitation of Sp1 and phospho-tau was observed in AD and PiD cases. Since Sp1 binding sites are present in the promoters of several genes involved in amyloid and tau, and Sp1 is regulated by oxidative stress, the present findings suggest that Sp1 deposition in hyper-phosphorylated tau deposits may have functional consequences in the pathology of AD and other tauopathies.

Transcriptional Regulation of ATP2C1 Gene by Sp1 and YY1 and Reduced Function of its Promoter in Hailey–Hailey Disease Keratinocytes

Hailey-Hailey disease (HHD) is a blistering skin disease caused by malfunction of the Ca2+-dependent ATPase, ATP2C1. In this study, key regulatory regions necessary for the expression of the gene encoding human ATP2C1 were investigated. The transient reporter assay demonstrated that region +21/+57 was necessary for activation of the ATP2C1 promoter, and the electrophoretic mobility shift assay demonstrated that the region was recognized by the transcription factors, Sp1 and YY1. In accordance with this result, when Sp1 or YY1 was overexpressed in keratinocytes, an obvious increase in ATP2C1 promoter activity was observed, which was in contrast with the case where a mutant promoter lacking the binding sites for Sp1 and YY1 was used as the reporter. Ca2+-stimulation signal increased nuclear Sp1 proteins and ATP2C1 mRNA levels in normal keratinocytes. In contrast, both these increases were suppressed in keratinocytes from HHD patients. These results indicate that Sp1 and YY1 transactivate the human ATP2C1 promoter via cis-enhancing elements and that incomplete upregulation of ATP2C1 transcription contributes to the keratinocyte-specific pathogenesis of HHD. This is a report describing the regulation of the expression of ATP2C1.

Expression of endothelial nitric oxide synthase correlates with the angiogenic phenotype of and predicts poor prognosis in human gastric cancer

Angiogenesis is a critical aspect of cancer biology and is subject to regulation by multiple molecular pathways. We evaluated the expression of nitric oxide synthase (NOS) III and its relationship with the angiogenic phenotype and expression of the transcription factor Sp1, as well as their effect on survival in patients with gastric cancer. The NOS III expression level and tumor microvessel density (MVD) status were determined via immunohistochemistry, using archived tissue specimens from 86 resected gastric cancer cases; these findings were then correlated with Sp1 expression and clinicopathological features. NOS III protein expression was significantly higher in both primary tumors and lymph node metastases than in normal gastric mucosa. In primary tumors, NOS III expression correlated highly with Sp1 expression (P = 0.001) and MVD status (P = 0.001). Patients with strong Sp1 expression were more likely to have strong NOS III expression (15 times) and a high MVD (7 times) than were those with negative Sp1 expression. In univariate survival analyses, strong NOS III expression (P = 0.042), strong Sp1 expression (P = 0.007), and a high MVD (P = 0.036) were associated with worse survival. However, when patients' NOS III and Sp1 expression levels, MVD status, disease stage, completeness of resection, Lauren's classification, and age were entered in a Cox proportional hazards model, only strong NOS III (P = 0.019) and Sp1 expression (P = 0.029) and advanced disease stage (P = 0.006) were independently prognostic of poor survival. Our results indicated that the expression of NOS III, a potential downstream effector of Sp1, may play an important role in tumor angiogenesis and aggressiveness in gastric cancer.

Physical and Functional Interactions between USF and Sp1 Proteins Regulate Human Deoxycytidine Kinase Promoter Activity

Deoxycytidine kinase (EC 2.7.1.74, dCK) is central to drug activity of anticancer and antiviral agents such as cytosine arabinoside (araC) and gemcitabine. HepG2 hepatocellular carcinoma cells were used to study the transcriptional regulation of dCK. 5'-Deletion and site-directed mutagenesis of the dCK upstream region (positions -464 to -27) confirmed the importance of two GC-boxes (positions -317 to -309 and -213 to -206) and two E-boxes (positions -302 to -297 and -278 to -273). In vitro electromobility shift assays with HepG2 nuclear extracts and in vivo chromatin immunoprecipitation assays with HepG2 chromatin extracts confirmed the presence of bound Sp1/Sp3 and USF1/2. Co-transfections in HepG2 cells showed that USF1 and USF2a stimulated and Sp1 repressed promoter activity from a dCK-luciferase reporter gene construct. In Sp- and USF-null Drosophila Mel-2 cells, both Sp1 and USF1 stimulated dCK promoter activity in a dose-dependent manner, however, both Sp3 and USF2a were effectively inert. Combined Sp1 and USF1 showed additive transactivation at lower concentrations of Sp1. Sp1 was inhibitory at higher levels. Stimulation by combined USF1/USF2a with Sp1 was similar to that for USF1 alone with Sp1, whereas transactivation by Sp1 and USF2a without USF1 was synergistic. Physical interactions between USF and Sp proteins were confirmed by immunoprecipitations with Sp- and USF-specific antibodies. Domain mapping of USF1 and USF2a localized the functional interactions between USF and Sp proteins to the DNA binding domain of USF. Identifying the physical and functional interactions between Sp and USF proteins may lead to a better understanding of the basis for differential expression of the dCK gene in tumor cells and may foster strategies for up-regulating dCK gene expression and improving chemotherapy with araC and gemcitabine.