Identification of a novel SP3 binding site in the promoter of human IGFBP4 gene: role of SP3 and AP-1 in regulating promoter activity in CaCo2 cells

Insulin-like growth factor binding protein 4 (IGFBP4/BP4) gene expression plays an important role in the transition from proliferation to differentiation of a human colon cancer cell line, CaCo2. We recently cloned and identified multiple cis elements (including putative binding sites for activator protein 1 (AP-1) and specificity proteins (Sps) ) in the promoter of human BP4 gene, and measured a significant upregulation of the promoter activity in response to c-Jun. We therefore examined the role of the single AP-1 site (-869/-863) and other cis elements, in regulating the expression of hBP4 gene, in the current studies. Deletion of a 25 bp sequence from -872 to -848, which contains the AP-1 site, significantly reduced BP4 promoter activity by approximately 50%. Surprisingly, mutation of the AP-1 site did not produce significant alteration in the activity of the BP4 promoter. However, mutation of 7 bp (5'-TGCTGCA) at the 3' end of the AP-1 site resulted in significantly decreasing the promoter activity by >50%. Proteins bound to the 25 bp probe (-872/-848) could be supershifted by antibodies specific for JunD and Sp3 in an EMSA. JunD binding was abolished on mutation of the AP-1 site and Sp3 binding was abolished on mutation of the 7 bp at -861/-855; binding of the purified Sp3 protein to the 25 bp probe was similarly abolished on mutation of the newly discovered Sp3 binding site (TGCTGCA). BP4 promoter activity was upregulated in insect cells in response to Sp3 expression, confirming a functional importance of the novel Sp3 binding site. These studies suggest that the Sp3 binding site, rather than the AP-1 site, may be playing a significant role in regulating the expression of IGFBP4 gene in CaCo2 cells.     

Polymorphism in the nuclear factor kappa-B binding promoter region of cyclooxygenase-2 is associated with an increased risk of bladder cancer

Cyclooxygenase-2 (COX-2) expression is mediated by constitutive nuclear factor (NF)-kappaB. The aim of this study was to investigate the association between the germline alteration of the NF-kappaB binding site of COX-2 and the risk of developing various types of human cancers. Using PCR and DNA sequence analysis, we performed a hospital-based case-control study involving various types of human cancers, namely cervical, breast, lung, and bladder cancer. The COX-2 gene was sequenced in 217 Korean individuals (122 cancer patients; 95 non-cancer patients). We identified 2 novel polymorphisms -1166 C-->G and -1186 T-->G, in the NF-kappaB binding promoter region of COX-2. A polymorphism in nucleotide 1186 was found to be associated with an increased risk of bladder cancer (P=0.038). However, in the case of the other cancers, no significant association was found between polymorphisms in the COX-2 promoter region and the risk of cancer. In conclusion, our results suggest that polymorphisms in nucleotide -1186, which is in the NF-kappaB binding promoter region of the COX-2 gene, may be associated with an increased risk of bladder cancer. Further research is needed to investigate the functional implications of the polymorphisms of the COX-2 promoter gene in human cancer.     

人类生存蛋白启动子的克隆及其在淋巴瘤细胞中的转录活性研究

  目的　克隆人类生存蛋白（Survivin）核心启动子,研究Survivin启动子在人类淋巴瘤细胞Ramos和健康人肝脏细胞Chang Liver中的转录活性。方法　以人类肠基因组DNA为模板,PCR扩增Survivin启动子987 bp片段,将其通过酶切位点连入pGL3-Basic载体构建pGL3-Survivin荧光素酶报告基因载体,通过脂质体转染法转染Ramos和Chang Liver 细胞,通过检测荧光素酶表达水平,比较Survivin启动子在这两种细胞中的转录活性。结果　成功克隆出987 bp的Survivin启动子;双酶切、PCR检测和DNA测序证实pGL3-Survivin载体构建成功;荧光素酶活性检查显示：Survivin启动子在Ramos细胞中的转录活性为阳性对照CMV启动子活性的4.5 %,明显高于在Chang Liver细胞中的0.19 %,在淋巴瘤细胞中具有较高特异性,且在淋巴瘤细胞中的活性明显高于阴性对照pGL3-Basic的0.086 %。结论　Survivin启动子在淋巴瘤细胞中具有较高的特异性,可以作为淋巴瘤细胞基因转染的肿瘤特异性启动子使用。     

Reduced expression of insulin-like growth factor binding protein-3 and its promoter hypermethylation in human hepatocellular carcinoma.

Insulin-like growth factor binding protein-3 (IGFBP-3) is postulated to be a mediator of growth suppression signals. Reduced expression of the IGFBP-3 was observed in nine out of 12 human hepatocellular carcinomas (HCC) (75%). Promoter hypermethylation of the IGFBP-3 was detected in four out of 12 HCCs (33%) although mutations were not identified. The expression of IGFBP-3 was restored by the demethylating agent 5-aza-2'-deoxycytidine in HCC cell line with promoter hypermethylation (HepG2). As IGFBP-3 functions like a tumor suppressor gene, it may be used as a therapeutic target for HCC.     

Partial characterization of specific nuclear triiodothyronine binding sites in two transplantable Morris hepatomas in the rat

Binding sites for 3,3' -5-triiodo-L-thyronine are shown to be present in nuclei prepared from either the 5123tc or the 7777 minimal-deviation murine hepatomas. Certain of the apparent in vivo characteristics of these binding proteins in the hepatomas were found to differ from those seen in host liver nuclei. The maximal binding capacities of these binding sites in the tumors were found to be 60% of that in host rat liver nuclei, and the percent-occupancy in vivo somewhat elevated in the tumors. On the other hand, intrinsic affinity constants (Ka) were found similar when comparing the liver and hepatoma nuclei. Also, using DEAE-Sephadex column chromatography, the binding sites in the 7777 tumor were found to co-elute with similar proteins derived from host liver nuclei. It is concluded then that any differences noted between the characteristics of these binding sites in the liver and hepatoma nuclei are on a functional rather than on a structural basis. A possible connection between the lowered levels of these binding sites in hepatoma nuclei and the proliferative rates of these tumor cells is suggested.     

Sensitivity of wild type and mutant ras alleles to Ras specific exchange factors: Identification of factor specific requirements

We have investigated the productive interaction between the four mammalian Ras proteins (H-, N-, KA- and KB-Ras) and their activators, the mammalian exchange factors mSos1, GRF1 and GRP, by using a modified Saccharomyces cerevisiae whose growth is dependent on activation of a mammalian Ras protein by its activator. All four mammalian Ras proteins were activated with similar efficiencies by the individual exchange factors. The H-Ras mutant V103E, which is competent for membrane localization, nucleotide binding, intrinsic and stimulated GTPase activity as well as intrinsic exchange, was defective for activation by all factors tested, suggesting that the integrity of this residue is necessary for catalyzed exchange. However, when other H-Ras mutants were studied, some distinct sensitivities to the exchange factors were observed. GRP-mediated, but not mSos1-mediated, exchange was blocked in additional mutants, suggesting different structural requirements for GRP. Analysis of Ras-mediated gene activation in murine fibroblasts confirmed these results.     

OCT4: A sensitive and specific biomarker for intratubular germ cell neoplasia of the testis.

PURPOSE: OCT4 (POU5F1, OCT3) immunostaining highlights pluripotent cells (embryonal carcinoma and seminoma) in primary testicular germ cell tumors, but its relative usefulness in diagnosing intratubular germ cell neoplasia, unclassified (IGCNU) is not well established. The present study aimed to establish OCT4 as a sensitive and specific maker for IGCNU, a putative precursor for adult germ cell tumors. EXPERIMENTAL DESIGN: We evaluated OCT4 immunostaining in 44 cases of IGCNU from patients who had testicular germ cell tumors. In addition, 27 of the 44 IGCNU sections were also examined with antibodies to placenta-like alkaline phosphatase, the most frequently used immunohistochemical marker for intratubular germ cell neoplasia. Sections from the testes of 10 patients who had undergone orchiectomy for hormonal treatment of prostate cancer and from autopsies of 10 patients without histories of germ cell tumors were also examined for OCT4 immunostaining. The immunoreactivity of the autopsy tissues was determined with vimentin staining, and all were reactive. RESULTS: In all 44 of the cases, antibody to OCT4 marked the nuclei of nearly all of the dysplastic cells of intratubular germ cell neoplasia but not non-neoplastic testicular cells. The staining intensity was strong in every case, and there was little or no background staining. All 20 of the control specimens (10 orchiectomy specimens from prostate cancer patients and 10 testes from autopsies) were completely negative for OCT4. The 27 cases that were stained with antiplacenta-like alkaline phosphatase antibodies showed staining of variable intensity in the areas of intratubular germ cell neoplasia, and there was a high level of background staining artifact. CONCLUSIONS: OCT4 is a sensitive and specific maker for intratubular germ cell neoplasia.     

hTERT启动子的克隆及在MCF7乳腺癌细胞中的特异转录活性

背景与目的：利用肿瘤特异性启动子增强肿瘤细胞中治疗基因的转录选择性是达到基因治疗靶向性的有效途径。人端粒酶催化亚基（hTERT）在大部分肿瘤细胞中呈特异性高表达，有可能成为肿瘤特异性启动子。本文扩增了hTERT基因启动子并分别克隆于报告基因重组质粒pEGFP-1和pGL3-Basic，检测并证实hTERT启动子在乳腺癌细胞MCF7中的特异转录活性。方法：采用套式PCR法扩增hTERT启动子，将其分别克隆到含报告基因增强绿色荧光蛋白（EGFP）的重组质粒pEGFP-1和含Luciferase的重组质粒pGL3-Basic，经脂质体分别转染人乳腺正常上皮细胞系HBL100和乳腺癌细胞MCF7，荧光显微镜观察EGFP的表达情况，并测定两种细胞中荧光素酶转录表达差异。结果：与正常细胞HBL100相比，hTERT启动子在MCF7细胞呈现强的绿色荧光表达；荧光素酶活性检测与其一致，hTERT启动子在MCF7乳腺癌细胞的荧光素酶相对活性RLU／U值（33784）约相当于HBL100细胞中（2400）的15倍。结论：hTERT启动子在乳腺癌MCF7细胞中具有很强的特异性，为进一步开发肿瘤的特异性基因治疗奠定实验基础。     

Studies on the mechanism by which a tumor promoter inhibits binding of epidermal growth factor to cellular receptors

This study analyzes the mechanism by which the tumor promoter 12-0-tetradecanoyl-phorbol 13-acetate (TPA) inhibits binding of epidermal growth factor (EGF) to its membrane receptors in HeLa cells. Kinetic studies indicate that inhibition of EGF binding occurs within a few minutes after exposure of cells to TPA; delayed addition of TPA causes a reduction in previously bound EGF. With prolonged exposure to TPA, the cells become refractory to TPA inhibition of EGF binding. Evidence was obtained that TPA acts by causing the dissociation of EGF-receptor complexes present on the cell surface and not by increasing the proteolytic degradation or internalization of EGF. Evidence that the TPA inhibition of EGF-receptor binding is not a direct consequence of TPA binding to the "active site" of EGF receptors was obtained by the differential effects of pH, temperature or exposure time and that TPA does not inhibit EGF binding when added to isolated plasma membrane fragments. Studies with a variety of inhibitors suggest that the TPA inhibition of EGF-receptor binding does not require macromolecular synthesis, energy metabolism, or cytoskeletal changes. Taken together, our results suggest that TPA inhibition of EGF-receptor binding results from TPA-induced changes in the membrane microenvironment of EGF receptors.