Publications (8)26.06 Total impact
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Article: A half-site of the p53-binding site on the keratin 14 promoter is specifically activated by p63.
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ABSTRACT: Three members of p53 family, p53, p63 and p73, can transactivate their specific target genes through a p53 consensus sequence-binding motif which consists with direct repeats of PuPuPuC(T/A)(T/A)GPyPyPy as a whole-site of p53-binding site. p63, an epidermal stem cells marker, can regulate epidermal development and differentiation, but p53 has no similar biological activity. One isoform of p63, TAp63α, can active an epidermal basal cell marker, keratin 14. However, the p53-binding site does not exist as a whole-site in the K14 promoter region, although it contains three putative p53 half-binding sites at -269 to -1 of the K14 promoter. Two of three putative half-sites of the p53-binding site can be bound by p63α by electrophoresis mobility shift assay and DNA affinity purification assay. Only mutation of the p53 half-binding site at -140 to -131, the TAp63α induced K14 promoter activity can be abolished. This half-site was specifically activated by p63, but not by p53. Once we extend this p53 half-site to a whole p53-binding site in K14 promoter, both p53 and p63 expression vectors can activate its activity. Therefore, we propose that the different length of p53-binding site would determinate the gene regulated by different p53 family proteins.Journal of biochemistry 05/2012; 152(1):99-110. · 1.95 Impact Factor -
Article: p53 acts as a co-repressor to regulate keratin 14 expression during epidermal cell differentiation.
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ABSTRACT: During epidermal cell differentiation, keratin 14 (K14) expression is down-regulated, p53 expression varies, and the expression of the p53 target genes, p21 and 14-3-3σ, increases. These trends suggest that the relative transcriptional activity of p53 is increased during epidermal cell differentiation. To determine the relationship between K14 and p53, we constructed K14 promoters of various sizes and found that wild-type p53 could repress the promoter activity of all of the K14 promoter constructs in H1299 cells. K14-p160 contains an SP1 binding site mutation that prevents p53 from repressing K14 expression. Using a DNA affinity precipitation assay, we confirmed that p53 forms a complex with SP1 at the SP1 binding site between nucleotides -48 and -43 on the K14 promoter. Thus, our data indicate that p53 acts as a co-repressor to down-regulate K14 expression by binding to SP1. Next, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal cell differentiation model to examine the inhibition of K14 expression caused by increased p53 activity. Human ovarian teratocarcinoma C9 cells were treated with TPA to induce differentiation. Over-expression of the dominant negative p53 mutant ΔTAp53, which inhibits p53 activity, prevented the TPA-induced K14 down-regulation in C9 cells. Furthermore, treatment of normal primary human foreskin keratinocytes (PHFK) with the p53 inhibitor pifithrin-α (PFT-α) showed that the inhibition of p53 activity relieves K14 repression during epidermal cell differentiation. Finally, we found that TPA induces the phosphorylation of p53 at residue 378, which enhances the affinity of p53 to bind to Sp1 and repress K14 expression.PLoS ONE 01/2012; 7(7):e41742. · 4.09 Impact Factor -
Article: Functional four-base A/T gap core sequence CATTAG of P53 response elements specifically bound tetrameric P53 differently than two-base A/T gap core sequence CATG bound both dimeric and tetrameric P53.
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ABSTRACT: The consensus sequence of p53 is repeated half sites of PuPuPuC(A/T)(A/T)GPyPyPy. GtAGCAttAGCCCAGACATGTCC is a 14-3-3sigma promoter p53 regulation site; the first core sequence is CAttAG, and the second is CATG. Both mutants GtAGgAttAGCCCAGACATGTCC and GtAGCAttAGCCCAGACATcTCC can be activated by p53 as a 1.5-fold half site. The original p53 regulated site on the 14-3-3sigma promoter is a whole site, and CATTAG is a functional core sequence. The p53-binding affinity and the activity of CATTAG were lower than for the mutant CATATG core sequence. Wild-type p53 acts as a tetramer to bind to the whole site; however, it also can bind to a half site by one of its dimers. Wild-type p53 can only bind to a half site with core sequence CATG but not to CATATG. The 1.5-fold half site or whole site with core sequence CATATG can be bound by wild-type p53. A p53 mutant, A344, forms dimeric p53; it can only bind to CATG, and not to CATATG. Therefore, tetrameric and dimeric p53 can bind to a two-base A/T gap core sequence, but only tetrameric p53 can bind to a four-base A/T gap core sequence.Nucleic Acids Research 03/2009; 37(6):1984-90. · 8.03 Impact Factor -
Article: A method for quantifying melanosome transfer efficacy from melanocytes to keratinocytes in vitro.
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ABSTRACT: Several different in vivo and in vitro bioassays are used to evaluate melanosome transfer efficacy from melanocytes to keratinocytes. However, these methods are complicated and time consuming. Here, we report on a simple, rapid, direct, and reliable in vitro method for observing the process of melanosome transfer from melanocytes to keratinocytes. First, we selected and tested a melanoma cell line RPMI-7951 that can normally synthesize melanin and transfer from mature melanosomes to keratinocytes in vitro. We cocultured these cells with a human ovarian teratoma transformed epidermal carcinoma cell line, which is also capable of accepting melanosomes transferred from melanocytes, as in normal keratinocytes. The cells were cocultured for 24-72 h and double labeled with FITC-conjugated antibody against the melanosome-associated protein TRP-1, and with Cy5-conjugated antibody against the keratinocyte-specific marker keratin 14. The cells were examined by fluorescence microscope and flow cytometry. Melanosome transfer from melanocytes to keratinocytes increased in a time-dependent manner. To verify the accessibility of this method, the melanosome transfer inhibitor, a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and a melanosome transfer stimulator, alpha-melanocyte-stimulating hormone, were added. The serine protease inhibitor decreased melanosome transfer, and alpha-melanocyte-stimulating hormone increased melanosome transfer, in a dose-dependent manner. In conclusion, this is a simple, rapid, and effective model system to quantify the melanosome transfer efficacy from melanocytes to keratinocytes in vitro.Pigment Cell & Melanoma Research 08/2008; 21(5):559-64. · 5.06 Impact Factor -
Article: Intracellular acidification enhances neutrophil phagocytosis in chronic haemodialysis patients: possible role of CD11b/CD18.
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ABSTRACT: We have demonstrated that uraemic neutrophils that exhibit a low intracellular pH (pHi) display enhanced phagocytosis. However, the underlying cellular mechanism is unclear. We used neutrophils from three groups of haemodialysis (HD) patients before dialysis (Groups A, B and C) and also from age- and sex-matched healthy individuals to determine pHi, phagocytosis and expression of CD11b, CD18, CD14 and toll-like receptors (TLR)-2 and TLR-4. The patients were categorized based on three consecutive monthly pre-dialysis plasma bicarbonate concentrations(P(HCO3)) and pH values; Groups A, B and C had a constant pre-dialysis P(HCO3) of </=21, 21-26 and >/=26 mmol/L (mEq/L), respectively. We also studied the effects induced by the correction of metabolic acidosis and monoclonal antibodies (mAbs) against CD11b/CD18 on neutrophils in Group A. Furthermore, we investigated the effect of intracellular acidification on uraemic neutrophils ex vivo. We observed that the neutrophils in Group A exhibited significantly increased phagocytosis and expression of CD11b/CD18 compared with those in Groups B and C. Additionally, our ex vivo studies demonstrated that the mAbs against CD11b/CD18 partially blocked the enhancement of neutrophil phagocytosis in Group A. Moreover, the pHi of uraemic neutrophils is inversely correlated with phagocytosis and expression of CD11b/CD18. HD patients with a low P(HCO3) exhibited low neutrophil pHi that in turn increased the expression of CD11b/CD18 compared with neutrophils with a normal or high pHi. This increased expression of CD11b/CD18 on the uraemic neutrophils may contribute to the pHi-mediated phagocytosis.Nephrology Dialysis Transplantation 06/2008; 23(5):1642-9. · 3.40 Impact Factor -
Article: Autocrine effects of endothelin on in vitro proliferation of vascular smooth muscle cells from spontaneously hypertensive and normotensive rats.
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ABSTRACT: According to previous studies, endothelin-1 (ET-1) is the most potent growth factor in the regulation of vascular smooth muscle cell (VSMC) proliferation in spontaneously hypertensive rats (SHR). To evaluate if the dominant effect of ET-1-induced VSMC proliferation is achieved by autocrine regulation, aortic smooth muscle cells from four-week-old SHR and WKY (Wistar-Kyoto) rats were cultured in 24-well dishes, and the effects of ET-1 on VSMC proliferation were determined by (a) 3H-thymidine incorporation assays with different ET-1 blocking treatments, including a specific anti-ET-1 antibody; BQ-123, an ETA receptor blocker; and BQ-788, an ETB receptor blocker; and (b) examining the ET-1 blockade on the effects of treatment with other growth factors, including thrombin and angiotension II (AT-II). These results demonstrated that the anti-ET-1 antibody, BQ-123, BQ-788, and BQ-123 plus BQ-788 all caused dose-dependent inhibition of proliferation. A 90% inhibitory effect was observed at the maximum doses used except for BQ-123. The ET-1 receptor blockers inhibited thrombin-induced VSMC growth; however, they did not efficiently inhibit AT-II-induced VSMC growth. These results indicate that the autocrine effects of ET-1 play a predominant role in the proliferation of VSMCs from SHR and WKY rats. They also suggest that thrombin-induced VSMC growth is mediated by the autocrine effects of ET-1, and angiotensin II-induced VSMC growth is mediated by other signal pathways.Clinical and Experimental Hypertension 08/2006; 28(5):463-74. · 1.07 Impact Factor -
Article: Multiple epithelial and nonepithelial tumors in hereditary nonpolyposis colorectal cancer: characterization of germline and somatic mutations of the MSH2 gene and heterogeneity of replication error phenotypes.
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ABSTRACT: Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal inherited cancer syndrome characterized by germline plus somatic mutations of DNA mismatch repair genes and familial clustering of cancers of colorectum and other visceral organs. So far, to our knowledge, there has been no proof of nonepithelial tumors in association with HNPCC. Here we report on a MSH2 frameshift HNPCC family with a carrier found to have multiple primary tumors, including endometrial hyperplasia, ovarian adenocarcinoma, skin cavernous hemangioma, and skin dermatofibrosarcoma protuberans (DFSP). We studied the replication error (RER) phenotype in noncoding (Bat-26, Bat-25, D2S123, D5S346, and D17S250) and coding (MSH3, MSH6, BAX, and TGFBR2 genes) DNA sequences, and characterized the germline and somatic mutations of the MSH2 gene in the tumors described above and in endometrial carcinomas from two of her affected siblings. RER was observed in an order of hyperplasic endometrium (6/10 markers), ovarian carcinoma (5/10 markers), endometrial carcinomas (4/9 and 3/10), DFSP (2/9 markers), and cavernous hemangioma (2/10 markers). All the tumors showed the same germline mutation of G5-->G6 frameshift at 183-187 and polymorphism of C1168T in a heterozygous pattern. In an endometrial carcinoma, deletion of the second allele of MSH2 was evident. Heterogeneous RER patterns were noted in multiple primary tumors of the same individual and in premalignant and malignant endometrial tumors from different individuals. The study demonstrated the two hits of the hMSH(2) gene as well as intra- and interindividual variations of RER phenotypes in HNPCC. The first characterized nonepithelial tumors in HNPCC seem to carry a limited panel of RER, including a framesift at the (A)(10) tract of TGFBR2.Cancer Genetics and Cytogenetics 09/2004; 153(2):108-14. · 1.39 Impact Factor -
Article: Coculture of vascular endothelial cells and smooth muscle cells from spontaneously hypertensive rats.
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ABSTRACT: The effect of endothelium-released vasoactive factors on vascular smooth muscle cell (VSMC) proliferation was studied in a coculture system. Isolated aortic endothelial cells and smooth muscle cells from 4-week-old spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) rats were cocultured. After coculture, the VSMC proliferation rate was examined by 3H-thymidine incorporation assay and the levels of the vasoactive factors in medium were determined by enzyme immunoassay (EIA). The results indicate that the proliferation rate of VSMCs in SHR was significantly higher than in WKY rats when VSMCs were cultured alone. When SHR vascular endothelial cells (VECs) were cocultured with VSMCs, the proliferation rate of SHR VSMCs was enhanced; however, there was no growth promoting effect in WKY VSMCs. When WKY VECs were cocultured with VSMCs, no VSMC proliferation effect was observed. When VSMCs were cultured alone, the endothelin-1 (ET-1) secretion in SHR was significantly higher than in WKY rats. When VECs and VSMCs were cocultured, the ET-1 concentration increased in both SHR VEC and WKY VEC coculture groups in a similar manner; but the SHR VECs tended to release more thromboxaneA2 (TXA2) and less PGI2 than WKY VECs. These results suggest that some kind of interaction between SHR VSMCs and SHR VECs is responsible for the high proliferation of SHR VSMCs but not the effects of SHR VECs per se.Clinical and Experimental Hypertension 11/2003; 25(7):413-25. · 1.07 Impact Factor
Top co-authors
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Institutions
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2008–2009
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National Defense Medical Center
- Institute of Life Sciences
Taipei, Taipei, Taiwan -
Shin Kong Wu Ho-Su Memorial Hospital
Taipei, Taipei, Taiwan
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