Jia-Ching Shieh

The clinical implications of MMP-11 and CK-20 expression in human breast cancer

BACKGROUND Tumor invasiveness and metastasis in cancer progression is manifested by epigenetic abnormality. However, it remains unknown whether transcription regulation of matrix metalloproteinase-11(MMP-11) and cytoskeleton-20 (CK-20) genes for the homoeostasis of epithelial/connective interface that can enhance cell dissemination and invasion may act as alternative mutators to tumor clinicopathology. METHODS Paired cancerous and tumor-adjacent normal tissues from 72 breast cancer patients were assayed for the expression of MMP-11 and CK-20 by using real-time RT-PCR. The expression profiles were evaluated for the association with clinicopathological factors. RESULTS Breast tumor tissues displayed higher expression levels of MMP-11 and CK-20 than those of the adjacent non-cancerous tissues. Overexpression of either MMP-11 or CK-20 correlated with patients having poorly differentiated tumors (P(MMP-11)=0.01 and P(CK-20)=0.05) and lymph node metastasis (LNM) (P(MMP-11)=0.004 and P(CK-20)=0.001). A synergistic effect between MMP-11 and CK-20 on risk elevation was significant in patients with advanced tumor stage (OR=2.03, 95%CI=1.10-3.77) and LNM (OR=2.83, 95%CI=1.20-4.71). Additionally, patients lacking progesterone receptor exhibited high expression of MMP-11 and CK-20. CONCLUSION We demonstrate that MMP-11 and CK-20 are probable prognostic markers whose expression reflects the stages of tumor differentiation and LNM of breast cancer.

Identification of Novel Mutations in the KCNQ4 Gene of Patients with Nonsyndromic Deafness from Taiwan

Ion channels play important roles in signal transduction and in the regulation of the ionic composition of intra- and extracellular fluids. Mutations in ion channels have long been thought to be responsible for some forms of hearing loss. Defects in KCNQ4, a voltage-gated potassium channel, are a cause of nonsyndromic sensorineural deafness type 2, an autosomal dominant form of progressive hearing loss. We present data of mutation analysis of KCNQ4 from 185 unrelated Taiwanese probands with nonsyndromic hearing loss. The analysis revealed three novel KCNQ4 mutations and many polymorphisms. The prevalence of KCNQ4 gene mutations in this study was 1.62% (3/185). The mutations include a missense mutation (F182L) and two silent mutations (R216R and T501T). The F182L missense mutation was located in the S3 domain of KCNQ4. The F182 residue of KCNQ4 is highly conserved in KCNQ4 among various species and is less conserved in all members of the KCNQ family. In addition, although R216R is a silent mutation and does not alter the content of amino acid residue, the neural network prediction system revealed that it can potentially create a novel splice donor site during transcription. This mutation might affect the protein structure of KCNQ4 and consequently the normal function of the K+ channel. Our data provide the first comprehensive analysis of the KCNQ4 gene in Taiwanese patients with nonsyndromic deafness.

Association analysis of GRIN1 and GRIN2B polymorphisms and Parkinson's disease in a hospital-based case-control study.

Hyperactivation of N-methyl-d-aspartate receptors (NMDARs) leads to neuronal excitotoxicity and is suggested to play a role in many brain disorders, including Alzheimers disease and schizophrenia. However, the association between polymorphisms in the genes that code for NMDAR subunits, N-methyl-d-aspartate 1 and 2B (GRIN1 and GRIN2B) and Parkinsons disease (PD) remains unclear. In a hospital-based case-control study of PD, DNA samples were collected from 101 PD patients and 205 healthy controls. Genotyping assays were used to screen for polymorphisms in the GRIN1 (rs2301364 T>C, rs28489906 T>C, and rs4880213 T>C) and GRIN2B (C366G, C2664T, and rs1805476 T>G) genes, and logistic regression analysis was then used to assess the association between these single nucleotide polymorphisms (SNPs) and PD susceptibility. None of the 6 SNPs were significantly associated with PD risk on their own. However, in conjunction with putative low-risk genotypes for the GRIN1 gene, the GRIN2BC366G variant was significantly associated with reduced PD risk compared with the homozygous genotype 366CC (OR=0.38, 95%CI=0.17-0.93, P=0.033). A synergistic effect on risk reduction was observed in subjects who carried multiple polymorphisms of GRIN1 and the GRIN2BC366G polymorphism (OR=0.78, 95%CI=0.59-1.02, P(trend)=0.073). Our results suggest that polymorphisms in the GRIN1 and GRIN2B genes may serve as potential biomarkers for a reduced risk of PD among the Chinese population in Taiwan.

Genotype polymorphisms of GGCX, NQO1, and VKORC1 genes associated with risk susceptibility in patients with large-artery atherosclerotic stroke

BACKGROUND Gamma-glutamyl carboxylation, a reaction essential for the biosynthesis of vitamin K-dependent coagulation factors, requires the participation of the gamma-glutamyl carboxylase (GGCX), vitamin K epoxide reductase (VKORC1), and NAD(P)H:quinone oxidoreductase (NQO1). We evaluated the role of these genotype polymorphisms in patients with large-artery atherosclerotic stroke. METHODS In this hospital-based case-control study, 117 patients who were categorized as having large-artery atherosclerotic stroke and 115 age- and gender-matched controls were recruited. Genotyping determination for the GGCX1 (Gln325Arg), NQO1 (Pro187Ser), and VKORC1 (rs9923231) polymorphisms was performed. The associations of genotype with ischemic stroke (IS) risk were examined. RESULTS A higher genotypic frequency of NQO1 C609T was found in the controls than in the patients, manifesting a 0.47-fold risk reduction in IS (95% CI=0.25-0.87). A tendency toward a reduced IS risk was statistically significant in those subjects who carried a greater number of the NQO1, GGCX, and VKORC1 polymorphisms (aOR=0.58, P(trend)=0.005). The synergistic effect of multiple genes on risk reduction was more significant in a subset of patients who were not alcoholics and who were non-smokers (P<0.05). CONCLUSIONS Compartmentation of coagulation factor metabolism may account for the preferential role of NQO1, GGCX, and VKORC1 polymorphisms to lower the risk for large-artery atherosclerotic stroke.

Association of MTHFR, MTR, and MTRR polymorphisms with Parkinson's disease among ethnic Chinese in Taiwan.

BACKGROUND Influence of folate/homocysteine conversion is considered to be important in the pathogenesis of Parkinsons disease (PD). However, association of the folate metabolic pathway gene polymorphisms with PD susceptibility remains unclear. METHODS To test this possibility in PD, we conducted a hospital-based case-control study constituting 211 patients and 218 age- and sex-matched controls of ethnic Chinese in Taiwan. Genotyping assay was performed to screen for polymorphisms of the methylenetetrahydrofolate reductase (MTHFR C677T), methyltetrahydrofolate-homocysteine methyltransferase (MTR A2756G), and 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR A1049G and C1783T) genes and assess the association between these genotype polymorphisms and PD risk using logistic regression analysis. RESULTS Of these four non-synonymous polymorphisms, the MTRR 1049GG variant was significantly associated with PD susceptibility (OR=3.17, 95%CI=1.08-9.35). Furthermore, we stratified our patients based on the MTHFR 677TT genotype in different strata, a significant association between the joint effect of polymorphisms and PD risk was observed in those patients whose genotypes were MTRR A1049G/MTR A2756G or MTRR C1783T/MTR A2756G (P<0.05). CONCLUSION Our findings provide support for the synergistic effects of polymorphisms in the folate metabolic pathway genes in PD susceptibility; the increased PD risk would be more significant in carriers with the polymorphisms of MTHFR, MTR, and MTRR genes.

Novel mutations in the connexin43 (GJA1) and GJA1 pseudogene may contribute to nonsyndromic hearing loss

Connexins (CXs), a large family of membrane proteins, are key components of gap junction channels. Among a cohort of patients with nonsyndromic hearing loss, we have recently identified three novel missense mutations in the GJA1 gene and GJA1 pseudogene (ρGJA1) as likely being causally related to hearing loss. However, the functional alteration of CX43 caused by the mutations of GJA1 and ρGJA1 gene remains unclear. This study compares the intracellular distribution and assembly of three CX43 mutants expressed in HeLa cells with their wild-type (WT) counterparts and the effects of the mutant proteins on those cells. Localization assay of WT CX43 reveals a typical punctuate fluorescence pattern of a gap junction channel between neighboring expression cells. Additionally, immunoblotting analysis of the transfectants confirms the production of mutant proteins, in which their distributions along appositional membranes are determined using immunofluorescent staining procedures. Furthermore, dye transfer assay results demonstrate that gap junctional intercellular communication is less in HeLa cells carrying mutant GJA1 or ρGJA1 gene than in WT-expressing cells. The results of this study suggest that the three mutations in GJA1 or ρGJA1 that we previously reported result in at least partial loss of normal functions carried out by CX43, which may form a basis for the mechanism contributing to hearing loss in patients.

The differential roles of Slit2-exon 15 splicing variants in angiogenesis and HUVEC permeability

Slit2, a secreted glycoprotein, is down-regulated in many cancers. Slit2/Robo signaling pathway plays an important, but controversial, role in angiogenesis. We identified splicing variants of Slit2 at exon 15, Slit2-WT and Slit2-ΔE15, with differential effects on proliferation and invasive capability of lung cancer cells. The aim of this study was to elucidate the differential roles of these exon 15 splicing variants in angiogenesis. Our results revealed that both Slit2-WT and Slit2-ΔE15 inhibit motility of human umbilical vein endothelial cells (HUVECs). The conditioned medium (CM) collected from CL1-5/VC or CL1-5/Slit2-WT lung adenocarcinoma cells blocked HUVEC tube formation and angiogenesis on chorioallantoic membrane (CAM) assay when compared with untreated HUVECs and CAM, respectively. However, CM of CL1-5/Slit2-ΔE15 restored the quality of tubes and the size of vessels. Although both Slit2-WT and Slit2-ΔE15 inhibited permeability induced by CM of cancer cells, Slit2-ΔE15 exhibited stronger effect. These results suggested that Slit2-ΔE15 plays important roles in normalization of blood vessels by enhancing tube quality and tightening endothelial cells, while Slit2-WT only enhances tightening of endothelial cells. It appears that Robo4 is responsible for Slit2 isoform-mediated inhibition of permeability, while neither Robo1 nor Robo4 is required for Slit2-ΔE15-enhanced tube quality. The results of this study suggest that Slit2-ΔE15 splicing form is a promising molecule for normalizing blood vessels around a tumor, which, in turn, may increase efficacy of chemotherapy and radiotherapy.

Dissection of the Candida albicans Cdc4 protein reveals the involvement of domains in morphogenesis and cell flocculation

BackgroundCDC4, which encodes an F-box protein that is a member of the Skp1-Cdc53/Cul1-F-box (SCF) ubiquitin E3 ligase, was initially identified in the budding yeast Saccharomyces cerevisiae as an essential gene for progression through G1-S transition of the cell cycle. Although Candida albicans CDC4 (CaCDC4) can release the mitotic defect caused by the loss of CDC4 in S. cerevisiae, CaCDC4 is nonessential and suppresses filamentation.ResultsTo further elucidate the function of CaCDC4, a C. albicans strain, with one CaCDC4 allele deleted and the other under the repressible C. albicans MET3 promoter (CaMET3 p) control, was made before introducing cassettes capable of doxycycline (Dox)-induced expression of various C. albicans Cdc4 (Ca Cdc4) domains. Cells from each strain could express a specific Ca Cdc4 domain under Dox-induced, but CaMET3-CaCDC4 repressed conditions. Cells expressing domains without either the F-box or WD40-repeat exhibited filamentation and flocculation similarly to those lacking CaCDC4 expression, indicating the functional essentiality of the F-box and WD40-repeat. Notably, cells expressing the N-terminal 85-amino acid truncated Ca Cdc4 partially reverse the filament-to-yeast and weaken the ability to flocculate compared to those expressing the full-length Ca Cdc4, suggesting that N-terminal 85-amino acid of Ca Cdc4 regulates both morphogenesis and flocculation.ConclusionsThe F-box and the WD40-repeat of Ca Cdc4 are essential in inhibiting yeast-to-filament transition and flocculation. The N-terminal region (1–85) of Ca Cdc4 also has a positive role for its function, lost of which impairs both the ability to flocculate and to reverse filamentous growth in C. albicans.

Differential effects of LY294002 and wortmannin on inducible nitric oxide synthase expression in glomerular mesangial cells.

Nitric oxide (NO) that is produced by inducible nitric oxide synthase (iNOS) is associated with the pathophysiology of glomerulonephritis. Numerous studies have focused on the regulation of NO production by iNOS to reduce NO-mediated cytotoxicity. In the present study, we demonstrated the differential effects of two phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, on lipopolysaccharide- (LPS) and interferon (IFN)-γ-induced NO production in a glomerular mesangial cell line, MES-13 cells. At dosages without affecting cell viability of MES-13 cells, 5μM LY294002 showed a more-significant inhibitory effect on LPS/IFN-γ-induced NO production, and iNOS protein and gene expressions than did 1μM wortmannin. Akt phosphorylation in MES-13 cells declined upon the addition of wortmannin, but not upon treatment with LY294002. Suppression of PI3K expression by small interfering (si)RNA exhibited no effect on LPS/IFN-γ-stimulated NO production or iNOS protein expression in MES-13 cells. Neither LY294002 nor wortmannin reduced IFN-γ-induced STAT-1α phosphorylation. LY294002 exhibited a more-significant inhibitory effect on NF-κB luciferase activities than wortmannin in LPS/IFN-γ-stimulated MES-13 cells. Moreover, LY294002, but not wortmannin, accelerated iNOS protein degradation and reduced the iNOS dimer/monomer ratio in MES-13 cells. Although both LY294002 and wortmannin are known as PI3K inhibitors, their differential effects on iNOS expression in MES-13 cells indicate that the effects of LY294002 on inhibiting NF-κB activation and accelerating iNOS protein degradation are through a mechanism independent of PI3K.

Construction of Candida albicans Tet‐on tagging vectors with a Ura‐blaster cassette

It has been difficult to develop molecular tools for studying the fungal pathogen Candida albicans because this species uses a non‐standard genetic code and is diploid without a complete sexual cycle. Vector systems with regulatable promoters to produce conditional mutants, epitope tags for protein detection and recyclable selection markers are useful for functional study of genes. However, most currently available vectors contain only a subset of desired properties, which limits their application. To combine several useful properties in one vector, the vector pTET25 was initially modified into pTET25M, so that the URA3 gene flanked by dpl200 could be used repetitively. To enable more choices for cloning, a multiple cloning site was introduced at both ends of GFP in pTET25M. GFP expression was induced by doxycycline in a dose‐ and time‐dependent manner when the plasmid was introduced into C. albicans with or without URA3. The applicability of the vectors was verified by constructing strains capable of expressing either the N‐terminal GFP fusion of Cdc10 or the C‐terminal GFP fusion of Cdc11. Additionally, by replacing the GFP gene of pTET25M with DNA sequence encoding Cdc10 with an epitope tag of six histidine residues at the C‐terminus, doxycycline‐induced expression of CDC10 was achieved when the expression vector was introduced into C. albicans. This new system allows for inducible expression of a desired C. albicans gene with the advantage of convenience of cloning. It also allows the presence of a recyclable URA3 marker and the detectable expression of fusion or epitope‐tagged protein. Copyright