Biomedical subjects

Yih-Horng Shiao

Publications and source records attributed to Yih-Horng Shiao.

8 recordsLinked to original sources

PubMed2007

Multiplex pyrosequencing for DNA variation analysis.

Pyrosequencing technique has been widely used to perform both single-nucleotide polymorphism detection and quantitative DNA methylation analysis. Simplex Pyrosequencing is sufficient to interrogate more than one polymorphic site if these gene variants are within the reach of the sequencing reaction. For polymorphisms far apart from each other or located on different genes, multiple simplex analyses are required. To reduce the number of simplex reactions, multiplex Pyrosequencing becomes a useful alternative method. The multiplex reaction is performed in the presence of single or multiple templates with several sequencing primers. Factors such as primer selection for the PCR and Pyrosequencing reaction, generation of optimal nucleotide dispensation order, use of internal and external controls, preparation of instrumentation, and Pyrogram interpretation are essential to the success of the multiplexing. In this chapter, the mouse 45S rRNA gene is used to present two general multiplex Pyrosequencing protocols for determining DNA methylation and allele frequency in the spacer promoter region of this gene.

Animals↗

PubMed2006

Effects of selenium supplementation on expression of glutathione peroxidase isoforms in cultured human lung adenocarcinoma cell lines.

Selenium is an essential nutrient, a component of several anti-oxidant enzymes, and a possible factor in cancer risk, including lung cancer. We determined the subtoxic range of selenium concentration (as sodium selenite) required to increase and maintain the expression of anti-oxidant selenoproteins gluthathione peroxidases GPX1 and GPX4 at a constant level in cultures of human lung adenocarcinoma cell lines (H460, H1703 and H1944) and in HPL1D, a non-transformed lung epithelial cell line. Selenium dose-dependently increased GPX1 protein expression 1.8-fold in HPL1D cells and approximately 40-fold in H460 and H1944 cancer cells, with maximum effects at 20-40 nM. GPX4 protein was also increased, but more so in HPL1D (five-fold) than in H460 or H1944 cells (two- to three-fold). GPX1 mRNA showed similar patterns but differences of lesser magnitude. GPX1 protein and activity level was not consistently detectable in H1703 cells, with or without Se supplementation; its mRNA was present but very low. GPX4 protein level was also low in H1703 cells, but was markedly increased by selenium supplementation (48-fold). These results confirm a role for selenium in risk of lung cancer and the independent regulation of GPX1 and GPX4. Characterization of individual tumors with regard to GPX1 and GPX4 levels and regulation might be useful for interpretation of clinical studies on effects of selenium in lung cancer risk.

Adenocarcinoma↗

PubMed2004

Allele-specific germ cell epimutation in the spacer promoter of the 45S ribosomal RNA gene after Cr(III) exposure.

Paternal exposure of mice to Cr(III) causes increased tumor risk in offspring; an epigenetic mechanism has been hypothesized. Representational difference analysis of gene methylation in sperm revealed hypomethylation in the 45S ribosomal RNA (rRNA) gene after Cr(III) exposure, compared with controls. The most striking effects were seen in the rRNA spacer promoter, a region in the intergenic region of rRNA gene clusters that can influence transcription. Methylation of the rRNA spacer promoter has not been studied heretofore. Sperm DNAs from Cr(III)-treated and control mice were modified by the bisulfite method followed by PCR amplification of the spacer promoter, including 27 CpG sites. Cloning and dideoxy sequencing identified sequence variants (T or G at base -2214) in the spacer promoter. The T allele had less DNA methylation than the G allele in control mice (17 of 17 clones vs. 42 of 72 clones, P = 0.0004). In spite of diversity of sperm DNA methylation patterns, the DNA clones from Cr(III)-exposed mice had fewer methylated CpG sites, by an average of 19% (P < 0.0001). This difference was limited to the G allele. The pyrosequencing technique was applied to quantify the percentage of methylation directly from amplified PCR products. Strikingly, for nine CpG sites including the spacer promoter core region, hypomethylation was highly significant in the Cr(III)-treated group (paired T test, P < 0.0001). Thus, one allele of the 45S rRNA spacer promoter is hypomethylated in sperm germ cells after Cr(III) exposure. This epimutation may lead to increase of tumor risk in the offspring.

Alleles↗

PubMed2004

Epigenetic and gene expression changes related to transgenerational carcinogenesis.

Transgenerational carcinogenesis refers to transmission of cancer risk to the untreated progeny of parents exposed to carcinogens before mating. Accumulated evidence suggests that the mechanism of this process is epigenetic, and might involve hormonal and gene expression changes in offspring. To begin to test this hypothesis, we utilized a mouse model (NIH Swiss) in which exposure of fathers to Cr(III) chloride 2 wk before mating can alter incidence of neoplastic and nonneoplastic changes in offspring tissues. Utilizing a MS-RDA approach, we found that the sperm of these fathers had a significantly higher percentage of undermethylated copies of the 45S ribosomal RNA gene (rRNA); this finding was confirmed by bisulfite sequencing. Because gene methylation is a known mechanism of expression control in germ cells, and ribosomal RNA levels have been linked to cancer, these findings are consistent with the hypothesis. Secondly, we observed that offspring of Cr(III)-treated fathers were significantly heavier than controls, and had higher levels of serum T3. Possible effects of T3 levels on gene expression in the offspring were examined by microarray analysis of cDNAs from liver. A total of 58 genes, including 25 named genes, had expression ratios that correlated significantly with serum T3 ratios at P </= 0.001. Some of these genes have potential roles in growth and/or tumor suppression. These results also support the hypothesis of an epigenetic and/or gene expression-based mechanism for transgenerational carcinogenesis. Published 2004 Wiley-Liss, Inc.

Animals↗

PubMed2003

A new reverse transcription-polymerase chain reaction method for accurate quantification.

BACKGROUND: Reverse transcription-polymerase chain reaction (RT-PCR) is a very sensitive technique to measure and to compare mRNA levels among samples. However, it is extremely difficult to maintain linearity across the entire procedure, especially at the step of PCR amplification. Specific genes have been used as baseline controls to be co-amplified with target genes to normalize the amplification efficiency, but development or selection of reliable controls itself has created a new challenge. RESULTS: Here, we describe a new quantitative RT-PCR to compare two mRNA samples directly without the requirement of synthetic control DNAs for reference. First, chimeric RT primers carrying gene-specific and universal PCR priming sequences with or without a linker for size distinction were utilized to generate cDNAs. The size-different cDNAs were then combined in a single reaction for PCR amplification using the same primer set. The two amplified products were resolved and detected with gel electrophoresis and fluorescence imaging. Relative abundance of the two products was obtained after a baseline correction. CONCLUSION: This methodology is simple and accurate as indicated by equal amplification efficiency throughout PCR cycling. It is also easily implemented for many existing protocols. In addition, parameters affecting RT linearity are characterized in this report.

Animals↗

PubMed2003

VHL down-regulation and differential localization as mechanisms in tumorigenesis.

BACKGROUND: The von Hippel-Lindau (VHL) gene has been widely analyzed in many tumors. Early studies in animal tumors suggest that changes in VHL protein level and localization may be also important in tumorigenesis. In this study, we determined the role of VHL protein in human renal cell carcinomas. METHODS: Seventy-five human renal cell carcinomas, predominantly of clear cell type (60 of 75), were examined for VHL protein by immunohistochemistry. The level and pattern of protein expression were then compared to VHL mutations and tumor characteristics. RESULTS: An apparent decline of VHL level (positive in <50% of tumor cells) was observed in 49 (65%) tumors, a change more frequent than VHL mutations (28 of 75) (37%). In tumors, VHL was localized to the cytoplasm and/or the cell membrane. The occurrence of a predominantly membranous signal was significantly associated with missense mutations (9 of 14 tumors with missense mutations versus 14 of 61 tumors with no or nonmissense mutations, P = 0.0025) and tumor stage (23 of 60 tumors with stage TI versus 0 of 15 tumors with TII and TIII, P = 0.0034). CONCLUSION: This study provides the first evidence of the role of VHL protein level and intracellular localization in tumorigenesis in humans.

Antibodies↗

PubMed2003

The von Hippel-Lindau gene and protein in tumorigenesis and angiogenesis: a potential target for therapeutic designs.

The von Hippel-Lindau (VHL) protein is able to suppress tumor growth and to down-regulate many angiogenic factors, and is ubiquitously detected in adult and fetal tissues. This makes VHL an excellent target for therapeutic intervention. Observation of VHL alterations in sporadic tumors has been increasing as a result of examination of abnormalities other than intragenic mutations. These abnormalities include loss of chromosome 3p25, changes in the promoter, down-regulation of transcript, and changes in protein level. This article also presents the finding of differential expression of two common VHL proteins among rat tissues, suggesting tissue- and development-dependent functional difference between these two isoforms. Molecular pathways linking VHL to angiogenesis have been extensively characterized and mechanisms have been proposed to explain how altered VHL leads to tumorigenesis. VHL functions in the presence of oxygen and/or oxygen species. Two strategies are proposed here for anti-tumor and anti-angiogenic treatments of VHL-deficient tumors and those without detectable VHL intragenic mutations. One is to restore wild-type VHL function in VHL-deficient tumors and the other is to enhance wild-type VHL expression and activity in tumors under hypoxic conditions.

Animals↗

PubMed2002

Mutations in the VHL gene from potassium bromate-induced rat clear cell renal tumors.

Potassium bromate (KBrO(3)) is a rat renal carcinogen and a major drinking water disinfection by-product in water disinfected with ozone. Clear cell renal tumors, the most common form of human renal epithelial neoplasm, are rare in animals but are inducible by KBrO(3) in F344 rats. Detection of cytoplasmic periodic acid-Schiff-positive granules in clear cell tumors, indicative of glycogen accumulation, provides evidence of their biochemical similarity to human counterparts. Mutation in the coding region of the von Hippel-Lindau (VHL) gene is frequently detected in human clear cell renal carcinomas. Detection of VHL mutations in KBrO(3)-induced rat renal tumors could enhance the relevancy of these rat renal tumors for human health risk assessment. Formalin-fixed paraffin-embedded control tissues and renal tumors from male F344 rats exposed to KBrO(3) in the drinking water for 2 years were examined microscopically and were microdissected for DNA extraction. The coding sequence and a promoter region of the VHL gene were examined by polymerase chain reaction-single strand conformation polymorphism and/or DNA sequencing. Two of nine clear cell renal tumors carried the same C to T mutation at the core region of the Sp1 transcription factor binding motif in the VHL promoter and one of four untreated animals had C to T mutation outside the highly conserved core region. Mutation in the VHL coding sequence was only detected in one tumor. No VHL mutations were observed in three chromophilic tumors. KBrO(3)-induced rat renal tumors are morphologically similar to their human counterpart but the genetic basis of tumorigenesis is different.

Adenocarcinoma, Clear Cell↗