Molecular characterization of G6PD mutations identifies new mutations and a high frequency of intronic variants in Thai females.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked enzymopathy caused by mutations in the G6PD gene. A medical concern associated with G6PD deficiency is acute hemolytic anemia induced by certain foods, drugs, and infections. Although phenotypic tests can correctly identify hemizygous males, as well as homozygous and compound heterozygous females, heterozygous females with a wide range of G6PD activity may be misclassified as normal. This study aimed to develop multiplex high-resolution melting (HRM) analyses to enable the accurate detection of G6PD mutations, especially among females with heterozygous deficiency. Multiplex HRM assays were developed to detect six G6PD variants, i.e., G6PD Gaohe (c.95A>G), G6PD Chinese-4 (c.392G>T), G6PD Mahidol (c.487G>A), G6PD Viangchan (c.871G>A), G6PD Chinese-5 (c.1024C>T), and G6PD Union (c.1360C>T) in two reactions. The assays were validated and then applied to genotype G6PD mutations in 248 Thai females. The sensitivity of the HRM assays developed was 100% [95% confidence interval (CI): 94.40%-100%] with a specificity of 100% (95% CI: 88.78%-100%) for detecting these six mutations. The prevalence of G6PD deficiency was estimated as 3.63% (9/248) for G6PD deficiency and 31.05% (77/248) for intermediate deficiency by phenotypic assay. The developed HRM assays identified three participants with normal enzyme activity as heterozygous for G6PD Viangchan. Interestingly, a deletion in intron 5 nucleotide position 637/638 (c.486-34delT) was also detected by the developed HRM assays. G6PD genotyping revealed a total of 12 G6PD genotypes, with a high prevalence of intronic variants. Our results suggested that HRM analysis-based genotyping is a simple and reliable approach for detecting G6PD mutations, and could be used to prevent the misdiagnosis of heterozygous females by phenotypic assay. This study also sheds light on the possibility of overlooking intronic variants, which could affect G6PD expression and contribute to enzyme deficiency.

Identification of Novel Candidate Biomarkers for Oral Squamous Cell Carcinoma Based on Whole Gene Expression Profiling.

This study aimed to determine the whole gene expression profiles and to ascertain potential biomarkers for 22 oral squamous cell carcinoma (OSCC) among Thai patients using the Illumina Human HT-12, V4.0 Expression BeadChip array. Result indicated 2,724 differential expressed genes composed of 1,560 up-regulated and 1,164 down-regulated genes (unpaired t-test, p-value <0.05; fold change ≥2.0 and ≤2.0). The top 9 up-regulated genes were validated in 39 OSCC cases using TaqMan real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay. Among these, the up-regulation of peptidase inhibitor 3 (PI3) and keratin 17 (KRT17) genes was harbored in all 39 OSCC patients (100%). Likewise, statistical analysis indicated that gene expression in 8 selective genes including keratin 16 (KRT16), keratin 14 (KRT14), keratinocyte differentiation-associated protein (KRTDAP), keratin 6B (KRT6B), PI3, S100 calcium binding protein A7 (S100A7), stratifin (SFN) and keratin 5 (KRT5) was significantly associated with well differentiated OSCC (p-value <0.05). Moreover, high level of KRT17 protein was significantly associated with well differentiated OSCC compared to moderately OSCC (p-value = 0.041). Notably, using nested-PCR analysis indicated all OSCC cases in this study were HPV-free. Especially, KRTDAP, PI3, SFN mRNA expression were first reported among patients with OSCC. Conclusion, the whole transcript expression study and TaqMan real-time qRT-PCR assay were relevant regarding the increase in gene expression in OSCC. In addition, the up-regulation of PI3 and KRT17 might constitute potential candidate molecular biomarkers to diagnose patients with OSCC.

Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorectal cancer risk in Thais: a case-control study.

Folate plays essential roles in DNA synthesis, repair, and methylation; thus, folate status may affect carcinogenesis. Genetics polymorphisms involved in folate metabolisms have been linked with colorectal cancer (CRC) development. Therefore, we hypothesized that low folate status and related genetic polymorphisms are associated with higher risk of CRC. This case-control study enrolled 105 new cases of CRC, 101 of colorectal adenoma (CRA), and 182 controls from hospitals in Bangkok, Thailand, to examine the association between folate status and methylenetetrahydrofolate reductase (MTHFR) 677C > T, methionine synthase (MTR) 2756A > G, and methionine synthase reductase (MTRR) 66A > G with the risk of CRC and CRA. Regarding CRC risk, the lowest quartile group of serum folate and folate intake had higher risk of CRC than the highest quartile group (odds ratio [OR] = 11.45, 95% confidence interval [CI] = 4.43-29.59) and (OR = 10.29, 95% CI = 4.17-25.41). The lowest quartile group of folate intake also had a higher risk of CRA (OR = 5.22, 95% CI = 2.19-6.09). Low red blood cell folate combined with MTHFR 677C > T polymorphism statistically increased CRC risk (OR = 10.00, 95% CI = 1.36-73.42). Low folate status combined with MTR 2756A > G significantly increased CRA risk (OR = 6.43, 95% CI = 1.38-29.94). Moreover, the risk of CRC was elevated with alcohol consumption and low exercise activity when combined with low folate status (P < .05). This study supported the hypothesis that, in Thais, low folate status is associated with a higher risk of CRC, particularly among those with polymorphisms of the MTHFR 677C > T and MTR 2756 A > G genes.

Down-regulation of mitochondrial NADH and cytochrome b gene associated with high tumor stages in head and neck squamous cell carcinoma.

OBJECTIVE: This study aimed to determine mitochondrial mRNA expression levels and the relationships between these expression levels and various adverse clinicopathological characteristics. METHODS: The mRNA expression levels of all 12 genes encoded protein, located on the heavy-strand of mitochondrial DNA including cytochrome b, NADH1, NADH2, NADH3, NADH4, NADH4L, NADH5, ATPase6, ATPase8, cytochrome c oxidase subunit 1, cytochrome c oxidase subunit 2, cytochrome c oxidase subunit 3 were analyzed in 30 head and neck squamous cell carcinoma (HNSCC) and the corresponding normal tissues using reverse transcriptase quantitative real time PCR. Pearson Chi-square test was used to determine the relationships between these expression levels and categorical parameters. RESULTS: The expression levels of 12 mitochondrial mRNAs were observed in all 30 HNSCC patients with down-regulation, ranging from 43.3% to 76.7% and up-regulation, ranging from 10.0% to 36.7%. Furthermore, the number of cases with down-regulations in all 6 NADH and cytochrome b mRNA with TMN stages III and IV were significantly higher than that in stages I and II (p=0.049 and 0.007, respectively). CONCLUSION: Down-regulation of all mitochondrial NADH mRNA as well as mitochondrial cytochrome b mRNA was associated with high tumor stage among HNSCC patients.

Characterization of Plasmodium falciparum ATP-dependent DNA helicase RuvB3.

BACKGROUND: Malaria is one of the most serious and widespread parasitic diseases affecting humans. Because of the spread of resistance in both parasites and the mosquito vectors to anti-malarial drugs and insecticides, controlling the spread of malaria is becoming difficult. Thus, identifying new drug targets is urgently needed. Helicases play key roles in a wide range of cellular activities involving DNA and RNA transactions, making them attractive anti-malarial drug targets. METHODS: ATP-dependent DNA helicase gene (PfRuvB3) of Plasmodium falciparum strain K1, a chloroquine and pyrimethamine-resistant strain, was inserted into pQE-TriSystem His-Strep 2 vector, heterologously expressed and affinity purified. Identity of recombinant PfRuvB3 was confirmed by western blotting coupled with tandem mass spectrometry. Helicase and ATPase activities were characterized as well as co-factors required for optimal function. RESULTS: Recombinant PfRuvB3 has molecular size of 59 kDa, showing both DNA helicase and ATPase activities. Its helicase activity is dependent on divalent cations (Cu2+, Mg2+, Ni+2 or Zn+2) and ATP or dATP but is inhibited by high NaCl concentration (>100 mM). PfPuvB3 is unable to act on blunt-ended duplex DNA, but manifests ATPase activity in the presence of either single- or double-stranded DNA. PfRuvB3.is inhibited by doxorubicin, daunorubicin and netropsin, known DNA helicase inhibitors. CONCLUSIONS: Purified recombinant PfRuvB3 contains both DNA helicase and ATPase activities. Differences in properties of RuvB between the malaria parasite obtained from the study and human host provide an avenue leading to the development of novel drugs targeting specifically the malaria form of RuvB family of DNA helicases.

Differential expression of matrix metalloproteinase-13 in association with invasion of breast cancer.

Matrix metalloproteinase-13 (MMP-13) has a potential role in tumour invasion and metastasis. However, its relevance to the prognosis of human breast cancer is poorly understood. The aim of this study is to investigate the expression patterns of MMP-13 protein and to determine its prognostic value in breast cancer, and to define its relation to the clinicopathological features. Immunohistochemistry analysis of MMP-13 was performed on formalin-fixed, paraffin-embedded sections of cancerous breast tissue (n = 76) and normal breast tissue (n = 20), all of which had clinicopathological information available. Based on the principle of immunoreactivity, the detection of MMP-13 on breast tissue was conducted using monoclonal antibodies against MMP-13. A semi-quantitative scoring system was used to assess the presence of, as well as the cellular localisation of MMP-13. MMP-13 expression was significantly greater in the cancerous breast tissues in comparison to those of normal breast tissues. In addition, high levels of MMP-13 expression were also found to be related to the positive detection of breast cancer cells in lymph nodes-amongst breast cancer patients. The results of this study showed that MMP-13 was frequently present in breast tumours, especially when tumours were accompanied by positive breast cancer cell detection in lymph nodes. This suggests that MMP-13 plays a potentially significant role in breast cancer invasion and metastasis.

Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ.

BACKGROUND: Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase δ is an essential enzyme required for chromosomal DNA replication and repair, and therefore may be a potential target for anti-malarial drug development. However, little is known of the characteristics and function of this P. falciparum enzyme. METHODS: The coding sequences of DNA polymerase δ catalytic subunit (PfPolδ-cat), DNA polymerase δ small subunit (PfPolδS) and proliferating cell nuclear antigen (PfPCNA) from chloroquine- and pyrimethamine-resistant P. falciparum strain K1 were amplified, cloned into an expression vector and expressed in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE and identified by LC-MS/MS. PfPolδ-cat was biochemically characterized. The roles of PfPolδS and PfPCNA in PfPolδ-cat function were investigated. In addition, inhibitory effects of 11 compounds were tested on PfPolδ-cat activity and on in vitro parasite growth using SYBR Green I assay. RESULTS: The purified recombinant protein PfPolδ-cat, PfPolδS and PfPCNA showed on SDS-PAGE the expected size of 143, 57 and 34 kDa, respectively. Predicted amino acid sequence of the PfPolδ-cat and PfPolδS had 59.2 and 24.7 % similarity respectively to that of the human counterpart. The PfPolδ-cat possessed both DNA polymerase and 3'-5' exonuclease activities. It used both Mg(2+) and Mn(2+) as cofactors and was inhibited by high KCl salt (>200 mM). PfPolδS stimulated PfPolδ-cat activity threefolds and up to fourfolds when PfPCNA was included in the assay. Only two compounds were potent inhibitors of PfPolδ-cat, namely, butylphenyl-dGTP (BuPdGTP; IC50 of 38 µM) and 7-acetoxypentyl-(3, 4 dichlorobenzyl) guanine (7-acetoxypentyl-DCBG; IC50 of 55 µM). The latter compound showed higher inhibition on parasite growth (IC50 of 4.1 µM). CONCLUSIONS: Recombinant PfPolδ-cat, PfPolδS and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA increased DNA polymerase activity of PfPolδ-cat. The high sensitivity of PfPolδ to BuPdGTP can be used to differentiate parasite enzyme from mammalian and human counterparts. Interestingly, 7-acetoxypentyl-DCBG showed inhibitory effects on both enzyme activity and parasite growth. Thus, 7-acetoxypentyl-DCBG is a potential candidate for future development of a new class of anti-malarial agents targeting parasite replicative DNA polymerase.

Up-regulation of AKAP13 and MAGT1 on cytoplasmic membrane in progressive hepatocellular carcinoma: a novel target for prognosis.

Hepatocellular carcinoma (HCC) is one of the most common cancers and is associated with high mortality worldwide. The current gold standards for HCC surveillance are detection of serum α-fetoprotein (AFP) and ultrasonography; however, non-specificity of AFP and ultrasonography has frequently been reported. Therefore, alternative tools, especially novel specific tumor markers, are required. In this study, cytoplasmic membrane proteins were isolated from phorbol 12-myristate 13-acetate (PMA)-induced invasive HepG2 cells and identified using nano-scale liquid chromatographic tandem mass spectrometry (NLC-MS/MS) with comparison to non-treated controls. The results showed that two proteins, magnesium transporter protein 1 (MAGT1) and A-kinase anchor protein 13 (AKAP13), were highly expressed in PMA-treated HepG2 cells. This up-regulation was confirmed by real-time RT-PCR, western blot analysis, and immunofluorescent staining studies. Furthermore, evaluation of MAGT1 and AKAP13 expression in clinical HCC tissues by immunohistochemistry suggested that both proteins were strongly expressed in tumor tissues with significantly higher average immunoreactive scores of Remmele and Stegner (IRS) than in non-tumor tissues (P ≤ 0.005). In conclusion, the expression levels of MAGT1 and AKAP13 in HCC may be potential biomarkers for the diagnosis and prognosis of this cancer.

ALCAM is a Novel Cytoplasmic Membrane Protein in TNF-α Stimulated Invasive Cholangiocarcinoma Cells.

BACKGROUND: Cholangiocarcinoma (CCA), or bile duct cancer, is incurable with a high mortality rate due to a lack of effective early diagnosis and treatment. Identifying cytoplasmic membrane proteins of invasive CCA that facilitate cancer progression would contribute toward the development of novel tumor markers and effective chemotherapy. MATERIALS AND METHODS: An invasive CCA cell line (KKU-100) was stimulated using TNF-α and then biotinylated and purified for mass spectrometry analysis. Novel proteins expressed were selected and their mRNAs expression levels were determined by real-time RT-PCR. In addition, the expression of ALCAM was selected for further observation by Western blot analysis, immunofluorescent imaging, and antibody neutralization assay. RESULTS: After comparing the proteomics profile of TNF-α induced invasive with non-treated control cells, over-expression of seven novel proteins was observed in the cytoplasmic membrane of TNF-α stimulated CCA cells. Among these, ALCAM is a novel candidate which showed significant higher mRNA- and protein levels. Immunofluorescent assay also supported that ALCAM was expressed on the cell membrane of the cancer, with increasing intensity associated with TNF-α. CONCLUSIONS: This study indicated that ALCAM may be a novel protein candidate expressed on cytoplasmic membranes of invasive CCA cells that could be used as a biomarker for development of diagnosis, prognosis, and drug or antibody-based targeted therapies in the future.

SLC35B2 expression is associated with a poor prognosis of invasive ductal breast carcinoma.

BACKGROUND: Breast cancer is the most common malignancy in women worldwide, including Thailand, and is a major cause of mortality and morbidity, despite advances in diagnosis and treatment. Novel gene expression in breast cancer is a focus in searches for prognostic biomarkers and new therapeutic targets. MATERIALS AND METHODS: The mRNA expression of novel B4GALT4, SLC35B2, and WDHD1 genes in breast cancer were examined in invasive ductal breast carcinoma (IDC) patients using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). RESULTS: Among these genes, increased expression of SLC35B2 mRNA was significantly associated with TNM stage III+IV of IDC (p<0.001). Hence, up-regulation of SLC35B2 may serve as a prognostic biomarker for poor prognosis, and is also a potential therapeutic target in breast cancer.

Determination of whole transcription profiles and specific pathways in invasive ductal breast carcinoma.

Breast cancer is the most common cancer affecting women worldwide including Thailand. Whole transcription profiles of invasive ductal breast carcinoma (IDC) obtained by oligonucleotide microarray should lead to a better understanding of the molecular basis of IDCs, allow for examination of specific markers for diagnosis, and provide novel targets for therapy. This study aimed to detect the whole transcript expression of approximately 35,000 target genes in Thai breast cancer patients, using Affymetrix GeneChip(®) Exon 1.0 Sense Target Arrays. Analysis revealed that the differential expression profiles of 928 genes (423 up-regulated and 505 down-regulated genes) were 2-fold or greater (unpaired t-test, p < 0.05) in invasive ductal breast cancer, compared with normal tissues. The Gene Ontology (GO) databases support important associations in 17 gene sets with p-value < 1E-10 and ≥ 4-fold changes, involving the tumorigenic pathways of cell cycles, extracellular regions, as well as cellular component organization. Likewise, the TGFBR and IL-6 pathways contain gene expression with statistically significant changes in IDC.

Hypermethylation of suppressor of cytokine signaling 1 in hepatocellular carcinoma patients.

Hepatocellular carcinoma (HCC), the most common primary hepatic tumor, is highly prevalent in the Asia-Pacific region, including Thailand. Many genetic and epigenetic alterations in HCC have been elucidated. The aim of this study was to determine whether aberrant methylation of the suppressor of cytokine signaling 1 gene (SOCS1) occurs in HCCs. Methylation specific-PCR assays were performed to identify the methylation status of SOCS1 in 29 tumors and their corresponding normal liver tissues. An abnormal methylation status was detected in 17 (59%), with a higher prevalence of aberrant SOCS1 methylation significantly correlating with HCC treated without chemotherapy (OR=0.04, 95%CI=0.01-0.31; P=0.001). This study suggests that epigenetic aberrant SOCS1 methylation may be a predictive marker for HCC patients.

Quantitative real-time RT-PCR of ITGA7, SVEP1, TNS1, LPHN3, SEMA3G, KLB and MMP13 mRNA expression in breast cancer.

Breast cancer is the leading cause of cancer deaths among women worldwide, including Thailand. In the present study, the differential mRNA expression of SVEP1, LPHN3, KLB, ITGA7, SEMA3G, TNS1 and MMP13 genes was examined in breast cancer using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). Among these genes, increased LPHN3 and MMP13 mRNA expression levels correlated with axillary-node metastasis (P=0.02). Multiple logistic regression analysis revealed that LPHN3 and MMP13 mRNA expression is significantly associated with axillary node status in breast cancer (P=0.04).

Histological type of intrahepatic cholangiocarcinoma differentiated by genetic alteration from AP-PCR fingerprint.

Cholangiocarcinoma (CCA), the malignant neoplasm of the biliary epithelium, is usually fatal due to difficulty in early diagnosis and lack of availability of effective therapy. The genetic mechanisms involved in the development of CCA are not well understood and only a few cytogenetic studies have been published. In this study, genomic instability in 30 Thai cases of intrahepatic cholangiocarcinoma (ICC) was assessed using an arbitrarily primed- polymerase chain reaction (AP-PCR) method. Genetic alterations were analyzed as banding pattern changes between tumors and corresponding normal DNA. The abnormal band present at the highest frequency (23/30 cases, 77%) appeared with the AO16 primer. Statistical analysis also showed that DNA alteration from this primer was significantly associated with the moderately to poorly differentiated histological type (P=0.038). Kaplan-Meier survival curves showed borderline significance for this DNA aberration (P=0.06 by the log-rank test). This DNA fragment may thus be of use to predict degree of malignancy of the disease.

Ubiquitin-specific protease 14 expression associated with intrahepatic cholangiocarcinoma cell differentiation.

The purpose of this study was to identify the gene alterations amplified from AO16 primer and examine whether the expression patterns of USP14 in clinical specimens from patients with intrahepatic cholangiocarcinoma (ICC) is associated with cancer cells. DNA from tumor and corresponding normal tissues of 52 patients was amplified with 33 arbitrary primers. The DNA fragment that altered most frequently in ICC was cloned, sequenced, and identified by comparison with known nucleotide sequences in the genome database. The DNA copy numbers of the allelic alterations in cholangiocarcinoma were determined by quantitative real-time PCR and interpreted as allelic loss or DNA amplification by comparison with the reference gene. Associations between allelic imbalance and clinicopathological parameters of ICC patients were evaluated by X²-tests. The Kaplan-Meier method was used to analyze survival rates. Immunohistochemically, USP14 showed weak cytoplasmic staining in normal bile duct epithelial cells. It was strongly detected in 21 cancer patients (43.8%). There were correlations between USP14 expression level and the clinicopathological features of ICC, histological grade (P < 0.05). However, there were no significant differences in age, gender, tumor size, metastasis, lymph node metastasis, and staging. USP14 expression was related to cholangiocarcinoma cell differentiation. Due to their emerging role in control of multiple signaling pathways and oncoproteins, USP14 inhibitors may be useful for anticancer agents.

Factors associated with regional bias of pfcrt (plasmodium falciparum chloroquine resistance transporter) haplotypes in Nepal.

Evidences of reappearance of chloroquine sensitive Plasmodium falciparum haplotypes after cessation of chloroquine in many countries provide a rationale for the search of chloroquine sensitive haplotypes in P. falciparum isolates in Nepal where the use of chloroquine for falciparum malaria treatment has been ceased since 1988. P. falciparum chloroquine resistant transporter gene (pfcrt) haplotypes were determined and the factors associated with pfcrt haplotypes in the Eastern and Central regions of Nepal were identified. Blood samples from 106 microscopy-positive falciparum malaria patients (62 from the Eastern and 44 from the Central region) were collected on filter paper. Pfcrt region covering codons 72-76 was amplified by PCR and sequenced. SVMNT haplotype was predominant in the Central region, whereas CVIET haplotype significantly more common in the Eastern region. In multivariable analysis of factors associated with CVIET haplotype, the Eastern region and parasite isolates from patients visiting India within one month are significant at 5% level of significance. These findings suggest that antimalarial pressure is different between Eastern and Central regions of Nepal and there is a need of an effective malaria control program in the border areas between India and Nepal.

Dengue virus type 2 recognizes the carbohydrate moiety of neutral glycosphingolipids in mammalian and mosquito cells.

Dengue viruses infect cells by attaching to a surface receptor which remains unknown. The putative receptor molecules of dengue virus type 2 on the surface of mosquito (AP-61) and mammalian (LLC-MK2) cell lines were investigated. The immunochemical detection and structural analysis of carbohydrates demonstrated that the neutral glycosphingolipids, L-3 (GlcNAcß1-3Manß1-4Glcß1-1'Cer) in AP-61 cells, and nLc(4) Cer (Galß1-4GlcNAcß1-3Galß1-4Glcß1-1'Cer) in LLC-MK2 cells were recognized by the virus. These findings strongly suggest that neutral glycosphingolipids share the key determinant for virus binding and that the ß-GlcNAc residue may play an important role in dengue virus binding to the host cell surface.

DNA amplification on chromosome 13q31.1 correlated with poor prognosis in colorectal cancer.

Colorectal cancer is a leading cause of cancer deaths worldwide. Genetic markers involved in prognosis of colorectal cancer are still being elucidated. In this study, genetic alterations associated with prognosis of colorectal cancer were determined using arbitrarily primed polymerase chain reaction (AP-PCR) and analyzed quantitatively by real-time PCR. Seven different DNA sequences, mapped on chromosomes 13q31.1, 9q31.1, 1q24, 4q31.3, 10q21, 11q13.4, and 13q13.3, were identified. Among these sequences, seven cases (23%) harbored DNA amplification in chromosome 13q31.1, and 9 (29%) and 7 (23%) presented genetic alterations in chromosome 1q24 and 11q13.4, respectively. Multivariate analysis showed that only DNA amplification in chromosome 13q31.1 was associated with poor survival among patients with colorectal cancer, with median survival time for chromosome 13q31.1 amplification versus no amplification of 64 versus 268 weeks (P = 0.001). This genetic alteration may have a prognostic role in colorectal cancer.

Genetic alteration in oral squamous cell carcinoma detected by arbitrarily primed polymerase chain reaction.

Oral cancer ranks as one of the top ten cancers in Thailand. Molecular carcinogenesis of this disease remains unknown. The purpose of this report was to identify the genetic alteration profile in Thai oral squamous cell carcinoma (OSCC) patients using arbitrarily primed PCR and to determine the association between genetic alterations and clinico-pathological characteristics. Band alteration profiles in the 32 OSCC tissues were compared with corresponding normal tissues amplified from 60 arbitrary primers using arbitrarily primed polymerase chain reaction (AP-PCR) were identified with 12 primers. Among these, 45 band patterns presented the alteration ranged from 36% to 88%. Primer AD15 at 750 base pairs (AD15-750 bp) was found to have both the highest band alteration (88%) and the highest band loss (37%). The highest DNA band amplification was found in primer AX11-1300 bp (56%). Primer AX-11 at 1300 base pairs at the altered frequency of 78% was significantly associated with smoking (p=0.007), and primer N20 at 800 base pairs showed association with low grade tumors (p=0.030). Our results indicate that AP-PCR is a useful technique for detect genetic alteration in oral squamous cell carcinoma and provide various genetic alternative data.

Promoter methylation and genetic polymorphism of glutathione S-transferase P1 gene (GSTP1) in Thai breast- cancer patients.

The GSTP1 gene encodes for a detoxification enzyme involved in protecting cells from carcinogens. In breast cancer, GSTP1 polymorphisms may produce lower effective enzyme detoxification properties and GSTP1 promoter hypermethylation may result in inactivation of GSTP1 expression. We therefore hypothesized an influence on progression of breast cancer. To study the effect of GSTP1 polymorphisms and CpG-island hypermethylation on GSTP1 promoter, PCR-RFLP and methylation-specific PCR techniques were used with 41 Thai breast-cancer patients. Associations between the codon 105 (A to G) genetic polymorphism, CpG-island hypermethylation, and clinico-pathological parameters were analyzed. GSTP1 hypermethylation was found in 26% of cases and the GSTP1 polymorphism in 14%. GSTP1 hypermethylation was significantly associated with breast cancer; lymph-node metastasis (P = 0.02) while GSTP1 polymorphism status significantly varied with progesterone receptor positivity (P = 0.04). No association was found between the GSTP1 polymorphism and methylation status. The results indicated that CpG-island hypermethylation of the GSTP1 promoter is associated with a biologically aggressive phenotype, but may not be related to the codon 105 (A to G) gene polymorphism in breast-cancer patients.