Association of glutathione S-transferase omega gene polymorphisms with progression of head and neck cancer.

This study investigated the influence of glutathione S-transferase omega 1 (GSTO1) and GSTO2 gene polymorphisms on susceptibility and aggressiveness of head and neck squamous cell carcinoma (HNSCC). A case-control study consisting of 300 HNSCC cases and 299 age and sex- matched normal control was performed. Genotyping of GSTO1*A140D and GSTO2*N142D polymorphisms was determined using the polymerase chain reaction-restriction fragment length polymorphism method. Our results revealed that the frequencies of GSTO1 and GSTO2 genotypes were not significantly different between HNSCC cases and controls. No significant differences were found in smoking or drinking status between cases and controls. However, HNSCC individuals with the GSTO1*D140 varient were significantly associated with nodal metastasis (OR = 0.53, 95 %CI = 0.31-0.91, P = 0.020) and advanced pathological stage (OR = 0.33,95 %CI = 0.15-0.70, P = 0.032), while no significant association was observed between GSTO2 genotype and clinicopathological features. Therefore, our findings suggest that the GSTO1*D140 variant genotype in individuals might play a protective role against the aggressiveness of HNSCC.

Characteristics and risk factors of Plasmodium falciparum malaria in Eastern and Central Nepal.

BACKGROUND: Very limited information is available on epidemiology of falciparum malaria in Nepal. Such information is very important for malaria control programmes. It is believed that malaria in Eastern region is imported from border districts of India and local transmission follows whereas it is indigenous in Central region. Therefore, the characteristics and risk factors of malaria are believed to be different in Eastern and Central Nepal. OBJECTIVE: The objective of the study is to describe and compare the characteristics and risk factors of falciparum malaria in Eastern and Central Nepal. MATERIALS AND METHODS: This cross-sectional study was conducted in falciparum malaria endemic districts of Eastern and Central Nepal, during the period 2007 to 2008. We identified and collected information from 106 patients (62 from Eastern and 44 from Central region). Patient examination, clinical and laboratory assessment were done and patients were interviewed using structured questionnaire for malaria related characteristics, risk factors and behaviours. RESULTS: There were significant differences in risk factors and characteristics of falciparum malaria in the Central than the Eastern region. In the Central region, male, illiteracy and thatched roof hut were significant risk factors of falciparum malaria patients as compared to the Eastern region. Visits outside within three months, previous malaria within three months, taking antimalarial before confirmatory diagnosis were significantly higher in patients of the Eastern region as compared to the Central region. CONCLUSION: Falciparum malaria in Nepal should not be seen as similar entity, and different strategies for prevention and control is needed for its diverse characteristics and endemicity.

Allelic loss on chromosome 5q34 is associated with poor prognosis in hepatocellular carcinoma.

PURPOSE: To identify and characterize novel genetic alterations in hepatocellular carcinoma (HCC). METHODS: DNA was extracted from 29 HCC and corresponding normal tissues and amplified with 59 different 10-base arbitrary primers. A 550 bp DNA fragment amplified using primer Q-9 and which was present in 19 of 29 cases (66%) was cloned, sequenced, and compared with known nucleotide sequences deposited in Genome database, and quantified by real-time PCR. RESULTS: DNA alterations were found on chromosomes 5q34, 6p25.2 and 8q12.1 in 11 of 29 cases (38%), 7 of 29 cases (24%), and 12 of 29 cases (41%), respectively. Multivariate analysis showed that the allelic loss on chromosome 5q34 was an independent prognostic factor for poor survival of HCC patients, with the median survival time of 19 weeks for allelic loss versus 109 weeks for no allelic loss (P = 0.001). CONCLUSIONS: This study indicates that allelic loss on chromosome 5q34 may be involved in the development of HCC and could be used as a prognostic indicator in HCC patients.

Novel DNA amplification on chromosomes 6q23-24 and 4p15.2 in breast cancer identified by arbitrarily primed polymerase chain reaction.

Breast cancer is the most common cancer among women worldwide. The molecular basis of breast cancer has not yet been fully elucidated. In this report, novel DNA amplification on chromosomes 6q23-24 and 4p15.2 were identified by arbitrarily primed polymerase chain reaction, gene cloning, nucleotide sequencing and identified by comparison with known sequences in genome data base, and quantitated by real-time PCR. Results revealed that 25 of 32 (78.1%) breast cancer cases harbored DNA amplification on chromosomes 6q23-24 and 4p15.2. There was a significant association between increase in tumor size (> 3cm) and DNA amplification on chromosome 6q23-24 (Odds ratio = 13.75, 95% CI = 1.26-350.38, P = 0.018). The results indicated that DNA amplification on chromosome 6q23-24 may be involved in the progression of breast cancer.

Novel hMSH2, hMSH6 and hMLH1 gene mutations and microsatellite instability in sporadic colorectal cancer.

PURPOSE: To detect the hMSH2, hMSH6 and hMLH1 DNA mismatch repair gene mutations and microsatellite instability in somatic colorectal cancer. PATIENTS AND METHODS: The mutations of hMSH2, hMSH6, and hMLH1 genes, including microsatellite instability of BAT-26, BAT-40, D2S123, D5S346 and D17S250 were analyzed in 31 patients with colorectal. RESULTS: The results revealed that eight cases (25.8%) harbored mutations in DNA mismatch repair genes. Of these, five novel mutations including I237V in exon 4 of hMSH2, ins T at codon 1196 in exon 7 of hMSH6, and ins G at codon 154 in exon 6, N158H in exon 6, and del A at codon 257 in exon 9 of hMLH1 were identified. Moreover, several intronic polymorphisms, including c-g transversion at IVS-1 nt211 + 9 of hMSH2, del T in poly T track at IVS-6 nt3559-5, ATCT duplicate in IVS-7 nt 3642 + 35 and t-g transversion at IVS-10 nt4080 + 185 of hMSH6 were demonstrated in these patients. In addition, seven cases (22.5%) exhibited microsatellite instability (MSI). CONCLUSION: These results suggested that the inactivation of DNA mismatch repair genes and microsatellite instability may play a minor role in somatic colorectal cancer development.

HMSH2 gene alterations associated with recurrence of oral squamous cell carcinoma.

Oral cancer, one of the ten most widespread cancers in Thailand, is a major public health problem. The aim of the study was to assess hMSH2 and hMLH1 gene mutations, microsatellite DNA alterations, and investigate the association between these alterations and clinicopathological features of oral squamous cell carcinomas (SCC) in a sample of Thai patients. Microsatellite alterations at D2S391, D3S647, D17S513, and D17S520 were detected at a frequency of 40.6%. Among these alterations, 12.5% exhibited loss of heterozygosity (LOH) at D3S647 and D17S513, while 34.4% exhibited microsatellite instability (MI) at D2S391, D17S513, and D17S520. Polymorphic change in the intronic region of hMSH2 at IVS 1 nt 211+9, c-->g was observed in 50% of cases. Significant correlation was observed between IVS 1 nt 211+9 polymorphism and the recurrence status of the patients (p = 0.030, OR = 10.67). This study demonstrated that the polymorphism of hMSH2 at IVS 1 nt 211+9 (c-->g) was associated with oral cancer recurrence status and could be used as a biomarker for prognosis and follow-up treatment of oral cancer.

Genetic alterations in chromosome 10q24.3 and glutathione S-transferase omega 2 gene polymorphism in ovarian cancer.

The molecular basis of ovarian cancer development has not been fully elucidated. In this study, genetic alterations in ovarian cancer were identified by arbitrarily primed polymerase chain reaction (AP-PCR). A gene in DNA fingerprinting, amplified from primer AE11, was cloned, sequenced, and identified by comparison with known genes in the genome database. Gene amplification in chromosome 10q24.3 was identified and measured by real-time PCR. Three out of 20 cases harbored this gene amplification. This amplified region was identified as IVS-4 of the glutathione-S-transferase Omega 2 (GSTO2) gene. Therefore, the mutations in all 6 exons of the GSTO2 gene were determined. The A to G transition at codon 142 in exon 4 (AAT to GAT, N142D) was observed. The frequency of GSTO2 gene polymorphism was analyzed in 20 ovarian cancers, compared with 41 normal individuals. The gene frequencies of D142 and N142 allele in ovarian cancer cases were 0.3 and 0.7, whereas in normal females, they were 0.2 and 0.8, respectively. The odds ratio of D142 allele in ovarian cancer was 1.73 (95% CI = 0.51-5.89), indicating that this GSTO2 gene polymorphism may be associated with the risk of ovarian cancer.

Purification and characterization of a novel 3'-5' DNA helicase from Plasmodium falciparum and its sensitivity to anthracycline antibiotics.

Plasmodium falciparum has developed resistance to most anti-malarials; therefore, an investigation of potential targets should be performed. DNA helicases are enzymes that catalyse the unwinding of double-stranded DNA to provide single-stranded templates for DNA replication, repair and recombination. In this study, a DNA helicase (PfDH A) was purified from a crude extract of Plasmodium falciparum. DNA helicase activity was measured by assaying unwinding activity. The apparent molecular weight of PfDH A as determined by SDS-PAGE was 90 kDa. PfDH A moved unidirectionally in the 3' -to- 5' direction along the bound strand and preferred a fork-like substrate structure and could not unwind blunt-ended duplex DNA. Unwinding activity required Mg2+ and could be inhibited by 200 mM NaCl or KCl and was dependent on hydrolysis of ATP or dATP. Anthracyclines, including daunorubicin, nogalamycin, doxorubicin, and aclarubicin, inhibited PfDH A activity with IC50 values of 2, 5, 8 and 9 microM, respectively. Based on the results, PfDH A differs from all known human DNA helicases. However, its function and roles in parasite DNA replication need to be elucidated in the future.

Novel DNA amplification on chromosomes 2p25.3 and 7q11.23 in cholangiocarcinoma identified by arbitrarily primed polymerase chain reaction.

PURPOSE: To detect and characterize amplified DNA sequences in cholangiocarcinoma (CCA). PATIENTS AND METHODS: We extracted DNA from tumor and corresponding normal tissues of 30 patients with CCA and amplified with 30 random ten-mer arbitrary primers by the arbitrarily primed polymerase chain reaction (AP-PCR) technique. RESULTS: Our results showed gains of genomic sequences at high frequency. Using the AX-11 arbitrary primer, we determined an amplified DNA fragment occurred frequently in the tumors analyzed. The DNA fragment was isolated and identified as two sequences mapped to chromosomes 2p25.3 and 7q11.23. Specific primers were designed employing these sequences and used for detecting amplification by real-time quantitative PCR. The amplification of the DNA sequences on chromosomes 2p25.3 and 7q11.23 was detected in 10 (33%) and 6 (20%) cases, respectively. Thirteen (43%) cases showed amplification on both or one of the chromosomes. In addition, amplification of the DNA on chromosome 2p25.3 was predominantly observed in poorly differentiated tumors. CONCLUSIONS: Our findings suggest that the novel amplified DNA on chromosomal regions at 2p25.3 and 7q11.23 might be involved in the development and progression of CCA.

Microsatellite alterations in non-small cell lung cancer.

Genetic alterations at 12 dinucleotide repeat loci located on human chromosomes 2, 3, 12, and 17 have been analyzed in non-small cell lung cancer from Thai patients. Seventeen out of 30 cases (57%) harbored the microsatellite alterations. Of the 30 cases, 19 patients had a history of tobacco smoking, of whom 14 (74%) were in the group with microsatellite alterations, whereas 3 out of 11 non-smokers (26%) had these alterations. The frequency of microsatellite alterations among smokers was significantly higher than it was in non-smokers (P = 0.01 Fisher's exact test; odds ratio; 7.47).

Stage specificity of Plasmodium falciparum telomerase and its inhibition by berberine.

Telomerase activity in synchronized Plasmodium falciparum during its erythrocytic cycle was examined using the TRAP assay. Telomerase activity was detected at all stages of the parasite intraerythrocyte development, with higher activity in trophozoite and schizont stages compared with ring form. Berberine, extracted from Arcangelisia flava (L.) Merr., inhibited telomerase activity in a dose-dependent manner over a range of 30-300 microM, indicating that P. falciparum telomerase might be a potential target for future malaria chemotherapy.

Detection of a novel point mutation in the p53 gene in grade II astrocytomas by PCR-SSCP analysis with additional Klenow treatment.

Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with additional Klenow treatment and direct sequencing mutations in the p53 tumor suppressor gene were analyzed from 21 cases of human astrocytomas. Three cases had p53 gene mutations: two of them were glioblastomas showing a point mutation, one in exon 5 and the other in 6. The last one was a gemistocytic astrocytoma showing a point mutation in exon 5. The frequency of p53 gene mutations in the astrocytomas examined was 14.3% (3 out of 21). No SSCP alterations were observed in any of the p53 fragments amplified from WHO grade I pilocytic astrocytomas and WHO grade III anaplastic astrocytomas. Further examination by direct sequencing showed that two mutations of glioblastomas had single-base substitutions resulting in silent and missense mutations, whereas one of the gemistocytic astrocytomas had a double-base substitution resulting in a missense mutation. The present studies revealed that all mutations were located outside the hot spots and, interestingly, one of them disclosed a missense mutation in exon 5 at codon 166, which was first detected in a grade II astrocytoma (gemistocytic type). It is possible that the missense mutation at this codon may be associated with special risk factors for the development of astrocytic tumors in Thai patients.

Molecular characterization of mitochondria in asexual and sexual blood stages of Plasmodium falciparum.

Molecular mechanisms that regulate gene expression during development of asexual stage to sexual stage of Plasmodium falciparum in the human erythrocyte are largely unknown. There were apparent variations in ultrastructural characteristics of the mitochondrion between the two developing stages. The asexual stage's mitochondrion had developed less than that of the sexual stage. The respiratory complexes of the mitochondrial electron transport system in the asexual stage were approximately 8-10 times less active than those in the sexual stage. Using quantitative polymerase chain reaction to amplify the cytochrome b gene encoding a subunit of mitochondrial cytochrome c reductase, the amount of the cytochrome b gene of the sexual stage was calculated to be approximately 3 times higher than that obtained from the asexual stage. Moreover, using quantitative reverse-transcription polymerase chain reaction, a relatively high level of approximately 1.3-kb transcript mRNA of the cytochrome b gene was observed in the sexual stage compared to the asexual stage. A known single-copy chromosomal dihydrofolate reductase gene was found to have a similar amount in the two stages. These results suggest that the copy number of the mitochondrial gene, including transcriptional and translational mechanisms, plays a major regulatory role in differential expression during the development of the asexual to sexual stage of P. falciparum in the human cell.

p53 gene mutations in non-small cell lung cancer from Thai patients.

Paraffin embedded tissues from twenty-two Thai patients with non-small cell lung cancer were studied for p53 gene mutations in exon 5 to 8 using polymerase chain reaction and single-stranded conformation polymorphism (PCR-SSCP) followed by thermal cycle sequencing. Results showed that point mutations in this region of p53 gene were present in 3 cases. One harboured the base change from GAC to AAC at codon 281, changing amino acid from aspartic acid to asparagine, whilst the other cases were transversion of AAA (lysine) to ACA (threonine) at codon 292. All subjects with p53 mutation had a past history of tobacco smoking.

K-ras oncogene and p53 gene mutations in cholangiocarcinoma from Thai patients.

Paraffin embedded tissues from twenty Thai patients with intrahepatic cholangiocarcinomas were studied for K-ras gene mutations at codon 12, 13 and 61 and for p53 gene mutations in exon 5 to 8 using polymerase chain reaction and thermal cycle sequencing. Results showed that point mutations at these regions in K-ras oncogene were not present in all the samples. One case harbored a p53 gene mutation in codon 282 in exon 8, CGG (arginine) to TGG (tryptophan), but the mutation was not found in other patient's tissues with similar histological features.

Cancer genes and cholangiocarcinoma.

Genes involved in cancer development include oncogenes and tumor suppressor genes. Ras oncogene and mutations in p53 tumor suppressor gene are commonly found in many types of cancer. In Thai patients with cholangiocarcinoma ras oncogenes occur less frequently than in other ethnic groups and furthermore, p53 mutations also occur with lower incidence when compared with Japanese subjects. It is unclear at this time the basis for these differences.

Mitochondrial cytochrome b gene in two developmental stages of human malarial parasite Plasmodium falciparum.

The cytochrome b gene of the mitochondrial ubiquinol-cytochrome c reductase (complex III of electron transport chain) was characterized in two developmental stages of human malarial parasite cultivated in vitro. The cytochrome b gene spanning the nucleotide position 4691 to 5930 in 6-kb mitochondrial DNA from gametocytic (sexual) and intraerythrocytic (asexual) stages of Plasmodium falciparum (a T9,94 mutant line) were in vitro amplified from total DNA using polymerase chain reaction (PCR). It was found that the parasites from both stages contained the PCR product approximately 1.2 kb in length that was localized in mitochondria. The nucleotide sequences of cytochrome b gene at Qi/quinone binding site from both stages were analyzed using thermal cycle sequencing and were found to be the same. The amount of this gene from both stages of the parasite were determined by using the quantitative PCR method. The results showed that the amount of the cytochrome b gene produced from the sexual stage was seven times higher than that obtained from the asexual stage. Our results would provide basic information on the regulation of cytochrome b and the 6-kb mitochondrial DNA during growth and development of the sexual and asexual stages of the malarial parasite in the mammalian host.

Molecular basis of beta (0)-thalassemia/HbE disease in Thailand.

The molecular basis of beta(0)-thalassemia/HbE disease in 30 Thai patients was investigated using DNA amplification and dot-blot hybridization with a number of allele specific oligonucleotide probes. The mutations identified were 17 cases of 4 base-pair deletion at codons 41-42, 4 cases of amber mutation at codon 17, and one case each of an ochre mutation at codon 35, a single base substitution at position 5 of IVS-1, and a single base substitution at position 654 of IVS-2.