Integrated Redox Proteomic Analysis Highlights New Mechanisms of Sensitivity to Silver Nanoparticles.

Silver nanoparticles (AgNPs) are widely used nanomaterials in both commercial and clinical biomedical applications, but the molecular mechanisms underlying their activity remain elusive. In this study we profiled proteomics and redox proteomics changes induced by AgNPs in two lung cancer cell lines: AgNPs-sensitive Calu-1 and AgNPs-resistant NCI-H358. We show that AgNPs induce changes in protein abundance and reversible oxidation in a time and cell-line-dependent manner impacting critical cellular processes such as protein translation and modification, lipid metabolism, bioenergetics, and mitochondrial dynamics. Supporting confocal microscopy and transmission electron microscopy (TEM) data further emphasize mitochondria as a target of AgNPs toxicity differentially impacting mitochondrial networks and morphology in Calu-1 and NCI-H358 lung cells. Proteomics data are available via ProteomeXchange with identifier PXD021493.

Triphenylphosphonium-Derived Protein Sulfenic Acid Trapping Agents: Synthesis, Reactivity, and Effect on Mitochondrial Function.

Redox-mediated protein modifications control numerous processes in both normal and disease metabolism. Protein sulfenic acids, formed from the oxidation of protein cysteine residues, play a critical role in thiol-based redox signaling. The reactivity of protein sulfenic acids requires their identification through chemical trapping, and this paper describes the use of the triphenylphosphonium (TPP) ion to direct known sulfenic acid traps to the mitochondria, a verified source of cellular reactive oxygen species. Coupling of the TPP group with the 2,4-(dioxocyclohexyl)propoxy (DCP) unit and the bicyclo[6.1.0]nonyne (BCN) group produces two new probes, DCP-TPP and BCN-TPP. DCP-TPP and BCN-TPP react with C165A AhpC-SOH, a model protein sulfenic acid, to form the expected adducts with second-order rate constants of k = 1.1 M-1 s-1 and k = 5.99 M-1 s-1, respectively, as determined by electrospray ionization time-of-flight mass spectrometry. The TPP group does not alter the rate of DCP-TPP reaction with protein sulfenic acid compared to dimedone but slows the rate of BCN-TPP reaction compared to a non-TPP-containing BCN-OH control by 4.6-fold. The hydrophobic TPP group may interact with the protein, preventing an optimal reaction orientation for BCN-TPP. Unlike BCN-OH, BCN-TPP does not react with the protein persulfide, C165A AhpC-SSH. Extracellular flux measurements using A549 cells show that DCP-TPP and BCN-TPP influence mitochondrial energetics, with BCN-TPP producing a drastic decrease in basal respiration, perhaps due to its faster reaction kinetics with sulfenylated proteins. Further control experiments with BCN-OH, TPP-COOH, and dimedone provide strong evidence for mitochondrial localization and accumulation of DCP-TPP and BCN-TPP. These results reveal the compatibility of the TPP group with reactive sulfenic acid probes as a mitochondrial director and support the use of the TPP group in the design of sulfenic acid traps.

Genome-wide somatic mutation analysis of sinonasal adenocarcinoma with and without wood dust exposure.

BACKGROUND: Sinonasal adenocarcinoma is a rare cancer, encompassing two different entities, the intestinal-type sinonasal adenocarcinoma (ITAC) and the non-intestinal-type sinonasal adenocarcinoma (non-ITAC). Occurrence of ITAC is strongly associated with exposure to hardwood dusts. In countries with predominant exposure to softwood dust the occurrence of sinonasal adenocarcinomas is lower and the relative amount of non-ITACs to ITACs is higher. The molecular mechanisms behind the tumorigenic effects of wood dust remain largely unknown. METHODS: We carried out whole-genome sequencing of formalin-fixed paraffin-embedded (FFPE) samples of sinonasal adenocarcinomas from ten wood dust-exposed and six non-exposed individuals, with partial tobacco exposure data. Sequences were analyzed for the presence of mutational signatures matching COSMIC database signatures. Driver mutations and CN variant regions were characterized. RESULTS: Mutation burden was higher in samples of wood dust-exposed patients (p = 0.016). Reactive oxygen species (ROS) damage-related mutational signatures were almost exclusively identified in ITAC subtype samples (p = 0.00055). Tobacco smoke mutational signatures were observed in samples of patients with tobacco exposure or missing information, but not in samples from non-exposed patients. A tetraploidy copy number (CN) signature was enriched in ITAC subtype (p = 0.042). CN variation included recurrent gains in COSMIC Cancer Gene Census genes TERT, SDHA, RAC1, ETV1, PCM1, and MYC. Pathogenic variants were observed most frequently in TP53, NF1, CHD2, BRAF, APC, and LRP1B. Driver mutations and copy number gains did not segregate by subtype. CONCLUSIONS: Our analysis identified distinct mutational characteristics in ITAC and non-ITAC. Mutational signature analysis may eventually become useful for documentation of occupation-related cancer, while the exact mechanisms behind wood dust-driven carcinogenesis remain elusive. The presence of homologous recombination deficiency signatures implies a novel opportunity for treatment, but further studies are needed.

Gymnotic uptake of AntimiRs alter microRNA-34a levels in 2D and 3D epithelial cell culture.

MicroRNAs are attractive therapeutic targets in many diseases, including chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. Among microRNA inhibitors antimiRs have been proven successful in lowering aberrant microRNA levels in the clinic. We present a set of antimiRs targeting miR-34a, which has been shown to be dysregulated in chronic lung diseases. The tool compounds were taken up by a bronchial epithelial cell line and primary human bronchial epithelial cells, followed by efficient knockdown of miR-34a. Similar results were observed in 3D differentiated primary human bronchial epithelial cells cultured at the air-liquid interface. Varying chemical properties of antimiRs had significant impact on cellular uptake and potency, resulting in effective tool compounds for use in lung-relevant cellular systems. This report demonstrates gymnotic antimiR uptake and activity in 3D epithelial cell culture after apical administration, mimicking inhalation conditions.

Occurrence of Sinonasal Intestinal-Type Adenocarcinoma and Non-Intestinal-Type Adenocarcinoma in Two Countries with Different Patterns of Wood Dust Exposure.

Sinonasal intestinal-type adenocarcinoma is strongly associated with hardwood dust exposure. Non-intestinal-type adenocarcinoma is a rarer and less well-known subtype considered not to be related with wood dust exposure. We determined the relative numbers of these two tumor types in 56 sinonasal adenocarcinoma patients in France and Finland, relating them with carefully assessed wood dust exposure histories. Diagnostic workup including immunohistochemistry for the intestinal markers CDX2 and CK20 indicated that the proportions of the two tumors differed significantly between France and Finland. In Finnish samples non-intestinal adenocarcinomas were more common than intestinal-type adenocarcinomas (12 non-intestinal vs. nine intestinal), while in the French samples the reverse was true (six non-intestinal vs. 29 intestinal). Such remarkably dissimilar occurrence of these tumors in France and Finland presumably reflects different pathogenetic circumstances in the two countries, and perhaps their different patterns of wood dust exposure. In France the main source of wood dust is from hardwoods. In Finland it is derived from softwoods. This is the first systematic comparison of the occurrence of intestinal-type adenocarcinoma and non-intestinal-type adenocarcinoma in two countries with different wood usage. It appears to be the first systematic study on differences in wood dust exposure between intestinal-type adenocarcinoma and non-intestinal-type adenocarcinoma.

Mutations in TP53 tumor suppressor gene in wood dust-related sinonasal cancer.

The causal role of work-related exposure to wood dust in the development of sinonasal cancer has long been established by numerous epidemiologic studies. To study molecular changes in these tumors, we analyzed TP53 gene mutations in 358 sinonasal cancer cases with or without occupational exposure to wood dust, using capillary electrophoresis single-strand conformation polymorphism analysis and direct sequencing. A significant association between wood-dust exposure and adenocarcinoma histology was observed [adjusted odds ratio (OR) 12.6, 95% confidence interval (CI), 5.0-31.6]. TP53 mutations occurred in all histologies, with an overall frequency of 77%. TP53 mutation positive status was most common in adenocarcinoma (OR 2.0, 95% CI, 1.1-3.7; compared with squamous cell carcinoma), and mutation positivity showed an overall, nonsignificant association with wood-dust exposure (OR 1.6, 95% CI, 0.8-3.1). Risk of TP53 mutation was significantly increased in association with duration (> or =24 years, OR 5.1, 95% CI, 1.5-17.1), average level (>2 mg/m(3); OR 3.6, 95% CI, 1.2-10.8) and cumulative level (> or =30 mg/m(3) x years; OR 3.5, 95% CI, 1.2-10.7) of wood-dust exposure; adjustment for formaldehyde affected the ORs only slightly. Smoking did not influence the occurrence of TP53 mutation; however, it was associated with multiple mutations (p = 0.03). As far as we are aware, this is the first study to demonstrate a high prevalence of TP53 mutation-positive cases in a large collection of sinonasal cancers with data on occupational exposure. Our results indicate that mutational mechanisms, in particular TP53 mutations, are associated with work-related exposure to wood dust in sinonasal cancer.

Profile of TP53 gene mutations in sinonasal cancer.

Genetic alterations underlying the development of the cancer of the nose and paranasal sinuses (sinonasal cancer, SNC), a rare cancer that can be included in the group of head and neck cancers, are still largely unknown. We recently reported that TP53 mutations are a common feature of SNC, with an overall frequency of 77%, and they show association to adenocarcinoma and wood-dust exposure [15]. In this study, we report in detail the sequence change for 159 TP53 mutations identified by direct sequencing. More than half of the mutations (60%, 95/159) were missense mutations; there were also 28 (18%) frameshift or nonsense mutations, and 36 (23%) intronic or silent mutations. In coding region, the most common base change detected was C-->T transition (43/125; 34% of base changes in the coding region). G-->T transversions occurred at a frequency of 10% (12/125), which is less than reported in mutation databases for head and neck squamous cell carcinoma (24%). Characteristically, in our SNC series, the mutations were scattered over a large number of codons, codon 248 being the most frequent target of base substitution. Codon 135 was the second most frequently mutated codon; this nucleotide position has not been reported before as frequently mutated in head and neck cancer or human cancer in general. About half of all tumours with TP53 mutations carried more than one mutation. Interestingly, 86% (19/22) of the silent mutations detected had occurred in tumours with multiple mutations.

Relationship between TP53 tumour suppressor gene mutations and smoking-related bulky DNA adducts in a lung cancer study population from Hungary.

Lung cancer rate in Hungary is one of the highest in the world among men and also very high among women, for reasons not clearly understood yet. The aim of the study was to explore characteristics of DNA damage and TP53 gene mutations in lung cancer from Hungary. Tissue samples from 104 lung resections for lung cancer patients, both men and women, operated on for non-small cell lung cancer, specifically, primary squamous cell carcinoma or adenocarcinoma were studied. Of the cases, 37% smoked up to the surgery, 24% stopped smoking within 1 year before the surgery, 26% stopped smoking more than a year before the surgery and 13% never smoked. TP53 mutations were detected by denaturant gradient gel electrophoresis, automated capillary electrophoresis single-strand conformation polymorphism and sequencing. Bulky DNA adduct levels were determined by (32)P-post-labelling in non-tumorous lung tissue. In total, 45% (47/104) of the cases carried TP53 mutation. The prevalence of TP53 mutations was statistically significantly associated with duration of smoking, tumour histology and gender. Smokers had approximately twice as high bulky adduct level as the combined group of former- and never-smokers (10.9 +/- 6.5 versus 5.5 +/- 3.4 adducts/10(8) nucleotides). The common base change G --> T transversion (8/43; 19%) was detected exclusively in smokers. For the first time, we demonstrate that most carriers of G --> T transversions had also a high level of bulky DNA adducts in their non-tumourous lung tissue. Our study provides evidence for a high burden of molecular alterations occurring concurrently in the lung of lung cancer patients.

Silver Nanoparticles Induce Mitochondrial Protein Oxidation in Lung Cells Impacting Cell Cycle and Proliferation.

Silver nanoparticles (AgNPs) are widely used nanomaterials in both commercial and clinical biomedical applications, due to their antibacterial properties. AgNPs are also being explored for the treatment of cancer in particular in combination with ionizing radiation. In this work, we studied the effects of AgNPs and ionizing radiation on mitochondrial redox state and function in a panel of lung cell lines (A549, BEAS-2B, Calu-1 and NCI-H358). The exposure to AgNPs caused cell cycle arrest and decreased cell proliferation in A549, BEAS-2B and Calu-1, but not in NCI-H358. The mitochondrial reactive oxygen species (ROS) and protein oxidation increased in a time- and dose-dependent manner in the more sensitive cell lines with the AgNP exposure, but not in NCI-H358. While ionizing radiation also induced changes in the mitochondrial redox profiles, in general, these were not synergistic with the effects of AgNPs with the exception of NCI-H358 and only at a higher dose of radiation.

Targeting NAD+ Metabolism to Enhance Radiation Therapy Responses.

Nicotinamide adenine dinucleotide (NAD+) metabolism is integrally connected with the mechanisms of action of radiation therapy and is altered in many radiation-resistant tumors. This makes NAD+ metabolism an ideal target for therapies that increase radiation sensitivity and improve patient outcomes. This review provides an overview of NAD+ metabolism in the context of the cellular response to ionizing radiation, as well as current therapies that target NAD+ metabolism to enhance radiation therapy responses. Additionally, we summarize state-of-the-art methods for measuring, modeling, and manipulating NAD+ metabolism, which are being used to identify novel targets in the NAD+ metabolic network for therapeutic interventions in combination with radiation therapy.

Peroxiredoxins in Cancer and Response to Radiation Therapies.

Peroxiredoxins have a long-established cellular function as regulators of redox metabolism by catalyzing the reduction of peroxides (e.g., H2O2, lipid peroxides) with high catalytic efficiency. This activity is also critical to the initiation and relay of both phosphorylation and redox signaling in a broad range of pathophysiological contexts. Under normal physiological conditions, peroxiredoxins protect normal cells from oxidative damage that could promote oncogenesis (e.g., environmental stressors). In cancer, higher expression level of peroxiredoxins has been associated with both tumor growth and resistance to radiation therapies. However, this relationship between the expression of peroxiredoxins and the response to radiation is not evident from an analysis of data in The Cancer Genome Atlas (TCGA) or NCI60 panel of cancer cell lines. The focus of this review is to summarize the current experimental knowledge implicating this class of proteins in cancer, and to provide a perspective on the value of targeting peroxiredoxins in the management of cancer. Potential biases in the analysis of the TCGA data with respect to radiation resistance are also highlighted.

COX-2 and p53 in human sinonasal cancer: COX-2 expression is associated with adenocarcinoma histology and wood-dust exposure.

The causal role of wood-dust exposure in sinonasal cancer (SNC) has been established in epidemiological studies, but the mechanisms of SNC carcinogenesis are still largely unknown. Increased amounts of COX-2 are found in both premalignant and malignant tissues, and experimental evidence link COX-2 to development of cancer. Many signals that activate COX-2 also induce tumor suppressor p53, a transcription factor central in cellular stress response. We investigated COX-2 and p53 expressions by immunohistochemistry in 50 SNCs (23 adenocarcinomas, and 27 squamous cell carcinomas (SCC); 48 analyzed for COX-2; 41 for p53). Occupational histories and smoking habits were available for majority of the cases. Most of the adenocarcinoma cases with exposure history data had been exposed to wood dust at work in the past (88%, 14/16). For smokers, 63% (12/19) presented with SSC, whereas 64% (7/11) of nonsmokers displayed adenocarcinoma. COX-2 was expressed at higher levels in adenocarcinoma as compared to SSC (p < 0.001). COX-2 expression showed significant association with occupational exposure to wood dust (p = 0.024), and with nonsmoking status (p = 0.001). No statistically significant associations between the exposures and p53 accumulation were found; however, the p53 accumulation pattern (p = 0.062 for wood dust exposure) resembled that of COX-2 expression. In summary, our findings show increased COX-2 expression in SNC adenocarcinoma with wood dust exposure, suggesting a role for inflammatory components in the carcinogenesis process. In contrast, SCCs predominated among smokers and expressed COX-2 rarely; this may suggest at least partially different molecular mechanisms.

K-ras mutations in sinonasal cancers in relation to wood dust exposure.

BACKGROUND: Cancer in the sinonasal tract is rare, but persons who have been occupationally exposed to wood dust have a substantially increased risk. It has been estimated that approximately 3.6 million workers are exposed to inhalable wood dust in EU. In previous small studies of this cancer, ras mutations were suggested to be related to wood dust exposure, but these studies were too limited to detect statistically significant associations. METHODS: We examined 174 cases of sinonasal cancer diagnosed in Denmark in the period from 1991 to 2001. To ensure uniformity, all histological diagnoses were carefully reviewed pathologically before inclusion. Paraffin embedded tumour samples from 58 adenocarcinomas, 109 squamous cell carcinomas and 7 other carcinomas were analysed for K-ras codon 12, 13 and 61 point mutations by restriction fragment length polymorphisms and direct sequencing. Information on occupational exposure to wood dust and to potential confounders was obtained from telephone interviews and from registry data. RESULTS: Among the patients in this study, exposure to wood dust was associated with a 21-fold increased risk of having an adenocarcinoma than a squamous cell carcinoma compared to unexposed [OR = 21.0, CI = 8.0-55.0]. K-ras was mutated in 13% of the adenocarcinomas (seven patients) and in 1% of squamous cell carcinomas (one patient). Of these eight mutations, five mutations were located in the codon 12. The exact sequence change of remaining three could not be identified unambiguously. Among the five identified mutations, the G-->A transition was the most common, and it was present in tumour tissue from two wood dust exposed adenocarcinoma patients and one patient with unknown exposure. Previously published studies of sinonasal cancer also identify the GGT --> GAT transition as the most common and often related to wood dust exposure. CONCLUSION: Patients exposed to wood dust seemed more likely to develop adenocarcinoma compared to squamous cell carcinomas. K-ras mutations were detected in 13% of adenocarcinomas. In this study and previously published studies of sinonasal cancer the found K-ras mutations, were almost exclusively G --> A transitions. In conclusion, our study, based on a large representative collection of human SNC tumours, indicates that K-ras mutations are relatively infrequent, and most commonly occur in adenocarcinomas. Wood dust exposure alone was not found to be explanatory for the G-->A mutations, but combination of exposure to tobacco, wood dust, and possibly other occupational agents may be a more likely explanation. Overall, the study suggests a limited role for K-ras mutations in development of sinonasal cancer.

Mitochondria-targeted Probes for Imaging Protein Sulfenylation.

Mitochondrial reactive oxygen species (ROS) are essential regulators of cellular signaling, metabolism and epigenetics underlying the pathophysiology of numerous diseases. Despite the critical function of redox regulation in mitochondria, currently there are limited methods available to monitor protein oxidation in this key subcellular organelle. Here, we describe compounds for imaging sulfenylated proteins in mitochondria: DCP-NEt2-Coumarin (DCP-NEt2C) and rhodamine-based DCP-Rho1. Side-by-side comparison studies are presented on the reactivity of DCP-NEt2C and DCP-Rho1 with a model protein sulfenic acid (AhpC-SOH) and mitochondrial localization to identify optimized experimental conditions for labeling and visualization of protein sulfenylation that would be independent of mitochondria membrane potential and would not impact mitochondrial function. These probes are applied to image mitochondrial protein sulfenylation under conditions of serum starvation and in a cell culture model of lung cancer exposed to ionizing radiation and silver nanoparticles, agents serving dual functions as environmental stressors and cancer therapeutics.

Genomic analysis of head and neck cancer cases from two high incidence regions.

We investigated how somatic changes in HNSCC interact with environmental and host risk factors and whether they influence the risk of HNSCC occurrence and outcome. 180-paired samples diagnosed as HNSCC in two high incidence regions of Europe and South America underwent targeted sequencing (14 genes) and evaluation of copy number alterations (SCNAs). TP53, PIK3CA, NOTCH1, TP63 and CDKN2A were the most frequently mutated genes. Cases were characterized by a low copy number burden with recurrent focal amplification in 11q13.3 and deletion in 15q22. Cases with low SCNAs showed an improved overall survival. We found significant correlations with decreased overall survival between focal amplified regions 4p16, 10q22 and 22q11, and losses in 12p12, 15q14 and 15q22. The mutational landscape in our cases showed an association to both environmental exposures and clinical characteristics. We confirmed that somatic copy number alterations are an important predictor of HNSCC overall survival.

Targeted deep sequencing of plasma circulating cell-free DNA reveals Vimentin and Fibulin 1 as potential epigenetic biomarkers for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer death worldwide, but is still lacking sensitive and specific biomarkers for early diagnosis and prognosis. In this study, we applied targeted massively parallel semiconductor sequencing to assess methylation on a panel of genes (FBLN1, HINT2, LAMC1, LTBP1, LTBP2, PSMA2, PSMA7, PXDN, TGFB1, UBE2L3, VIM and YWHAZ) in plasma circulating cell-free DNA (cfDNA) and to evaluate the potential of these genes as HCC biomarkers in two different series, one from France (42 HCC cases and 42 controls) and one from Thailand (42 HCC cases, 26 chronic liver disease cases and 42 controls). We also analyzed a set of HCC and adjacent tissues and liver cell lines to further compare with 'The Cancer Genome Atlas' (TCGA) data. The methylation in cfDNA was detected for FBLN1, PSMA7, PXDN and VIM, with differences in methylation patterns between cases and controls for FBLN1 and VIM. The average methylation level across analyzed CpG-sites was associated with higher odds of HCC for VIM (1.48 [1.02, 2.16] for French cases and 2.18 [1.28, 3.72] for Thai cases), and lower odds of HCC for FBLN1 (0.89 [0.76, 1.03] for French cases and 0.75 [0.63, 0.88] for Thai cases). In conclusion, our study provides evidence that changes in VIM and FBLN1 methylation levels in cfDNA are associated with HCC and could represent useful plasma-based biomarkers. Also, the potential to investigate methylation patterns in cfDNA could bring new strategies for HCC detection and monitoring high-risk groups and response to treatment.

Targeted deep DNA methylation analysis of circulating cell-free DNA in plasma using massively parallel semiconductor sequencing.

AIM: To set up a targeted methylation analysis using semiconductor sequencing and evaluate the potential for studying methylation in circulating cell-free DNA (cfDNA). MATERIALS & METHODS: Methylation of VIM, FBLN1, LTBP2, HINT2, h19 and IGF2 was analyzed in plasma cfDNA and white blood cell DNA obtained from eight hepatocellular carcinoma patients and eight controls using Ion Torrent™ PGM sequencer. RESULTS: h19 and IGF2 showed consistent methylation levels and methylation was detected for VIM and FBLN1, whereas LTBP2 and HINT2 did not show methylation for target regions. VIM gene promoter methylation was higher in HCC cfDNA than in cfDNA of controls or white blood cell DNA. CONCLUSION: Semiconductor sequencing is a suitable method for analyzing methylation profiles in cfDNA. Furthermore, differences in cfDNA methylation can be detected between controls and hepatocellular carcinoma cases, even though due to the small sample set these results need further validation.

Translational cancer research: balancing prevention and treatment to combat cancer globally.

Cancer research is drawing on the human genome project to develop new molecular-targeted treatments. This is an exciting but insufficient response to the growing, global burden of cancer, particularly as the projected increase in new cases in the coming decades is increasingly falling on developing countries. The world is not able to treat its way out of the cancer problem. However, the mechanistic insights from basic science can be harnessed to better understand cancer causes and prevention, thus underpinning a complementary public health approach to cancer control. This manuscript focuses on how new knowledge about the molecular and cellular basis of cancer, and the associated high-throughput laboratory technologies for studying those pathways, can be applied to population-based epidemiological studies, particularly in the context of large prospective cohorts with associated biobanks to provide an evidence base for cancer prevention. This integrated approach should allow a more rapid and informed translation of the research into educational and policy interventions aimed at risk reduction across a population.

Identification of genetic polymorphisms of CYP2S1 in a Finnish Caucasian population.

CYP2S1 is a recently discovered member of the cytochrome P450 (CYP) gene superfamily. Interestingly, even though the DNA sequence identifies it as the sole member of the new CYP2S family, CYP2S1 exhibits many features typical to CYP1 family members, e.g. dioxin-inducibility mediated by the aryl hydrocarbon receptor (AHR) and the aryl hydrocarbon receptor nuclear translocator (ARNT). In addition, CYP2S1 metabolises some aromatic hydrocarbons as well as cellular substances. These characteristics, together with a wide extrahepatic tissue distribution, suggest that CYP2S1 may have an important role in both exogenous and endogenous metabolism. This is the first study characterising CYP2S1 alleles and naming them with the recommended CYP allele nomenclature. We used denaturing gradient gel electrophoresis (DGGE) and direct sequencing to investigate genetic variation of CYP2S1 in 100 male Finnish Caucasians. Those exons in which variation was found were examined in subsequent 100 subjects. The coding region of all of the nine exons, as well as a 449 bp fragment of the proximal promoter region, was analysed. This systematic investigation revealed eight single nucleotide polymorphisms (SNPs), which comprise nine different variant alleles (haplotypes), in addition to the wild-type allele. Seven of the SNPs occurred in the protein-coding areas and one in the proximal 3' untranslated region (3'UTR). Two of these sequence variations (10347C > T and 13106C > T) result in non-conservative amino acid substitutions, i.e. Arg380Cys and Pro466Leu, respectively. The respective allelic variants, CYP2S1*2 ([10347C > T]) and CYP2S1*3 (13106C > T; 13255A > G]), occurred in our study population at frequencies of 0.50 and 3.75%, respectively. The most common of the variant alleles was CYP2S1*1H (23.8%), harbouring a 13255A > G substitution located in the 3'UTR.

Analysis of TP53 gene mutations in human lung cancer: comparison of capillary electrophoresis single strand conformation polymorphism assay with denaturing gradient gel electrophoresis and direct sequencing.

INTRODUCTION: Automated capillary electrophoresis single strand conformation polymorphism (CE-SSCP), denaturant gradient gel electrophoresis (DGGE), and direct sequencing were compared to investigate the benefits and sensitivity of each of the methods for detection of unknown TP53 mutations in human lung cancer. METHODS: Twenty previously analyzed DNA samples from lung tumors were examined under dummy laboratory codes for occurrence of mutations of the TP53 gene. RESULTS: Mutations were found in 17 samples; 15 (88%) of them were detected by DGGE, 16 (94%) by CE-SSCP and 12 (71%) by direct sequencing. One of the two mutations that remained undetected in DGGE was in fact outside the sequence area covered by DGGE screening, thus rendering DGGE and CE-SSCP equally efficient in mutation detection. Direct sequencing performed less well in finding mutations than the two other assays, as also shown previously. CONCLUSION: The study showed that CE-SSCP is a fast and highly reproducible method, which is considerably less laborious compared to DGGE, for screening of unknown TP53 mutations.