Thallium(I) induces a prolonged inhibition of (6-4)photoproduct binding and UV damage excision repair activities in zebrafish (Danio rerio) embryos via protein inactivation.

Cyclobutane pyrimidine dimer (CPD) and (6-4)photoproduct (6-4 PP) are two major types of UV-induced DNA lesion and 6-4 PP is more mutagenic than CPD. Activated by lesion detection, nucleotide excision repair (NER) eliminates CPDs and 6-4 PPs. Thallium (Tl) is a toxic metal existing primarily as Tl+ in the aquatic environment. Ingestion of Tl+-contaminated foods and water is a major route of human poisoning. As Tl+ may inhibit enzyme activities via binding to sulfhydryl groups, this study explored if Tl+ could intensify UV mutagenicity by inactivating NER-linked damage recognition factors using zebrafish (Danio rerio) embryo as a model system. Incubation of Tl+ (as thallium nitrate) at 0.1-0.4 µg/mL with zebrafish extracts for 20 min caused a concentration-dependent inhibition of 6-4 PP binding activities as shown by a photolesion-specific band shift assay, while CPD binding activities were insensitive to Tl+. The ability of Tl+ to suppress 6-4 PP detection was stronger than that of Hg2+. Exposure of zebrafish embryos at 1 h post fertilization (hpf) to Tl+ at 0.4-1 µg/mL for 9 or 71 h also specifically inhibited 6-4 PP detection, indicating that Tl+ induced a prolonged inhibition of 6-4 PP sensing ability primarily via its direct interaction with damage recognition molecules. Tl+-mediated inhibition of 6-4 PP binding in embryos at distinct stages resulted in a suppression of NER capacity monitored by a transcription-based DNA repair assay. Our results revealed the potential of Tl+ to enhance UV mutagenicity by disturbing the removal of 6-4 PP through repressing the lesion detection step of NER.

Structural Variants and Ultralow Detection Ability for Tryptamine in Two Polymorphs of a Zincophosphite Framework.

Two organic-inorganic hybrid zinc phosphites incorporating 1,2,4,5-tetrakis(imidazol-1-ylmethyl)benzene (TIMB) molecules were synthesized under hydro(solvo)thermal methods and structurally characterized by single-crystal X-ray diffraction (SCXD). Interestingly, the solvent ratio of water to dimethylformamide induced the formation of a new compound of Zn2(TIMB)0.5(HPO3)2·3H2O (1) and our previously reported structure of Zn2(TIMB)0.5(HPO3)2·H2O (2). Additionally, their dehydrated crystals (1a and 2a) were prepared through heat treatment at 150 °C. SCXD and powder X-ray diffraction showed that all four compounds share the same framework formula of Zn2(TIMB)0.5(HPO3)2 but exhibit a huge difference in their inorganic components and final structures. In 1 and 1a, the inorganic units formed two-dimensional zincophosphite layers, while in 2 and 2a, they formed one-dimensional chains. The inorganic parts of 1 (1a) and 2 (2a) were bridged with TIMB linkers, resulting in 3D structures with rectangular and tubular windows, respectively. Furthermore, 1 was coated on the screen-printed carbon electron as a hybrid material, displaying excellent performance while having a linear relationship with an R2 value of 0.99 within the concentration range of 10-10 to 10-6 mol/L for detecting tryptamine (Try) molecules. Moreover, the results showed that 1 exhibits an ultralow limit of detection of 5.43 × 10-11 mol/L and high specificity toward Try over histamine, ascorbic acid, uric acid, and glucose. The synthesis, structural diversity, stability, and sensing ability are also discussed.

Susceptibility of DNA damage recognition activities linked to nucleotide excision and mismatch repair in zebrafish (Danio rerio) early and mid-early embryos to 2.5 to 4.5 °C heat stress.

Fish at early life stages are sensitive to temperature change because of their narrower temperature tolerance ranges. Initiated by damage detection, DNA mismatch repair (MMR) and nucleotide excision repair (NER) maintain genome integrity respectively by eliminating mismatched nucleotides and helix-distorting DNA lesions. As discharge of heated effluent from power plants may elevate water temperatures to only 2 to 6 °C higher than ambient, this study explored if temperatures within this range affected MMR and NER-linked damage detection activities in fish embryos using zebrafish (Danio rerio) embryo as a model organism. Exposure of early embryos at 10 h post fertilization (hpf) to a warmer temperature at + 4.5 °C for 30 min enhanced damage recognition activities targeting UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs) that distorted helical structures. Conversely, photolesions sensing activities were inhibited in 24 hpf mid-early embryos under the same stress conditions. A much higher temperature at + 8.5 °C imposed similar effects on UV damage detection. A mild heat stress at + 2.5 °C for 30 min, however, repressed both CPD and 6-4PP binding activities in 10 and 24 hpf embryos. Inhibition of damage recognition under mild heat stress impeded the overall NER capacity evidenced by a transcription-based repair assay. Warmer water temperatures at + 2.5 and + 4.5 °C also inhibited G-T mismatch binding activities in 10 and 24 hpf embryos, but G-T recognition was more sensitive to + 4.5 °C stress. Inhibition of G-T binding partially correlated with a downregulation of Sp1 transcription factor activity. Our results showed the potential of water temperature elevation within 2 to 4.5 °C to disturb DNA damage repair in fish at embryonic stages.

Aluminum (Al) causes a delayed suppression of nucleotide excision repair (NER) capacity in zebrafish (Danio rerio) embryos via disturbance of DNA lesion detection.

Aluminum (Al) is extensively used for making cooking utensils and its presence in the aquatic environment may occur through acid mine drainage and wastewater discharge. Al is known to induce genotoxicity in human cells, rodents, and fish. Nucleotide excision repair (NER) eliminates helix-twisting DNA lesions such as UV-induced dipyrimidine photoproducts. Because our earlier investigation revealed the operation of NER in zebrafish (Danio rerio) embryos, this study explored if inhibition of NER could be a mechanism of Al-induced genotoxicity using zebrafish embryo as a model system. An acute fish embryo toxicity test indicated that Al (as aluminum sulfate) at 2-15 mg/L were nonlethal to zebrafish embryos, yet exposure of embryos at 1 h post fertilization (hpf) to Al at 10-15 mg/L for 71 h significantly repressed their NER capacity monitored by a transcription-based DNA repair assay. Band shift analysis indicated a higher sensitivity of (6-4) photoproduct (6-4PP) than cyclobutane pyrimidine dimer (CPD) detecting activities to Al, reflecting the preferential influence of Al on the detection of strongly distorted DNA lesions. Time-course experiments showed a delayed response of NER to Al as repair machinery was unaffected by Al at 15 mg/L following a 35-h exposure, while Al treatment for the same period obviously inhibited 6-4PP binding activities although the gene expression of damage recognition factors remained active. Inhibition of 6-4PP detection blocked downstream lesion incision/excision detected by a terminal deoxy transferase-mediated end labeling assay. As the disturbance of damage sensing preceded that of the overall repair process, Al exposure was believed to downregulate NER capacity by inhibiting the activities of lesion detection proteins. Our results revealed the ability of Al to enhance its genotoxicity by suppressing NER capacity.

Heat stress modulates nucleotide excision repair capacity in zebrafish (Danio rerio) early and mid-early embryos via distinct mechanisms.

Discharge of heated effluent at 8-12 °C above ambient into water areas is known to retard the growth of aquatic organisms due to heat stress. Nucleotide excision repair (NER) maintains genome integrity by removing helix-distorting adducts such as UV-induced DNA lesions. This study explored how NER in zebrafish (Danio rerio) embryos at different hours post fertilization (hpf) responded to + 8.5 °C heat shock for 30 min. Our transcription-based repair assay monitoring the ability of zebrafish extracts to upregulate a UV-suppressed gene expression detected a 2-fold increase of NER capacity in 10 hpf early embryos after heat stress. In contrast, heat stress caused a mild inhibition of NER capacity in 24 hpf mid-early embryos. Heat-treated and untreated 10 hpf zebrafish extracts displayed similar levels of UV-damaged-DNA binding activities, while an apparently weaker (6-4) photoproduct (6-4 PP) binding activity was present in heat-stressed 24 hpf zebrafish extracts. Heat stress enhanced UV-induced NER in 10 hpf embryos by increasing the efficiency of damage incision/excision based on both genomic DNA electrophoresis and terminal deoxytransferase (TdT)-mediated end labeling assay. UV-irradiated embryos preexposed to heat stress produced a significantly larger amount of NER-associated DNA fragments about 20-30 nucleotides in length than embryos only heat-treated or irradiated. Correlated with its inhibitory effect on 6-4 PP damage recognition, heat stress downregulated damage incision/excision activities in 24 hpf embryos. Hence, thermal stress may positively or negatively modulate NER capacity in zebrafish embryos at different stages by targeting at the step of DNA incision/excision or damage recognition.

RAB38 is a potential prognostic factor for tumor recurrence in non-small cell lung cancer.

Ras-related protein Rab-38 (RAB38) is a member of the Ras small G protein family that regulates intracellular vesicular trafficking. Although the expression of RAB38 is reportedly deregulated in several types of cancer, its role in tumor biology remains to be elucidated. In the present study, the expression of RAB38 was analyzed in tumor specimens from patients with non-small cell lung cancer (NSCLC) with tumor recurrence within 4 years (Group R), and those remaining disease-free following initial surgery (Group NR), by reverse transcription-semi-quantitative PCR and subsequent semi-quantification using ImageJ v4.0 software. The results revealed that the expression of RAB38 in Group R and NR specimens was positively associated with tumor recurrence; a high expression level was also associated with poor survival rate in these patients. Using NSCLC cell lines, it was demonstrated that tumor cells with mutations in the active epidermal growth factor receptor (EGFR) gene expressed higher levels of RAB38 compared with those with the wild-type gene by reverse transcription-PCR and western blot analysis. Furthermore, following specific RAB38 gene knockdown by short hairpin RNA transfection, EGFR mutants exhibited markedly reduced invasiveness when compared with cells transfected with empty vector controls by Matrigel Transwell assays. These results suggest that RAB38 is an important prognostic factor in NSCLC, and may serve a critical role in NSCLC-associated tumor metastasis.

Heat stress upregulates G-T mismatch binding activities in zebrafish (Danio rerio) embryos preexposed and nonexposed to a sublethal level of cadmium (Cd).

G-T mispair frequently appears in eukaryotic DNA due to the spontaneous deamination of 5-methylcytosine paired with guanine and is therefore an important target for DNA mismatch repair (MMR). Our earlier studies showed the downregulation of G-T binding activities in cadmium (Cd)-exposed (Danio rerio) embryos. Since elevation of water temperature was reported to increase Cd toxicity in zebrafish, this study explored whether heat stress affected zebrafish mismatch binding capacity in the absence or presence of Cd. Heat stress (37 °C for 30 min) induced heat shock protein 70 mRNA expression in embryos at 10 and 24 h post fertilization (hpf). Heat stress weakly upregulated normal G-T sensing machinery and inhibited G-T recognition activity in embryos preexposed to 3 µM Cd for 9 h. Either heat shock or a 23-h Cd treatment alone caused a 1.7-fold stimulation of G-T binding capacity in 24 hpf embryos and heat stress of Cd-preexposed embryos further enhanced G-T binding activity to 2.5 fold of control. Normal and Cd-downregulated loop binding activities in 10 and 24 hpf embryos were almost unreactive to heat shock. Heat stress-upregulated G-T sensing in nonexposed, but not in Cd-preexposed, 24 hpf embryos correlated with stronger gene activities encoding MMR-linked mismatch detecting factors MutS homolog 2 and 6 plus a higher DNA binding activity of the transcription factor Sp1 that regulates msh2/msh6 expression. Our results suggested the importance of heat shock response in facilitating the correction of G-T mismatch in developing zebrafish even under Cd exposure.

A Series of Organic-Inorganic Hybrid Zinc Phosphites Containing Extra-Large Channels.

A series of organic-inorganic hybrid zinc phosphites with extra-large channels were synthesized and characterized by single-crystal X-ray diffraction. This is an unusual example of introducing 1,4-benzenedicarboxylate and/or biphenyl-4,4'-dicarboxylate ligands into the organically templated metal phosphite system to build extra-large-channel zeolite-related materials via hydro(solvo)thermal reactions. Those frameworks are composed of carboxylate linkers and inorganic tubes of zinc phosphites, translating their channel windows from a square shape (NTOU-1) to rhombus forms (NTOU-2 and NTOU-3) via the replacement of organic amines or ligands under synthesis conditions otherwise identical with those used to prepare NTOU-1. The synthesis, structural diversity, photoluminescence, and adsorption properties for dye molecules and lanthanide ions are also reported.

Mercury (II) impairs nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos by targeting primarily at the stage of DNA incision.

Mercuric ion (Hg2+) is the most prevalent form of inorganic Hg found in polluted aquatic environment. As inhibition of DNA damage repair has been proposed as one of the mechanisms of Hg2+-induced genotoxicity in aquatic animals and mammalian cells, this study explored the susceptibility of different stages of nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos to Hg2+ using UV-damaged DNA as the repair substrate. Exposure of embryos at 1h post fertilization (hpf) to HgCl2 at 0.1-2.5µM for 9h caused a concentration-dependent inhibition of NER capacity monitored by a transcription-based DNA repair assay. The extracts of embryos exposed to 2.5µM Hg2+ almost failed to up-regulate UV-suppressed marker cDNA transcription. No inhibition of ATP production was observed in all Hg2+-exposed embryos. Hg2+ exposure imposed either weak inhibitory or stimulating effects on the gene expression of NER factors, while band shift assay showed the inhibition of photolesion binding activities to about 40% of control in embryos treated with 1-2.5µM HgCl2. The damage incision stage of NER in zebrafish embryos was found to be more sensitive to Hg2+ than photolesion binding capacity due to the complete loss of damage incision activity in the extracts of embryos exposed to 1-2.5µM Hg2+. NER-related DNA incision was induced in UV-irradiated embryos based on the production of short DNA fragments matching the sizes of excision products generated by eukaryotic NER. Pre-exposure of embryos to Hg2+ at 0.1-2.5µM all suppressed DNA incision/excision in UV-irradiated embryos, reflecting a high sensitivity of DNA damage incision/excision to Hg2+. Our results showed the potential of Hg2+ at environmental relevant levels to disturb NER in zebrafish embryos by targeting primarily at the stage of DNA incision/excision.

Identification of low-molecular-weight vitellogenin 1 (Vg1)-like proteins as nucleotide excision repair (NER) factors in developing zebrafish (Danio rerio) using a transcription-based DNA repair assay.

Nucleotide excision repair (NER) removes helix-distorting DNA lesions such as UV-induced pyrimidine dimers and cisplatin-induced strand crosslinking. Our earlier studies have identified low-molecular-weight proteins homologous to the 150-kDa vitellogenin 1 (Vg1) as UV-damaged DNA-binding factors expressed in developing zebrafish (Danio rerio). This present study explored if Vg1-like proteins also participated in NER in zebrafish. Immunoblot analysis of affinity-captured 12 h post-fertilization (hpf) zebrafish extract proteins showed a transient binding of a 30-kDa Vg1-like polypeptide to UV-damaged DNA. A transcription-based in vitro repair assay revealed a significant up-regulation of UVC or cisplatin-suppressed transcriptional activity of a marker cDNA driven by a SP6 RNA polymerase-regulated promotor after incubating the damaged plasmid with the extracts of 12 hpf embryos or 96 hpf larvae. The up-regulation of UV or cisplatin-suppressed transcription was abolished in the presence of a monoclonal anti-zebrafish Vg1 antibody. The differential sensitivity of UV-induced repair in 12 and 96 hpf zebrafish extracts to exogenous ATP suggested a development-dependent expression of Vg1-like NER factors. A T4 endonuclease V digestion assay showed no inhibition of the anti-Vg1 antibody on the excision of UV-induced cyclobutane pyrimidine dimers. Our results identified the participation of Vg1-like factors in NER in developing zebrafish, and these factors may function at post-incison steps of NER.

Angiotensin-(1-7) Inhibits Thrombin-Induced Endothelial Phenotypic Changes and Reactive Oxygen Species Production via NADPH Oxidase 5 Downregulation.

Background and Aims: The angiotensin-(1-7)/angiotensin-converting enzyme 2/Mas receptor axis counter-regulates the detrimental effects of angiotensin II. Beneficial effects of angiotensin-(1-7), including anti-inflammation, oxidative stress reduction, and anti-thrombosis, have been reported. Previous studies documented that ramipril decreased thrombin generation in human hypertension and that the anti-thrombotic effects of captopril and losartan were angiotensin-(1-7)-dependent, suggesting an interaction between thrombin and angiotensin-(1-7). However, it is not clear whether angiotensin-(1-7) can alleviate the endothelial phenotypic changes induced by thrombin. We have previously documented cytoskeleton remodeling, cell adhesion, and cell migration as dominant altered phenotypes in thrombin-stimulated human aortic endothelial cells (HAECs). In this study, we investigated whether angiotensin-(1-7) can modulate thrombin-induced phenotypic changes. Furthermore, we investigated whether NAPDH oxidase 5 (Nox5)-produced reactive oxygen species (ROS) play a significant role in angiotensin-(1-7)-mediated phenotypic changes. Methods: HAECs were pretreated with 100 nM angiotensin-(1-7) for 1 h, followed by stimulation with 2 units/mL thrombin for different times. Immunofluorescent assay, monocyte adhesion assay, wound-healing assay, ROS assay, real-time PCR, Western blotting, and Nox5 siRNA transfection were conducted. HAECs were pretreated with the ROS scavenger N-acetylcysteine (NAC) to determine whether thrombin-induced phenotypic changes depended on ROS production. Results: Angiotensin-(1-7) prevented thrombin-induced actin cytoskeleton derangements, monocyte adhesion, and migratory impairment. Nox5 siRNA transfection confirmed that thrombin-induced Nox5 expression stimulated ROS production and increased HO-1/NQO-1/ICAM-1/VCAM-1 gene expression, all of which were decreased by angiotensin-(1-7). Phenotypic changes induced by thrombin were prevented by NAC pretreatment. Conclusion: Angiotensin-(1-7) prevents actin cytoskeleton derangement, monocyte adhesion, and migration impairment induced by thrombin via downregulation of ROS production. In addition, thrombin-induced Nox5 expression is involved in the production of ROS, and angiotensin-(1-7) decreases ROS through its inhibitory effect on Nox5 expression.

Oxidative stress intensity-related effects of cadmium (Cd) and paraquat (PQ) on UV-damaged-DNA binding and excision repair activities in zebrafish (Danio rerio) embryos.

Our earlier studies showed the inhibitory effects of cadmium (Cd) and paraquat (PQ) on the gene expression of DNA mismatch recognition proteins in zebrafish (Danio rerio) embryos. This study explored the effects of Cd and PQ on nucleotide excision repair (NER) capacity in zebrafish embryos. Exposure of embryos at 1 h post fertilization (hpf) to 3-5 µM Cd or 30-100 µM PQ for 9 h induced a 2-3-fold increase of oxidative stress, while a 6.5-fold increase of oxidative stress was induced by 200 µM PQ. Real-time RT-PCR detected a down-regulated xeroderma pigmentosum C (XPC) and an up-regulated UV-DDB2 gene expression in mildly-stressed embryos, whereas 8-oxoguanine DNA glycosylase (OGG1) gene expression increased with PQ exposure levels. NER of UV-damaged DNA was enhanced in weakly oxidant-stressed embryos as shown by a transcription-based DNA repair assay, yet repair activities of both UV and cisplatin-damaged DNA were inhibited in embryos exposed to 200 µM PQ. Band shift assay showed a suppression of cyclobutane pyrimidine dimer (CPD) binding activity in all stressed embryos. In contrast, (6-4) photoproduct (6-4PP) recognition activity was weakly stimulated except in embryos exposed to 200 µM PQ, revealing a link of NER capacity to 6-4PP binding. Our results showed that Cd and PQ imposed similar inducing effects on UV-DDB2 gene expression, NER of UV-damaged DNA and 6-4PP binding activity in zebrafish embryo under low levels of oxidative stress and NER capacity could be inhibited if the intensity of oxidative stress increased to a critical level.

A comparison of immunohistochemical and molecular methods used for analyzing the BRAF V600E gene mutation in malignant melanoma in Taiwan.

AIMS: The BRAF V600 mutation has been shown to be clinically meaningful in terms of both the prognosis and sensitivity of BRAF inhibitors in patients with metastatic melanoma. Recently, a BRAF V600E mutation-specific antibody, VE1, was generated for the detection of tumors bearing BRAF V600E mutations. To determine the clinical value of immunohistochemical testing, we compared the prevalence of mutant BRAF detected by VE1 with direct sequencing results. METHODS: Paraffin-embedded, formalin-fixed melanoma biopsies were analyzed for the BRAF mutation status by immunohistochemistry with the VE1 antibody. Sanger sequencing was applied to verify the immunohistochemical results. RESULTS: A total of 73 melanoma cases with tumor samples from primary lymph nodes and metastatic sites were selected for this study. Direct sequencing demonstrated that 18 of 73 cases (24.6%) harbored the BRAF V600 mutation: 17 with V600E and one with V600K. All 18 tumors shown to harbor the BRAF V600E/K mutations were VE1-positive. One additional case was false-positive for VE1. The sensitivity and specificity of VE1 was 100% (18/18) and 98% (54/55), respectively. The overall concordance between the immunohistochemical method and direct sequencing was excellent (98.6%). CONCLUSIONS: Our findings demonstrate that immunohistochemical analysis using VE1 constitutes a highly sensitive test for the detection of BRAF mutations and suggest that this cost-effective method is suitable as a rapid diagnostic approach complementary to molecular testing.

Comparative Effects of Mercury(II) and Cadmium on MutS Homolog 6(MSH6)-Mediated DNA Mismatch Binding Activities in Zebrafish (Danio rerio) Embryos.

MutS homolog 6 (MSH6) of the MSH2-MSH6 complex binds simple mispairs and small insertion-deletion loops (IDL) then initiates DNA mismatch repair in eukaryotes. We have shown the ability of Cd(2+) to downregulate msh2/msh6 expression in zebrafish embryos via oxidative stress. This study explored the effects of Cd(2+) and Hg(2+) on MSH6-mediated mismatch binding activities. MSH6-mediated G-T and IDL-specific binding activities were significantly inhibited at similar levels after exposing zebrafish embryos at 1 h postfertilization) to HgCl2 or CdCl2 at 1.0 to 2.5 µM for 9 h, but MSH6 synthesis was found to be less sensitive to Hg(2+) than to Cd(2+) . Real-time RT-PCR and in situ hybridization also detected a weaker susceptibility of MSH gene transcription to Hg(2+) . The weaker response of MSH gene activities to Hg(2+) correlated with the lower oxidative stress-inducing potential of Hg(2+) . Hence, Hg(2+) targets mismatch sensing capacity at protein function rather than at transcription level.

Cytoplasmic and nuclear parathyroid hormone-related proteins are opposing prognostic factors in patients with non-small-cell lung cancer who have undergone curative resection.

OBJECTIVE: Parathyroid hormone-like related protein was a prognostic factor for non-small-cell lung cancer, but the results were conflicting. The present study was to examine the role of cytoplasmic and nuclear parathyroid hormone-like related protein in patients with non-small-cell lung cancer who have undergone surgical therapy. METHODS: The expression of parathyroid hormone-like related protein was examined by immunohistochemical staining in 56 patients with resectable non-small-cell lung cancer. The impact of parathyroid hormone-like related protein expression on cancer recurrence and survival was assessed in combination with clinicopathologic features. RESULTS: Patients with a high expression of cytoplasmic parathyroid hormone-like related protein had a significantly unfavorable prognosis in both disease-free survival (median 16.7 vs. 58.0 months, P = 0.029) and overall survival (median 31.6 months vs. not reached, P = 0.046). In contrast, the patients with high expression of nuclear parathyroid hormone-like related protein had favorable disease-free survival (median 35.1 vs. 19.9 months, P = 0.069) and a significantly better overall survival (median not reached vs. 36.9 months, P = 0.033). There was no correlation between the expression of cytoplasmic and nuclear parathyroid hormone-like related protein (P = 1.00). Furthermore, multivariate analysis using a Cox regression model confirmed that high expression of cytoplasmic parathyroid hormone-like related protein (disease-free survival, hazard ratio: 1.973, P = 0.079; overall survival, hazard ratio: 2.461, P = 0.067) and nuclear parathyroid hormone-like related protein (disease-free survival, hazard ratio: 0.436, P = 0.029; overall survival, hazard ratio: 0.375, P = 0.018) were independently prognostic factors for disease-free survival and overall survival. CONCLUSION: Cytoplasmic and nuclear parathyroid hormone-like related protein play opposing prognostic roles for the disease-free survival and overall survival of patients with early non-small-cell lung cancer who have undergone curative resection.

Cadmium(Cd)-induced oxidative stress down-regulates the gene expression of DNA mismatch recognition proteins MutS homolog 2 (MSH2) and MSH6 in zebrafish (Danio rerio) embryos.

DNA mismatch repair (MMR) of simple base mismatches and small insertion-deletion loops in eukaryotes is initiated by the binding of the MutS homolog 2 (MSH2)-MSH6 heterodimer to mismatched DNA. Cadmium (Cd) is a genotoxic heavy metal that has been recognized as a human carcinogen. Oxidant stress and inhibition of DNA repair have been proposed as major factors underlying Cd genotoxicity. Our previous studies indicated the ability of Cd to disturb the gene expression of MSH6 in zebrafish (Danio rerio) embryos. This study was undertaken to explore if Cd-induced oxidative stress down-regulated MSH gene activities. Following the exposure of zebrafish embryos at 1 h post fertilization (hpf) to sublethal concentrations of Cd at 3-5 µM for 4 or 9 h, a parallel down-regulation of MSH2, MSH6 and Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene expression was detected by real-time RT-PCR and the expression levels were 40-50% of control after a 9-h exposure. Cd exposure also induced oxidative stress, yet no inhibition of catalase gene activity was observed. Whole mount in situ hybridization revealed a wide distribution of msh6 mRNA in the head regions of 10 hpf embryos and pretreatment of embryos with antioxidants butylhydroxytoluene (BHT), d-mannitol or N-acetylcysteine (NAC) at 1-10 µM restored Cd-suppressed msh6 expression. QPCR confirmed the protective effects of antioxidants on Cd-suppressed msh2/msh6 mRNA production. Down-regulated MSH gene activities reaching about 50% of control were also induced in embryos exposed to paraquat, a reactive oxygen species (ROS)-generating herbicide, or hydrogen peroxide at 200 µM. Hence, Cd at sublethal levels down-regulates msh2/msh6 expression primarily via ROS as signaling molecules. The transcriptional activation of human msh6 is known to be fully dependent on the specificity factor 1 (Sp1). Cd failed to inhibit the DNA binding activity of zebrafish Sp1 unless at lethal concentrations based on band shift assay, therefore excluding the involvement of Sp1 inactivation in Cd-induced MSH gene inhibition in zebrafish embryos.

Comparison of genomic signatures of non-small cell lung cancer recurrence between two microarray platforms.

BACKGROUND: Cancer genomic signatures may vary using different platforms. We compared the differential gene expression in non-small cell lung cancer (NSCLC) between two platforms in order to find the most relevant genomic signatures of tumor recurrence. MATERIALS AND METHODS: We analyzed gene expression in frozen lung cancer tissue from 59 selected patients who had undergone surgical resection of NSCLC. These patients were divided into two groups: group R, patients who had a tumor recurrence within four years, n=37; group NR, patients who remained disease-free four years following initial surgery, n=22. Each RNA sample was assayed twice using both Affymetrix and Illumina GeneChip. Data were analyzed by principal component analysis and leave-one-out cross-validation. RESULTS: Using the same filtering criteria, 13 genes that were differentially expressed between R and NR were identified by Affymetrix, while 21 genes were identified by Illumina GeneChip. In common, a total of six genes were detected by both systems. Using univariate analysis, four (lipocalin 2, LCN2; parathyroid hormone-like hormone, PTHLH; ras-related protein Rab-38, RAB38; and four jointed box 1, FJX1) of these six genes were associated with survival. A risk score of survival was calculated according to the four-gene expression. There was a significant difference in overall survival between low- and high-risk groups. CONCLUSION: A four-gene signature is associated with survival among patients with early-stage NSCLC. Further validation of these findings is warranted.

Erlotinib treatment in patients with advanced lung adenocarcinoma with CISH-positive and CISH-negative EGFR gene alterations.

BACKGROUND: Epidermal growth factor receptor (EGFR) positivity as assessed by chromogenic in situ hybridization (CISH) has been demonstrated to be associated with EGFR mutation status. This study was conducted to compare the responsiveness of CISH-positive and CISH-negative lung adenocarcinomas to erlotinib. PATIENTS AND METHODS: Patients received erlotinib (150 mg/day) alone until disease progression or intolerable toxicity. EGFR gene status was examined by CISH. The response rate (RR), progression-free survival (PFS), overall survival (OS) and toxicity profiles were assessed. RESULTS: Thirty-one patients underwent response evaluations and CISH analyses, 12 of whom harboured CISH-positive adenocarcinomas. The overall RR (p=0.035), median PFS (p=0.091) and median OS (p=0.408) were higher in the CISH-positive group. No difference in toxicity profiles was observed between these two groups. CONCLUSION: EGFR status as assessed by CISH can predict the response to erlotinib in patients with advanced lung adenocarcinoma.

Affinity isolation and mass spectral analysis of 1,10-phenanthroline (OP)-stimulated UV-damaged-DNA binding proteins expressed in zebrafish (Danio rerio) embryos.

Our earlier studies indicated the high expression of a UV-damaged-DNA binding activity in zebrafish (Danio rerio) embryos at 12 h postfertilization (hpf). Two 30- to 35-kDa polypeptides homologous to the N-terminal lipovitellin 1 (Lv1) domain of the 150-kDa zebrafish vitellogenin 1 (zfVg1) were identified as the damage recognition factors in zebrafish extracts, and the metal-chelating agent 1,10-phenanthroline (OP) was found to inhibit the embryonic UV-damaged-DNA binding activity. This study further explored the DNA damage-sensing components in 12 hpf zebrafish extracts. UV-damaged-DNA binding proteins were enriched from zebrafish extracts by isoelectrofocusing. Both OP-sensitive and OP-stimulated, UV-damaged-DNA binding activities were detected in fractionated zebrafish extracts. Two-dimensional gel electrophoresis of proteins captured by an immobilized oligonucleotide carrying a UV-induced (6-4)photoproduct (6-4PP) revealed a 25-kDa polypeptide as the major 6-4PP-binding factor in an OP-stimulated fraction. Three 25-kDa factors that bound weakly to 6-4PPs were also isolated. The four polypeptides having pIs between 7.0 and 7.3 were unreactive to an anti-zfVg1 antibody targeting the Lv1 domain. Mass spectral analysis showed the appearance of amino acid sequences LPIIVTTYAK and IPEITMSK in all 25-kDa polypeptides and sequences exactly matching those contained in the four factors exist only in the C-terminal Lv2 domain of zfVg1, reflecting the origination of these factors from enzymatic cleavage of the Lv2 domain at slightly different positions. The OP-stimulated fraction produced a much stronger UV-dependent DNA incision activity in the presence than in the absence of OP, suggesting the association of these factors with DNA damage repair under metal-deficient conditions.

Epidermal growth factor receptor mutations in patients with oral cavity cancer in a betel nut chewing-prevalent area.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutations exist in patients with oral cavity squamous cell carcinoma (OSCC), but few data about mutation patterns with clinical outcomes were reported. METHODS: Fifty-six formalin-fixed paraffin-embedded tumor samples were obtained surgically from OSCC patients. Direct sequencing of EGFR was carried out using nested polymerase chain reaction. The relationship between EGFR status and clinical courses was analyzed. RESULTS: Two (3.56%) missense mutations (G857R; L862Q) in exon 20 were identified. Two types of silent mutation, A859A in exon 21 (1.79%) and Q787Q mutations in exon 20 (30.36%), were also found. No mutation was detected in exons 18 and 19. No significant difference in disease-free survival and locoregional control rate was shown between patients with and without Q787Q mutation. CONCLUSIONS: We identified a high frequency of Q787Q mutation and a less prevalent active EGFR mutation in OSCC patients in Taiwan where betel nut is commonly chewed.