Construction and identification of antisense c-Jun N-terminal kinase 1 eukaryotic fluorescent expressing plasmids and JNK1-/- human embryo lung fibroblasts cell line / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To construct antisense c-Jun N-terminal kinase 1 (JNK1) eukaryotic fluorescent expressing vector and JNK1-/- human embryo lung fibroblasts cell line.</p><p><b>METHODS</b>Trizol reagent was used to extract total RNA in HELF. The proper primers of JNK1 were chosen and synthesized. RT-PCR and gene recombinant techniques were used to construct the fragment of JNK1. After purification, the PCR products were cut, and JNK1 were inserted reversely into eukaryotic fluorescent expressing vector pEGFP-C1. Enzyme-cutting and DNA auto-sequencing were used to prove the successful construction of JNK1 eukaryotic expressing vector. Then plasmids were extracted and transfected into HELF cells and screen by G418 24 h later. Monoclone was chosen and cultured. Fluorescent imaging and Western blot were used to identify the JNK1-/- HELF cell line.</p><p><b>RESULTS</b>Sequence analysis of pEGFP-C1-as JNK1 plasmids was same as expected. The expression level of JNK1 was inhibited markedly.</p><p><b>CONCLUSION</b>Construction of antisense JNK1 eukaryotic fluorescent expressing vectors and JNK1-/- HELF cell line is successful.</p>

The relationship between hormesis of proliferation and oxidative stress induced by sodium arsenite in human embryo lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the hormesis of proliferation and oxidative stress induced by sodium arsenite (Na(2)AsO(2)) in human embryo lung fibroblasts (HELF).</p><p><b>METHODS</b>HELF were treated with Na(2)AsO(2) of 0.0, 0.1, 0.5, 1.0, 5.0 and 10.0 micromol/L for 4 hours or 24 hours, respectively. The cell proliferation, the reactive oxygen species (ROS) level, the malondialdehyde (MDA) content and the activity of glutathione peroxide (GSH-Px) and the superoxide dismutase (SOD) in HELF were detected respectively.</p><p><b>RESULTS</b>The HELF proliferation induced by 0.1 and 0.5 micromol/L Na(2)AsO(2) was significantly higher than that in the control group (P < 0.01). The HELF proliferation induced by 5.0 and 10.0 micromol/L Na(2)AsO(2) was significantly lower than that in the control group (P < 0.01) with the dose-effect relation of an inverted U curve. The ROS level induced by Na(2)AsO(2) of between 0.5 and 10.0 micromol/L was significantly increased (P < 0.05, P < 0.01). The positive correlation was found between the ROS level and the exposure dose of Na(2)AsO(2) (r = 0.934, P < 0.01). The 5.0 and 10.0 micromol/L Na(2)AsO(2) induced the significant increase of the MDA contents (P < 0.01) and the significant decrease of the GSH-Px activity compared to those in the control group (P < 0.01). The SOD activity in 0.5 micromol/L Na(2)AsO(2) group was significantly higher than that in the control group (P < 0.01) while the SOD activity induced by 5.0 and 10.0 micromol/L Na(2)AsO(2) was significantly decreased (P < 0.01) if compared with the control group with the dose-effect relation of an inverted U curve.</p><p><b>CONCLUSION</b>The sodium arsenite can induce the hormesis of proliferation in HELF with the dose-effect relation of an inverted U curve. The mechanisms probably relates to different levels of oxidative stress induced by sodium arsenite of different concentrations.</p>

Expression deficiency of JWA enhanced DNA damage and delayed DNA repair in HeLa cells induced by benzo (a) pyrene exposure / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the role of JWA gene in benzo (a) pyrene [B (a) P] induced DNA damage and repair effects in HeLa cells.</p><p><b>METHODS</b>The antisense JWA express vector (pEGFP-C1-asJWA) was constructed and stably transfected into HeLa cells. JWA deficient HeLa cells (asJWA-HeLa) was then screened and established. The general characteristics of asJWA-HeLa cells were investigated. DNA damage and repair cell culture model was conducted by treating the cells with 50 micromol/L B (a) P plus S9 for 3 hours and then the cells were maintained further 0, 1, 3, and 24 hours for DNA repairing. The damaged DNA was detected by single cell gel electrophoresis assay (comet assay).</p><p><b>RESULTS</b>JWA deficient HeLa cells (with a 31% of JWA protein expression as compared with the control) were obtained successfully. Compared with the empty vector transfected cells (C1-HeLa) and the untransfected HeLa cells, asJWA-HeLa cells were more sensitive to B (a) P exposure and with a delayed DNA repair process.</p><p><b>CONCLUSION</b>The JWA determined might function as a potential effective environmental responsive gene and actively participate the process of B (a) P exposure associated with intracellular signal pathways of DNA damage and repair.</p>

Expression of novel environmental responsive protein JWA involved in the oxidative stress responsiveness in MCF-7 cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the expression and the possible role of JWA protein in oxidative stress-induced damage of MCF-7 cells, especially the relationship between JWA and heat shock proteins (HSPs).</p><p><b>METHODS</b>MCF-7 cells were exposed to different concentration of H(2)O(2) (0.01,0.10, 1.00 mmol/L) for different time (10, 30, 60 and 180 min) respectively. DNA damage was detected by using DNA gel electrophoresis. The MTT assay was used to analyze the effect of H(2)O(2) on the cytotoxicity and relative cell proliferation ratio of the cells. The expressions of JWA, HSP70, HSP27 and HSF1 were determined by Western-blot.</p><p><b>RESULTS</b>The inhibitory effect on MCF-7 cells viability induced by H(2)O(2) was shown a dose-and time-dependent manner and MCF-7 cells proliferation, and was almost completely inhibited by the exposure of H(2)O(2) at 1.00 mmol/L for 180 min. Hydrogen peroxide treatment of MCF-7 cells caused oxidative stress which up-regulated the expressions of JWA, HSP70 and heat shock factor 1 (HSF1) in a dose-dependent manner, and the expression pattern of JWA was very similar to those of HSP70 and HSF1 but not to HSP27.</p><p><b>CONCLUSION</b>JWA might enhance intracellular defenses against H(2)O(2)-induced oxidative damage in human breast carcinoma cells. JWA is determined functioning as an effective environmental responsive protein and as a parallel molecule of HSP70 actively participates in the signal pathways of oxidative damage which might be regulated by HSF1.</p>

JWA gene acts as sensitive molecule responsive to oxidative stress and apoptosis in K562 cells / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the expression of JWA gene, heat shock proteins (hsp70 and hsp27) and p53, and to explore the role and the possible mechanism of JWA gene involved in H2O2-induced oxidative stress of K562 cells.</p><p><b>METHODS</b>0.01, 0.1, 1 mmol/L H2O2 treated K562 cells at 10, 30, 60 and 180 min to established the models of DNA damage. Furthermore, K562 cells were induced apoptosis by 0.1 mmol/L H2O2 at different time (6-48 h) and different concentration (0.5-1,000 micromol/L) of H2O2 at 48 h. DNA damage and cell apoptosis were detected by DNA gel electrophoresis. And the immunoblotting assay was used for detecting expressions of JWA protein and correlated genes (hsp27, hsp70 and p53).</p><p><b>RESULTS</b>During the DNA damage, JWA was much more sensitive to H2O2 than those heat shock proteins, and its expression pattern was very similar to that of hsp70. And at low concentration of H2O2-exposure (0.01 mmol/L), the expressions of JWA and heat shock proteins were all increased greatly. In addition, JWA, hsp70, hsp27 and p53 overexpressed and their expression pattern were similar during cell apoptosis.</p><p><b>CONCLUSION</b>JWA should be functioning as an effective environmental responsive gene and should actively participate the signal pathways of oxidative stress which might be associated with hsp70 and p53.</p>

A case-control study on JWA promoter -76G-->C polymorphism and the susceptibility of bladder cancer / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>This case-control study was aimed to detect the single nucleotide polymorphisms (SNPs) in JWA promoter region, to assess the effect of SNP on transcriptional activity, and to probe the relationship between SNP and the risk of bladder cancer.</p><p><b>METHODS</b>The design of one control per case was adopted. The JWA gene promoter region in 155 patients with bladder cancer and in 155 cancer-free controls was amplified by PCR-SSCP technique, and the SNP were confirmed by direct DNA sequencing. The recombinant plasmids of JWA promoter fragment which contain the SNP were constructed as CAT reporter gene and were transfected transiently into NIH 3T3 cells for disclosing whether SNP changes the transcriptional activity of the promoter.</p><p><b>RESULTS</b>A novel SNP -76 G-->C at promoter region of JWA gene was found. The frequencies of the C allele and GC genotype at JWA promoter -76G-->C in bladder cancer group (10.00% and 20.00% respectively) were significantly higher than those in control group (5.16% and 10.32% respectively) (P < 0.05). The transcriptional activity of -76GC allele genotype was significantly down-regulated as compared with that of -76GG allele genotype (P < 0.01). Multivariate logistic regression analysis revealed that JWA polymorphism at promoter -76G-->C is an independent novel risk factor for bladder cancer.</p><p><b>CONCLUSION</b>The JWA -76G-->C variant genotype may play an important role in transcription regulation of JWA gene and in the susceptibility to bladder cancer.</p>

Effects of hydroquinone on DNA and nucleus damage in human embryo lung fibroblasts / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study DNA and nucleus damage in human embryo lung fibroblast (HLF) exposed to hydroquinone (HQ) and its genotoxicity.</p><p><b>METHODS</b>HLF were treated with HQ (0, 10, 20, 40, 80 micro mol/L, respectively) for 3 h and DNA damage was detected by comet assay. HLF was also treated with the same concentrations of HQ for 1 h and micronucleus test was performed after they were cultured for 24 h.</p><p><b>RESULTS</b>Comet assay showed that percentage of cells with tails in each groups treated with varied doses of HQ was 12%, 19%, 42%, 79% and 95%, respectively, with mean tail length of 7.87, 9.35, 11.03, 19.28 and 23.32 micro m, respectively, in an obvious dose-dependent manner (P < 0.05). Very significant increase in percentage of cells with tails and length of their comet tail were observed in those groups treated with HQ of 20, 40 and 80 micro mol/L (P < 0.01). And, proportion of high and severe DNA damage increased with dose of HQ. HQ could also induce formation of micronucleus and abnormal nucleus in all groups treated by varied doses of HQ, with rates of micronucleus and abnormal nucleus of 2%, 3%, 10%, 9% and 15%, and 6%, 7%, 16%, 27% and 28%, respectively, in a significant dose-dependent manner. There was significant increase in rates of micronuclei and abnormal nuclei in cells treated with HQ at doses of 20, 40 and 80 micro mol/L (P < 0.05).</p><p><b>CONCLUSIONS</b>Exposure to HQ could cause DNA and nucleus damage inducing genotoxic effects on HLF.</p>

Construction of eukaryotic expression vector of hMTH1 gene antisense RNA / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To construct pEGFP-C1-T vector, an eukaryotic expression plasmid of hMTH1 gene antisense RNA.</p><p><b>METHODS</b>The conservative region of hMTH1 gene was amplified by RT-PCR after total RNA being extracted from human embryo lung fibroblast (HLF) and then cloned into pGEM-T vector. After the recombinant plasmid was certified by DNA sequencing, the conservative region of hMTH1 gene was inserted into pEGFP-C1 vector reversedly and pEGFP-C1-T vector was constructed. The efficiency of antisense inhibition was verified by Western blotting after cell transfection.</p><p><b>RESULTS</b>423 bp fragment including conservative region of hMTH1 gene was obtained by RT-PCR. After cloned by pGEM-T vector and certified by DNA sequencing, pEGFP-C1-T vector was successfully constructed by means of recombinant DNA technology. Additionally pEGFP-C1-T vector could efficiently decrease hMTH1 protein level by 46%.</p><p><b>CONCLUSION</b>The efficient expression vector of hMTH1 gene antisense RNA, pEGFP-C1-T has been constructed successfully.</p>

Construction of double-strand break repair protein hKu70 deficient cell strain and its biologic characters / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To construct DNA double-strand break (DSB) repair protein hKu70 deficient cell strain and to observe its biological characters for studying the functions of hKu70 gene and the effects of occupational harmfulness factors on DSB repair.</p><p><b>METHODS</b>Human lung fibroblasts (HLF) were transfected with the eukaryotic expression plasmids of hKu70 gene antisense RNA (pEGFP-C1-K) to construct hKu70 protein deficient cells (named as "HLFK"). The protein expression levels of hKu70 gene in HLFC and HLFK were detected by the Western blotting to estimate the effects of antisense inhibition. Morphology, growth character and growth status in soft agar of transfected HLFK were observed.</p><p><b>RESULTS</b>pEGFP-C1-K vector was successfully expressed in HLF. The protein expression level of hKu70 gene in HLFK was decreased by 42% as compared with that in HLFC. No obvious changes of the biologic characters were observed in HLFK.</p><p><b>CONCLUSION</b>The hKu70 protein deficient cell strain was successfully constructed. The hKu70 protein deficiency alone didn't induce obvious changes of the biological characters in HLFK.</p>