ABSTRACT
<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>
Subject(s)
Humans , Benzo(a)pyrene , Toxicity , DNA Damage , DNA Repair , DNA-Formamidopyrimidine Glycosylase , Genetics , Gene Expression , HeLa Cells , Heat-Shock Proteins , Genetics , Intracellular Signaling Peptides and Proteins , GeneticsABSTRACT
<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>
Subject(s)
Female , Humans , Cell Line, Tumor , DNA Damage , DNA-Binding Proteins , Dose-Response Relationship, Drug , HSP27 Heat-Shock Proteins , HSP70 Heat-Shock Proteins , Heat Shock Transcription Factors , Heat-Shock Proteins , Hydrogen Peroxide , Intracellular Signaling Peptides and Proteins , Neoplasm Proteins , Oxidative Stress , Transcription Factors , Up-RegulationABSTRACT
<p><b>OBJECTIVE</b>To investigate the expressions of JWA gene and heat shock protein 70 (hsp70) in human embryonic lung (cccHPF-1) cells after exposure to activated benzo(a)pyrene (B(a)P), and to explore the role and the possible mechanism of JWA gene involved in B(a)P-induced DNA damage and repair.</p><p><b>METHODS</b>The models of DNA damage of ccc-HPF-1 cells were established by treatment of cells with B(a)P plus S9 at 10 to 100 micromol/L for 3 hours with or without 24 hours recovery for DNA repairing. The DNA damage was detected by single cell gel electrophoresis (SCGE) assay (comet assay). And the immuno-blotting assay was used for detecting expressions of JWA and hsp70.</p><p><b>RESULTS</b>JWA expression was actively modulated by B(a)P exposure. The expressions of both JWA and hsp70 were increased greatly at 50 micromol/L and 100 micromol/L B(a)P treated cells and maintained at over expressed levels treated by 10-100 micromol/L B(a)P during the restored time. In addition, JWA expression pattern was similar to that of hsp70.</p><p><b>CONCLUSION</b>JWA is determined in this study by its functioning as an effective environmental responsive gene and actively participating in the signal pathways of DNA damage and repair which might be associated with excision repair.</p>