ABSTRACT
<p><b>OBJECTIVE</b>To develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence.</p><p><b>METHODS</b>Genomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and subsequently a pair of primer having no CpG sites was designed for amplification target DNA containing methylated or unmethylated CpG sites. The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes.</p><p><b>RESULTS</b>The percentage of methylation of artificial mixtures DNA showed a linear relation. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines.</p><p><b>CONCLUSION</b>Compared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.</p>
Subject(s)
Humans , Cell Line, Tumor , CpG Islands , DNA Methylation , Genes, p16 , Luminescent Measurements , Nucleic Acid Hybridization , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , SulfitesABSTRACT
<p><b>OBJECTIVE</b>To study the expression levels of ERCC2, UDG, and PCNA in cisplatin-treated A549 cell line.</p><p><b>METHOD</b>Comet assay, RT-PCR, and western blot were used to study the mRNA and protein expression levels of ERCC2, UDG, and PCNA.</p><p><b>RESULTS</b>When treated with IC(20) cisplatin, the DNA damage level increased as the cisplatin treated time increased within 24 h of cisplatin treatment. The tail state 12 h and 24 h after treatment was 5.02 +/- 0.68 and 7.22 +/- 0.53 respectively, which was significantly higher than those of the controls (2.73 +/- 0.29). The tail state 24 h after treatment was not significantly different from that of the controls. The DNA damage level decreased to normal after cisplatin treatment in 24 h (tail state 3.64 +/- 0.7). The expression levels of ERCC2, UDG, PCNA protein (4.37 +/- 0.57, 5.47 +/- 0.46, 2.21 +/- 0.47 respectively) and mRNA (0.71 +/- 0.08, 0.74 +/- 0.06, 0.82 +/- 0.09) were increased significantly within 24 h exposure and decreased to normal 24 h after cisplatin treatment. The 3 enzymes' mRNA and protein expression increased when treated with cisplatin, but the changes of protein level were slower than those of mRNA levels.</p><p><b>CONCLUSIONS</b>The DNA repair capability in A549 cells increases after cisplatin treatment. Cisplatin was a positive regulation of ERCC2, UDG, PCNA expression levels, which causes the increase of mRNA, and protein. The positive regulation only works in a short time and returns normal after 24 h of cisplatin treatment.</p>
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Biomarkers, Tumor , Cell Line, Tumor , Cisplatin , Pharmacology , Comet Assay , DNA Glycosylases , Genetics , DNA Helicases , DNA Repair , DNA-Binding Proteins , Gene Expression Regulation, Neoplastic , Lung Neoplasms , Genetics , Metabolism , Pathology , Proliferating Cell Nuclear Antigen , Genetics , Protein Biosynthesis , Proteins , Genetics , RNA, Messenger , Transcription Factors , Xeroderma Pigmentosum Group D ProteinABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of ERCC1 gene on the repair capability of damaged DNA in lung cancer A549 cells induced by benzo[a]pyrene.</p><p><b>METHODS</b>Recombinant plasmid expressing ERCC1 antisense RNA was constructed and transfected into A549 cells by Lipofectin reagent. The stable-transfected cell colonies were selected by hygromycin. Cell viability was determined by the MTT assay. The level of ERCC1 mRNA was measured by Northern Blot analysis. Single cell gel electrophoresis assay was applied to determine the cellular DNA damage and fifty cells for each group were counted.</p><p><b>RESULTS</b>Seven positive colonies expressing ERCC1 antisense RNA were screened. There was no growth rate difference between the antisense-transfected cells and the parental cells. The endogenous mRNA level in transfected colonies decreased in varied degrees, i.e. 12% approximately 86% of that of the parental cells in Northern Blot assay. After 24 h treatment of 10 micro mol/l benzo[a]pyrene, the repair capability for DNA damage in transfected colonies was reduced to 29% approximately 71% of that of the parental cells. Also, a statistically significant correlation was observed between expression of ERCC1 mRNA and repair capability (r = 0.84).</p><p><b>CONCLUSION</b>Antisense ERCC1 RNA decreased the repair capability for damaged DNA in lung cancer cells induced by benzo[a]pyrene.</p>