RESUMO
We have used a modification of the Southern hybridization method to analyze the removal of UV-induced pyrimidine cyclobutane dimers from the domain of the c-myc oncogene. The study was performed in human COLO320HSR cells, which exhibit a 30- to 40-fold amplification of c-myc that is maintained in a marker chromosome as a homogeneously staining region. Intron 2 and the region upstream from the gene showed better dimer removal than intron 1 or the region downstream from the c-myc gene. Regions showing less repair coincide with regions that are hotspots for mutations and chromosome translocations. Therefore, it is proposed that the inefficiency of DNA repair may play an important role in the origin of c-myc rearrangements.
Assuntos
Reparo do DNA , Genes myc/efeitos da radiação , Raios Ultravioleta , Sequência de Bases , Southern Blotting , Carcinoma/genética , Mapeamento Cromossômico , Neoplasias do Colo/genética , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Ágar , Humanos , Dados de Sequência Molecular , Dímeros de Pirimidina/genéticaRESUMO
Growing monolayer COLO320 human cells having a 30- to 40-fold amplification of a MYC domain were pulse treated for 15 min with increasing doses of bleomycin (BLM). Cellular DNA was extracted at the end of the BLM treatment or after an interval of 30 min, during which the cells were allowed to repair the DNA damage at 37 degrees C in culture medium without BLM. Damage and repair in total cellular DNA was assessed by alkaline unwinding and by neutral and alkaline gel electrophoresis. The response to BLM in the domain of MYC oncogene was evaluated by Southern blotting of EcoRI-digested DNAs separated by neutral or alkaline gel electrophoresis. We found that MYC domains from COLO320HSR showed a higher frequency of double-strand DNA breaks than MYC domains from COLO320DM cells. At the level of total cellular DNA, both cell lines showed the same frequency of double-strand nicks. No repair of double-strand breaks was observed. Total DNA from COLO320HSR cells was more sensitive to single-strand breakage than DNA from COLO320DM. At the gene level, the frequency of single-strand scissions was higher in COLO320DM than in COLO320HSR MYC domains. Both cell lines had good capability to close single-strand scissions. However, at high BLM doses the damage was more efficiently repaired by COLO320DM cells. We propose that chromatin organization in total cellular DNA and in the amplified MYC domain probably plays an important role in the DNA response to BLM.
Assuntos
Bleomicina/farmacologia , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Amplificação de Genes , Oncogenes/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacos , Adenocarcinoma , Linhagem Celular , Neoplasias do Colo , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Humanos , Cinética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mycRESUMO
COLO320DM and COLO320HSR are two human cell lines with 30-40-fold c-myc amplification. Half of the c-myc gene copies in COLO320DM are truncated and expressed as the predominant mRNA, while half are normal. Most c-myc copies in COLO320HSR are normal and expressed. Truncated c-myc genes are fully methylated, while the normal ones are fully demethylated irrespective of their stage of expression. The normal transcriptionally active c-myc from fibroblast cells is fully methylated, while c-myc from granulocytes (probably downregulated) is almost fully demethylated. These results indicate a lack of correlation between expression and the state of methylation for human c-myc oncogenes. Furthermore, exons 1 and 2 and intron 1 of c-myc are CpG-rich islands. Since these islands are constitutively demethylated, it is assumed that demethylation is the constitutive state and methylation the facultative state of the c-myc oncogene.