Stable expression in rat glioma cells of sense and antisense nucleic acids to a human multifunctional DNA repair enzyme, APEX nuclease.
We have cloned mouse and human cDNAs for a multifunctional DNA repair enzyme (APEX nuclease) having apurinic/apyrimidinic (AP) endonuclease, 3',5' exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities. To investigate the biological role of APEX nuclease, sense or antisense APEX RNA was stably expressed at a high level in cultured rat glioma cells by introducing plasmids (pABWN-HAPX1F for expression of sense RNA or pABWN-HAPX2R for expression of antisense RNA) constructed from the human APEX cDNA and an expression vector pABWN. Multiple copies of the construct were integrated into the glioma cells transfected with pABWN-HAPX1F or pABWN-HAPX2R. These transfectants showed markedly high expression of RNA hybridizable to human APEX cDNA, indicating the expression of the sense or antisense RNA. Activity blotting analyses of salt extracts of these transfectants showed that the sense RNA-expressed cells had higher AP endonuclease activity and that the antisense RNA-expressed cells had extremely lower AP endonuclease activity than the control cells. The APEX nuclease-depressed glioma cells became more sensitive to methyl methanesulfonate and hydrogen peroxide than the control cells or the APEX nuclease-overexpressed cells. The results indicate that APEX nuclease plays an important role in repair of DNA damage caused by these genotoxic agents. The present stable expression systems for the sense and antisense APEX RNAs should be useful for analyzing the biological functions such as an antimutagenic function of the enzyme.
Ono
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Y
Department of Neurological Surgery, Okayama University Medical School, Japan.
Furuta
T
T
Ohmoto
T
T
Akiyama
K
K
Seki
S
S
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000235:genetics* / Q000473:pathology; D019757:Carbon-Oxygen Lyases; D002455:Cell Division; D002470:Cell Survival; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005910:Glioma / Q000235:genetics* / Q000473:pathology; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism*; D010957:Plasmids; D012313:RNA / Q000235:genetics*; D016372:RNA, Antisense / Q000235:genetics*; D051381:Rats; D014407:Tumor Cells, Cultured
D009097:Multienzyme Complexes; D016372:RNA, Antisense; D012313:RNA; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; C470319:Apex1 protein, rat; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase