Fe(II)-induced DNA damage in alpha-synuclein-transfected human dopaminergic BE(2)-M17 neuroblastoma cells: detection by the Comet assay.
Lewy bodies in the brains of patients with Parkinson's disease (PD) contain aggregates of alpha-synuclein (alpha-syn). Missense mutations (A53T or A30P) in the gene encoding alpha-syn are responsible for rare, inherited forms of PD. In this study, we explored the susceptibility of untransfected human dopaminergic BE(2)-M17 neuroblastoma cells, cells transfected with vector only, or cells transfected with wild-type alpha-syn, A30P alpha-syn or A53T alpha-syn to Fe(II)-induced DNA damage in the form of single-strand breaks (SSBs). DNA SSBs were detected following 2-h treatments with various concentrations of Fe(II) (0.01-100.0 microm), using the alkaline single cell-gel electrophoresis ('Comet') assay and quantified by measuring comet tail length (CTL) microm). Fe(II) treatment induced significant increases in CTL in cells transfected with A30P alpha-syn or A53T alpha-syn, even at the lowest concentrations of Fe(II) tested. In comparison, untransfected cells, vector control cells or cells transfected with wild-type alpha-syn exhibited increases in SSBs only when exposed to concentrations of 1.0 microm Fe(II) and above. Even when exposed to higher concentrations (10.0-100.0 microm) of Fe(II), untransfected cells, vector control cells or cells transfected with wild-type alpha-syn were less susceptible to DNA-damage induction than cells transfected with A30P alpha-syn or A53T alpha-syn. Incorporation of DNA-repair inhibitors, hydroxyurea and cytosine arabinoside, enhanced the sensitivity of DNA damage detection. Susceptibility to Fe(II)-induced DNA damage appeared to be dependent on alpha-syn status because cells transfected with wild-type alpha-syn or A53T alpha-syn were equally susceptible to the damaging effects of the mitochondrial respiratory chain inhibitor rotenone. Overall, our data are suggestive of an enhanced susceptibility to the toxic effects of Fe(II) in neuroblastoma cells transfected with mutant alpha-syn associated with inherited forms of PD.
Martin
Francis L
FL
Department of Biological Sciences, I.E.N.S., Lancaster University, Lancaster, UK. f.martin@lancaster.ac.uk
Williamson
Sally J M
SJ
Paleologou
Katerina E
KE
Hewitt
Rebecca
R
El-Agnaf
Omar M A
OM
Allsop
David
D
D019943:Amino Acid Substitution; D045744:Cell Line, Tumor; D020552:Comet Assay; D003561:Cytarabine / Q000494:pharmacology; D004247:DNA / Q000032:analysis / Q000187:drug effects; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects; D004298:Dopamine / Q000378:metabolism*; D004579:Electron Transport / Q000187:drug effects; D005296:Ferrous Compounds / Q000633:toxicity*; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D051843:Synucleins; D014162:Transfection; D014475:Uncoupling Agents / Q000494:pharmacology; D051844:alpha-Synuclein
D005296:Ferrous Compounds; D009419:Nerve Tissue Proteins; C497604:SNCA protein, human; D051843:Synucleins; D014475:Uncoupling Agents; D051844:alpha-Synuclein; D012402:Rotenone; D003561:Cytarabine; D004247:DNA; C029451:ferrous chloride; D004298:Dopamine; D006918:Hydroxyurea