In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies.
The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derived DNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N(2)-yl)-2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF-nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF. Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells.
Iwamoto
Taka-aki
TA
Radioisotope Research Center, Department of Dermatology, Nara Medical University, Kashihara, Nara 634-8521, Japan.
Kobayashi
Nobuhiko
N
Imoto
Kyoko
K
Yamamoto
Aya
A
Nakamura
Yu
Y
Yamauchi
Yukika
Y
Okumura
Hiromi
H
Tanaka
Akiko
A
Hanaoka
Fumio
F
Shibutani
Shinya
S
Miyagawa
Sachiko
S
Mori
Toshio
T
D000099:Acetoxyacetylaminofluorene / Q000032:analysis* / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002460:Cell Line; D018736:DNA Adducts / Q000032:analysis* / Q000276:immunology*; D004249:DNA Damage; D004260:DNA Repair; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008856:Microscopy, Fluorescence; D014983:Xeroderma Pigmentosum / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018736:DNA Adducts; D000099:Acetoxyacetylaminofluorene