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.

N-acetoxy-N-2-acetylaminofluorene-induced damage on SV40 DNA: inhibition of DNA replication and visualization of DNA lesions.

The chemical carcinogen, N-acetoxy-N-2-acetylaminofluorene (AAAF), which induces well characterized DNA lesions, strongly inhibits Simian virus 40 (SV40) DNA replication. By using SV40 mutants which were temperature-sensitive for replication initiation, we were able to synchronize SV40 DNA replication and therefore to introduce AAAF-induced lesions only on unreplicating SV40 molecules. One to two acetylaminofluorene (AAF)-adducts per SV40 genome inhibit more than 90% of normal semi-conservative DNA synthesis. SV40 replicative intermediates (RIs) from AAAF-treated infected cells, purified through neutral sucrose gradients and BND-cellulose column, possess a structure different from the usual Cairns molecules found in the untreated cultures. Both by neutral and alkaline sucrose gradients and by electron microscopy, the RIs isolated from treated cells appear as complex molecules with single-stranded portions and sometimes with a tailed structure. Moreover, the newly synthesized strands found in these molecules are equal in size to the average distance between AAF-adducts on the template strand, indicating that AAF-adducts represent a block for the SV40 DNA replication. By using specific anti Guo-AAF antibodies and electron microscopy, we show the presence of an AAF adduct at halted replication forks, i.e. showing a DNA replication block in a mammalian replicon for the first time. We therefore assume that AAF-adducts severely block the progression of the replication forks by inhibiting, at least, the in vivo polymerization of the leading strand.

An ELISA for detection of DNA-bound carcinogen using a monoclonal antibody to N-acetoxy-2-acetylaminofluorene-modified DNA.

We have produced and characterized a murine monoclonal antibody that recognizes DNA modified with N-acetoxy-2-acetylaminofluorene. The effectiveness of competitive binding inhibitors in an ELISA indicates that this antibody binds most strongly to N-(guanin-8-yl)-2-acetylaminofluorene. It also binds to N-(guanin-8-yl)-2-aminofluorene, albeit some 20-fold less avidly. Thus the monoclonal antibody displays specificity generally similar to the polyclonal antisera elicited by other laboratories but having the advantage of stable and precisely defined specificities. We employed a biotin-streptavidin ELISA to demonstrate that the antibody can detect less than 10 fmol of N-(guanin-8-yl)-2-acetylaminofluorene. N-(guanin-8-yl)-2-acetylaminofluorene is a more effective competitive binding inhibitor of the antibody than is N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene or calf thymus DNA modified with N-acetoxy-2-acetylaminofluorene. Thus the antibody should be most useful in quantifying the persistence of N-acetoxy-2-acetylaminofluorene adducts in DNA hydrolyzed with trifluoroacetic acid.

Chemically modified nucleic acids as immunodetectable probes in hybridization experiments.

Guanine residues in nucleic acids can be modified by treatment with N-acetoxy-N-2-acetylaminofluorene and its 7-iodo derivative in an in vitro nonenzymatic reaction. The modified nucleic acids (ribo or deoxyribo, single or double stranded) are recognized by specific antibodies. They can be immunoprecipitated or used as probes in hybridization experiments and detected by immunochemical techniques.

Monoclonal antibodies to DNA modified by the carcinogen N-acetoxy-N-2 acetyl aminofluorene.

BALB/c mice were immunized against acetyl aminofluorene-substituted guanosine (AAF.Guo). Hybridomas were generated from the immune splenocytes fused with the SP2/0.Ag8 cell line. Four monoclonal antibodies (mAb) were selected. Two of them recognize mainly AAF. substituted DNA but also native DNA, while the others (derived from the hybridoma 16) are specific for the modified DNA. Using a solid phase radioimmunoassay, the fine specificity of mAb 16 was examined. The interaction between purified mAb 16 an AAF.DNA is inhibited by either AAF.GMP, AAF.Guo, or AAF.DNA. Moreover this interaction is slightly inhibited by aminofluorene-substituted guanosine (AF.Guo). By control, free AAF, fluorenamine, guanosine, desoxy GMP and native DNA do not compete in the assay. This mAb 16, which specifically recognizes the AAF-nucleotide complex may be a very useful tool to analyze the carcinogenic processes. Such an antibody can also be used for chromosomal localization of genes by AAF-substituted probes.

Electron microscopic visualization of N-acetoxy-N-2-acetylaminofluorene binding sites in ColE1 DNA by means of specific antibodies.

ColE1 DNA has been allowed to react in vitro with N-acetoxy-N-2-[14C]acetylaminofluorene in the range of 0-15 N-2-[14C]acetylaminofluorene residues bound per molecule of DNA, at the C8 of guanine residues. Purified rabbit antibodies to both N-2-(guanosine-8-yl)-acetylaminofluorene and native DNA that had reacted with N-acetoxy-N-2-acetylaminofluorene were shown by electron microscopy to recognize specifically the acetylaminofluorene-modified ColE1 DNA. The antibodies bound to DNA were visualized either per se or after reaction with goat anti-rabbit immunoglobulins coupled with ferritin. There was a linear relationship between the average number of antibodies bound per DNA molecule and the number of N-2-(deoxyguanosine-8yl)-acetylaminofluorene residues per DNA molecule. The slope of this straight line was equal to 0.4. Due to the bivalence of the immunoglobulins one would expect a value of 0.5; we actually observed an important fraction of the bound antibodies crosslinking two parts of the same (or of another) DNA molecule.