QUANTIFICATION OF SPECIFIC DNA O-ALKYLATION PRODUCTS IN INDIVIDUAL CELLS BY MONOCLONAL-ANTIBODIES AND DIGITAL IMAGING OF INTENSIFIED NUCLEAR FLUORESCENCE
F. Seiler et al., QUANTIFICATION OF SPECIFIC DNA O-ALKYLATION PRODUCTS IN INDIVIDUAL CELLS BY MONOCLONAL-ANTIBODIES AND DIGITAL IMAGING OF INTENSIFIED NUCLEAR FLUORESCENCE, Carcinogenesis, 14(9), 1993, pp. 1907-1913
We report the establishment of a standardized, monoclonal antibody (Ma
b)-based immunocytological assay (quantitative ICA) for the visualizat
ion and quantification of low levels of specific DNA O-alkylation prod
ucts in individual cells by electronically intensified, indirect or di
rect immunofluorescence. In terms of specific binding to alkali-denatu
red nuclear DNA and low background noise, 10 Mabs from a collection of
154 Mabs specific for O6-methyl-2'-deoxyguanosine (O6-MedGuo), O6-eth
yl-2'-deoxyguanosine (O6-EtdGuo), O6-n-butyl-2'-deoxyguanosine (O6-Bud
Guo) and O4-ethyl-2'-deoxythymidine (O4-EtdThd) with antibody affinity
constants ranging between 1.0 x 10(-6)-3.0 x 10(10)/mol were found to
be best suited for ICA. At present, greater-than-or-equal-to 200 O6-E
tdGuo residues (corresponding to an O6-EtdGuo/dGuo molar ratio in DNA
of greater-than-or-equal-to 8.4 x 10(-8)), greater-than-or-equal-to 40
0 O6-BudGuo residues (O6-BudGuo/dGuo, greater-than-or-equal-to 1.7 x 1
0(-7)), greater-than-or-equal-to 1800 O4-EtdThd residues (O4-EtdThd/dT
hd, greater-than-or-equal-to 7.5 x 10(-7)) and greater-than-or-equal-t
o 4800 O6-MedGuo residues (O6-MedGuo/dGuo, greater-than-or-equal-to 2.
0 x 10(-6)), can be quantified per diploid genome. Using a SIT video c
amera in combination with multiparameter image digital analysis, DNA a
dduct-specific rhodamine fluorescence signals are measured relative to
nuclear DNA content (DAPI fluorescence). Adduct-specific fluorescence
recordings in three different rat cell lines (BT3Ca, Fao and NO) were
in excellent agreement with the data obtained by competitive radioimm
unoassay (RIA) for hydrolysates of DNA isolated from the respective ce
lls exposed in parallel to the same alkylating carcinogens (N-methyl-,
N-ethyl- and N-[n-butyl]-N-nitrosourea). Accordingly, the kinetics of
O6-EtdGuo repair, as determined by ICA and RIA, respectively, were su
perimposable. Cell-specific, quantitative ICA can, therefore, be used
for the quantification of specific, stable DNA adducts induced by alky
lating carcinogens or chemotherapeutic agents and for DNA repair measu
rements in individual (e.g. human) cells. Work is currently underway t
o extend the spectrum of carcinogen - DNA adduct-specific Mabs suited
for quantitative ICA.