Flow cytometric analysis of 8-methoxypsoralen-DNA photoadducts in human keratinocytes.
Flow cytometric methods have been developed for the analysis of 8-methoxypsoralen-DNA adducts in SV40-transformed human keratinocytes (svk-14). Monoclonal antibodies which specifically recognize these adducts were used in conjunction with fluorescein isothiocyanate-labeled second antibodies and propidium iodide staining to simultaneously determine 8-methoxypsoralen-DNA adduct levels and DNA content, respectively. The sensitivity of the method was determined by quantitation of adduct levels in DNA isolated from treated cells by enzyme-linked immunosorbent assay. Adduct formation during various stages of the cell cycle was also investigated. Two separate cell populations with a DNA content indicative of cells in G1 but with different levels of fluorescein isothiocyanate labeling were observed. Pretreatment of cells with aphidicolin decreased the number of cells in S phase, and only one fluorescein isothiocyanate-staining G1 population was observed. There is an indication of greater adduct formation in S-phase cells when immunofluorescence is corrected for DNA content. These results suggest that adduct formation is not simply linear with respect to DNA content.
Yang
X Y
XY
Comprehensive Cancer Center, School of Public Health, Columbia University, New York, New York 10032.
Delohery
T
T
Santella
R M
RM
D000911:Antibodies, Monoclonal; D004247:DNA / Q000032:analysis*; D018736:DNA Adducts; D004797:Enzyme-Linked Immunosorbent Assay; D004817:Epidermis / Q000032:analysis*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006801:Humans; D007633:Keratins; D008730:Methoxsalen / Q000032:analysis*; D013539:Simian virus 40; D013861:Thiocyanates
C057863:8-methoxypsoralen-DNA; D000911:Antibodies, Monoclonal; D018736:DNA Adducts; D005452:Fluoresceins; D013861:Thiocyanates; D007633:Keratins; D004247:DNA; D016650:Fluorescein-5-isothiocyanate; D008730:Methoxsalen