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Flow Cytometric Analysis of 8-Methoxypsoralen-DNA Photoadducts in Human Keratinocytes¹

Comprehensive Cancer Center and Division of Environmental Sciences, School of Public Health, Columbia University, New York, New York 10032

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.

¹ This work was supported by Grants ES03881 from the National Institute of Environmental Health Sciences and CA21111.

² To whom requests for reprints should be addressed, at Division of Environment Sciences, Columbia University, 650 W. 168th St., New York, NY 10032.

Received 4/19/88. Revised 8/29/88. Accepted 9/ 8/88.