FLOW CYTOMETRIC QUANTIFICATION OF UV-INDUCED CELL-DEATH IN A HUMAN SQUAMOUS-CELL CARCINOMA-DERIVED CELL-LINE - DOSE AND KINETIC-STUDIES

Exposure to ultraviolet (UV) radiation and photochemotherapy induces a poptotic cell death in epidermal cells. In this study annexin V bindin g and propidium iodide (PI) uptake have been measured by flow cytometr y to evaluate W-induced cell death in the human squamous cell carcinom a-derived cell line A 431. Physiological and therapeutical relevant do ses of UVA, UVA1, UVB, narrow-band UVB (311 nm) and photochemotherapy using 100 ng/ml of 8-methoxypsoralen (8-MOP) with WA or UVA1 (PUVA or PUVA1) have been applied. Doses ranged from g to 96 J/cm(2) for UVA1 a nd WA, from 8 to 128 mJ/cm(2) for WE, from 256 to 4096 mJ/cm(2) for na rrow-band UVB (311 nm) and from 1 to 16 J/cm(2) for photochemotherapy. Results show that the amount of annexin V binding, a measure of early apoptosis, as well as PI uptake, a parameter of ultimate cell death, are strictly correlated with the applied UV dose. Peak values of annex in V-positive cells are noted 12 h after UV exposure in all protocols and are followed by an increase of PI-uptaking cells with peak values at 24 h after UVA and UVA1, and 48 h after PUVA, PUVA1, UVB and narrow -band UVB. To compare the effect of different wavelengths and light so urces, dose equivalents are calculated based on the induction of 50% c ell death las determined by PI uptake). The equivalents are 96 J/cm(2) for UVA and UVA1, 16 J/cm(2) for PUVA and PUVA1, 256 mJ/cm(2) for UVB and 2048 mJ/cm(2) for narrow-band UVB. Our results establish annexin V/PI double staining as an appropriate method for the quantification o f UV-induced cell death. Moreover, they provide a basis for further in vestigations concerning mechanisms and modifications of UV-induced apo ptosis. (C) 1998 Elsevier Science S.A. All rights reserved.