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 apoptotic cell death in epidermal cells. In this study annexin V binding and propidium iodide (PI) uptake have been measured by flow cytometry to evaluate UV-induced cell death in the human squamous cell carcinoma-derived cell line A 431. Physiological and therapeutical relevant doses of UVA, UVA1, UVB, narrow-band UVB (311 nm) and photochemotherapy using 100 ng/ml of 8-methoxypsoralen (8-MOP) with UVA or UVA1 (PUVA or PUVA1) have been applied. Doses ranged from 8 to 96 J/cm2 for UVA1 and UVA, from 8 to 128 mJ/cm2 for UVB, from 256 to 4096 mJ/cm2 for narrow-band UVB (311 nm) and from 1 to 16J/cm2 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 annexin 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 sources, dose equivalents are calculated based on the induction of 50% cell death (as determined by PI uptake). The equivalents are 96 J/cm2 for UVA and UVA1, 16 J/cm2 for PUVA and PUVA1, 256 mJ/cm2 for UVB and 2048 mJ/cm2 for narrow-band UVB. Our results establish annexin V/PI double staining as an appropriate method for the quantification of UV-induced cell death. Moreover, they provide a basis for further investigations concerning mechanisms and modifications of UV-induced apoptosis.
Schindl
A
A
Department of Dermatology, University of Vienna Medical School, Austria. andreas.schindl@akh-wien.ac.at
Klosner
G
G
Hönigsmann
H
H
Jori
G
G
Calzavara-Pinton
P C
PC
Trautinger
F
F
D017304:Annexin A5 / Q000493:pharmacokinetics; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000378:metabolism / Q000532:radiotherapy*; D016923:Cell Death / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D008730:Methoxsalen / Q000627:therapeutic use; D010778:Photochemotherapy; D011419:Propidium / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured; D014467:Ultraviolet Therapy
D017304:Annexin A5; D011419:Propidium; D008730:Methoxsalen