Quantitative analysis of UVB-induced apoptosis in human epidermis.
The quantitative measurement of the induction of apoptosis in cells grown in vitro can be accomplished using a variety of proven methods. However, the quantitative assay of apoptosis within an intact tissue is very laborious and the results can be misleading. We have established a method to quantitatively analyze the induction of apoptosis in human epidermis following UVB irradiation. The assay is based on the activation of the apoptotically induced enzyme caspase 3, using a synthetic caspase 3 substrate. The activation of caspase 3 was shown to correlate with the induction of apoptosis in human keratinocytes cultures as a monolayer. We then demonstrated that the activation of caspase 3 could be measured from UVB-irradiated whole skin. The induction of apoptosis was confirmed by cellular morphology and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling. Therefore, we concluded that the measurement of caspase 3 specific activity in UVB-irradiated human epidermis was an efficient, inexpensive, and accurate method to quantitate UVB-induced apoptosis in vivo.
Hurwitz
S A
SA
Department of Dermatology, Indiana University School of Medicine, Indianapolis 46202-5121, USA.
Spandau
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DF
D017209:Apoptosis / Q000528:radiation effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation / Q000528:radiation effects; D000078404:Epidermal Cells; D004817:Epidermis / Q000201:enzymology / Q000528:radiation effects*; D006801:Humans; D020287:In Situ Nick-End Labeling; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology / Q000528:radiation effects; D014466:Ultraviolet Rays / Q000009:adverse effects*
C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases