FIG. 7.

Caspase 3 activation pattern in primary and transformed keratinocytes. (A) DEVD-specific caspase activity in cellular lysates was analyzed 3 h after TRAIL stimulation in primary keratinocytes (PK) and transformed HaCaT keratinocytes (TK). DEVDase activity of p19, p17, and p15 (upper panel) was detectable in transformed keratinocytes treated with TRAIL, whereas the same concentrations of TRAIL did not induce detectable DEVDase activity of cleaved fragments of caspase 3 in primary keratinocytes. Induction of apoptosis was monitored in parallel plates by hypodiploidy analysis. Higher concentrations of TRAIL sufficient to induce partial cell death in primary keratinocytes led to a dose-dependent increase in full-length caspase 3 labeling, indicative of some enzymatic activity of full-length caspase 3. Subsequent reprobing of blots with caspase 3-specific antibody confirmed that low concentrations of TRAIL were sufficient to induce the slower-migrating caspase 3 form (p20) in primary keratinocytes. In contrast, mainly the mature cleaved forms p17 and a smaller fragment (p15) were detected in transformed keratinocytes, even at low concentrations of TRAIL. Ponceau Red staining of the membrane confirmed comparable loading of proteins. HRP, horseradish peroxidase.