Figure 4.

ΔEGFR expression causes reduced activation of caspase-3-like proteases in response to CDDP treatment. (A) Total cell lysates were prepared 2 days after treatment with 5 μg/ml of CDDP. Samples were assayed for protease activity by using the peptide substrate Ac-DEVD-pNA. For inhibition of the protease activity, 10 μM Ac-DEVD-CHO was added to the reaction mixture before the addition of substrate. Cells also were treated with the plasma membrane-soluble caspase inhibitor Z-Asp-CH₂-DCB (200 μM) before and during CDDP treatment. □, U87MG; ▪, U87MG.ΔEGFR; ▨, U87MG.DK; ▧, U87MG.wtEGFR. Values represent the means of seven independent experiments (two for the Z-Asp-CH₂-DCB experiment). ∗∗∗, Significantly different (P < 0.001) from the value for U87MG.ΔEGFR cells treated with CDDP. (Bars = SE.) (B) Proteolytic cleavage of PARP after CDDP treatment. For each sample, 20 μg total clarified protein lysate was loaded onto SDS/PAGE gels, electrophoresed, transferred to membranes, and probed with anti-PARP mAbs. FL, full-length; CF, cleaved fragment.