GDP dissociation inhibitor D4-GDI (Rho-GDI 2), but not the homologous Rho-GDI 1, is cleaved by caspase-3 during drug-induced apoptosis

Abstract

Different cytotoxic drugs induce cell death by activating the apoptotic programme; a family of cysteinyl aspartate proteases named caspases has been shown to be involved in the initiation as well as the execution of this kind of cell death. In the present study, cleavage of D4-GDI (Rho-GDI 2), an abundant haemopoietic-cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, was demonstrated after treatment of BJAB Burkitt-like lymphoma cells with taxol or epirubicin. The cleavage of D4-GDI occurred simultaneously with the activation of caspase-3 but preceded DNA fragmentation and the morphological changes associated with apoptotic cell death. By using high-resolution two-dimensional gel electrophoresis it was shown that this cleavage is specific: whereas the level of the homologous protein Rho-GDI 1 was not significantly altered during drug-induced apoptosis and in cytochrome c/dATP-activated cellular extracts, D4-GDI disappeared owing to proteolytic cleavage. Inhibitor experiments with Z-DEVD-fmk (in which Z stands for benzyloxycarbonyl and fmk for fluoromethyl ketone) and microsequencing of the D4-GDI fragment revealed that this occurs at the caspase-3 cleavage site. Our results strongly suggest the differential regulation of the homologous GDP dissociation inhibitors Rho-GDI 1 and D4-GDI during drug-induced apoptosis by proteolysis mediated by caspase-3 but not by caspase-1. Owing to their crucial role as modulators of Rho GTPases, this might in turn have a significant impact on the mechanisms that induce the cytoskeletal and morphological changes in apoptotic cells.

Overexpression of caspase-3 restores sensitivity for drug-induced apoptosis in breast cancer cell lines with acquired drug resistance

“…Several anticancer drugs have been demonstrated to induce apoptotic cell death and to activate caspases in a variety of cellular systems (Essmann et al, 2000;Ibrado et al, 1996a,b;Suzuki et al, 1998;Wesselborg et al, 1999). Although these cytotoxic agents head for di erent primary targets it has become obvious that in most cases drug-induced apoptosis converges on a common pathway, causing mitochondrial membrane permeabilization (Kroemer and Reed, 2000) and subsequent activation of downstream caspases, such as caspase-3.…”

Adenine deoxynucleotides fludarabine and cladribine induce apoptosis in a CD95/Fas receptor, FADD and caspase-8-independent manner by activation of the mitochondrial cell death pathway

“…Apoptosis was determined on the single cell level by measuring the DNA content of individual cells by flow cytometry as described (Essmann et al, 2000). Cellular DNA content was measured with a logarithmic amplification in the FL-3 channel of a FACScan flow cytometer (Becton Dickinson; Heidelberg, Germany) equipped with the CellQuest software.…”

“…Western blot analyses were performed as described (Essmann et al, 2000). Protein bands were visualized using the enhanced chemiluminescence (ECL) system and exposed to Hyperfilm (both Amersham-Buchler; Braunschweig, Germany).…”

Induction of apoptosis by enediyne antibiotic calicheamicin ϑII proceeds through a caspase-mediated mitochondrial amplification loop in an entirely Bax-dependent manner

“…DNA fragmentation was measured essentially as described (Daniel et al, 1999;Essmann et al, 2000). After treatment with different concentrations of calicheamicin, cells were detached by trypsination and collected by centrifugation at 300 g for 5 min.…”