The Contribution of Apoptosis-inducing Factor, Caspase-activated DNase, and Inhibitor of Caspase-activated DNase to the Nuclear Phenotype and DNA Degradation during Apoptosis

Victor J Yuste,Isabel Sánchez-López,Carme Solé,Rana S Moubarak,José R Bayascas,Xavier Dolcet,Mario Encinas,Santos A Susin,Joan X Comella

doi:10.1074/jbc.m504015200

Abstract

We have assessed the contribution of apoptosis-inducing factor (AIF) and inhibitor of caspase-activated DNase (ICAD) to the nuclear morphology and DNA degradation pattern in staurosporine-induced apoptosis. Expression of D117E ICAD, a mutant that is resistant to caspase cleavage at residue 117, prevented low molecular weight (LMW) DNA fragmentation, stage II nuclear morphology, and detection of terminal deoxynucleotidyl transferase staining. However, high molecular weight (HMW) DNA fragmentation and stage I nuclear morphology remained unaffected. On the other hand, expression of either D224E or wild type ICAD had no effect on DNA fragmentation or nuclear morphology. In addition, both HMW and LMW DNA degradation required functional executor caspases. Interestingly, silencing of endogenous AIF abolished type I nuclear morphology without any effect on HMW or LMW DNA fragmentation. Together, these results demonstrate that AIF is responsible for stage I nuclear morphology and suggest that HMW DNA degradation is a caspase-activated DNase and AIF-independent process.

Highlights

Quently, DNA is degraded into smaller fragments of oligonucleosomal size, known as low molecular weight (LMW) DNA degradation or DNA ladder [2]
We have previously demonstrated that IMR-5 neuroblastoma cells failed to display LMW DNA fragmentation due to a defect in the inhibitor of caspaseactivated DNase (CAD) (ICAD)/ CAD system that results in a non-functional CAD [9]
We demonstrate that overexpression of the ICAD-D117E single mutant prevents LMW DNA fragmentation, stage II chromatin condensation, and detection of TUNEL staining, whereas high molecular weight (HMW) DNA degradation and stage I nuclear morphology remain unaltered

Summary

Introduction

Quently, DNA is degraded into smaller fragments of oligonucleosomal size, known as low molecular weight (LMW) DNA degradation or DNA ladder [2]. Human lymphoma cell lines overexpressing caspase-resistant ICAD lack both LMW and HMW DNA fragmentation [23]. These results suggest that CAD is the enzyme responsible for both HMW and LMW DNA fragmentation. CAD-deficient chicken DT40 cells failed to undergo LMW degradation and stage II chromatin condensation but displayed normal HMW DNA degradation and stage I morphology. These data suggest that factors other than CAD might be involved [24]. It has been suggested that endonucleases, other than CAD, could be responsible for HMW DNA fragmentation (24, 28 –33)

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