Loss of Caspase-9 Provides Genetic Evidence for the Type I/II Concept of CD95-mediated Apoptosis

Ajoy K Samraj,Eric Keil,Nana Ueffing,Klaus Schulze-Osthoff,Ingo Schmitz

doi:10.1074/jbc.m603487200

Ajoy K Samraj, Eric Keil + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m603487200

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Abstract

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.

Highlights

Jurkat cells were incubated with different concentrations of anti-CD95 antibody for 18 h, and apoptosis was measured by quantification of subdiploid DNA in a flow cytometer
We observed that one Jurkat clone, designated JMR, exhibited a profound resistance toward CD95-mediated apoptosis when compared with J16 or other Jurkat clones (Fig. 1A)
At a concentration of 100 ng/ml of anti-CD95, J16 cells showed 41.8% Ϯ 7.3% apoptosis compared with 11.6% Ϯ 3.6% in JMR cells

Summary

Introduction

J16 and JMR cells as well as Jurkat cells overexpressing Bcl-2 or a vector control were stimulated for the indicated time with 1 ␮g/ml anti-CD95 and 10 ng/ml protein A and analyzed for cell death by the propidium iodide dye exclusion assay. These clones showed comparable sensitivity toward antiCD95 and CD95L treatment as J16 cells suggesting that caspase-9 deficiency is the only defect within the CD95 pathway in JMR cells (Fig. 4B).

Results

Conclusion