Activation of the CPP32 apoptotic protease by distinct signaling pathways with differential sensitivity to Bcl-xL

Area of Science:

• Cell biology
• Molecular biology
• Biochemistry

Background:

• Mammalian cells undergo apoptosis without growth factors.
• Growth factors enhance cell survival via phosphatidylinositol 3-kinase (PI 3-kinase) activation.
• Apoptotic pathways share common elements but can be differentially regulated.

Purpose of the Study:

• To compare downstream apoptotic pathways activated by PI 3-kinase inhibitors and other stimuli.
• To investigate the role of CPP32 activation and Bcl-xL in distinct cell death signaling.
• To differentiate cell death mechanisms induced by PI 3-kinase inhibition versus TNFalpha.

Main Methods:

• Treatment of U937 cells with PI 3-kinase inhibitors (wortmannin, LY294002), etoposide, and TNFalpha.
• Analysis of CPP32 apoptotic protease activation (cleavage to p17 and p20 subunits).
• Overexpression of Bcl-xL to assess its effect on DNA fragmentation, CPP32 activation, and poly(ADP-ribose) polymerase cleavage.

Main Results:

• Both PI 3-kinase inhibitors and etoposide induced CPP32 activation via p17 subunit cleavage.
• TNFalpha treatment resulted in a distinct active CPP32 subunit, p20.
• Bcl-xL overexpression inhibited apoptosis induced by PI 3-kinase inhibitors and etoposide, but not by TNFalpha.

Conclusions:

• Distinct patterns of CPP32 activation differentiate cell death pathways.
• Differential sensitivity to Bcl-xL distinguishes PI 3-kinase/DNA damage-induced apoptosis from TNFalpha-induced apoptosis.
• These findings elucidate distinct molecular mechanisms underlying growth factor-dependent and -independent cell survival pathways.