A delay prior to mitotic entry triggers caspase 8-dependent cell death in p53-deficient Hela and HCT-116 cells

Abstract

Stathmin/Oncoprotein 18, a microtubule destabilizing protein, is required for survival of p53-deficient cells. Stathmin-depleted cells are slower to enter mitosis, but whether delayed mitotic entry triggers cell death or whether stathmin has a separate pro-survival function was unknown. To test these possibilities, we abrogated the cell cycle delay by inhibiting Wee1 in synchronized, stathmin-depleted cells and found that apoptosis was reduced to control levels. Synchronized cells treated with a 4 hour pulse of inhibitors to CDK1 or both Aurora A and PLK1 delayed mitotic entry and apoptosis was triggered only in p53-deficient cells. We did not detect mitotic defects downstream of the delayed mitotic entry, indicating that cell death is activated by a mechanism distinct from those activated by prolonged mitotic arrest. Cell death is triggered by initiator caspase 8, based on its cleavage to the active form and by rescue of viability after caspase 8 depletion or treatment with a caspase 8 inhibitor. In contrast, initiator caspase 9, activated by prolonged mitotic arrest, is not activated and is not required for apoptosis under our experimental conditions. P53 upregulates expression of cFLIP_L, a protein that blocks caspase 8 activation. cFLIP_L levels are lower in cells lacking p53 and these levels are reduced to a greater extent after stathmin depletion. Expression of FLAG-tagged cFLIP_L in p53-deficient cells rescues them from apoptosis triggered by stathmin depletion or CDK1 inhibition during G2. These data indicate that a cell cycle delay in G2 activates caspase 8 to initiate apoptosis specifically in p53-deficient cells.

Keywords:

• apoptosis
• caspase 8
• cFLIP
• mitotic entry delay
• p53
• stathmin

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors wish to thank Drs. Bob Skibbens, Linda Lowe-Krentz, Frank Luca and Maureen Murphy for helpful discussions and insightful comments. We also thank Dr. B. Vogelstein, Johns Hopkins School of Medicine for HCT116 cell lines and Dr. Marcus Peter, Feinberg School of Medicine, Northwestern University, for a plasmid encoding FLAG-tagged cFLIP.

Funding

This work supported by a grant from the National Institutes of Health, GM100381, to LC.