Commitment and effector phases of the physiological cell death pathway elucidated with respect to Bcl-2 caspase, and cyclin-dependent kinase activities

Abstract

Physiological cell deaths occur ubiquitously throughout biology and have common attributes, including apoptotic morphology with mitosis-like chromatin condensation and prelytic genome digestion. The fundamental question is whether a common mechanism of dying underlies these common hallmarks of death. Here we describe evidence of such a conserved mechanism in different cells induced by distinct stimuli to undergo physiological cell death. Our genetic and quantitative biochemical analyses of T- and B-cell deaths reveal a conserved pattern of requisite components. We have dissected the role of cysteine proteases (caspases) in cell death to reflect two obligate classes of cytoplasmic activities functioning in an amplifying cascade, with upstream interleukin-1beta-converting enzyme-like proteases activating downstream caspase 3-like caspases. Bcl-2 spares cells from death by punctuating this cascade, preventing the activation of downstream caspases while leaving upstream activity undisturbed. This observation permits an operational definition of the stages of the cell death process. Upstream steps, which are necessary but not themselves lethal, are modulators of the death process. Downstream steps are effectors of, and not dissociable from, actual death; the irreversible commitment to cell death reflects the initiation of this downstream phase. In addition to caspase 3-like proteases, the effector phase of death involves the activation in the nucleus of cell cycle kinases of the cyclin-dependent kinase (Cdk) family. Nuclear recruitment and activation of Cdk components is dependent on the caspase cascade, suggesting that catastrophic Cdk activity may be the actual effector of cell death. The conservation of the cell death mechanism is not reflected in the molecular identity of its individual components, however. For example, we have detected different cyclin-Cdk pairs in different instances of cell death. The ordered course of events that we have observed in distinct cases reflects essential thematic elements of a conserved sequence of modulatory and effector activities comprising a common pathway of physiological cell death.

FIG. 1

In B cells, physiological cell death and associated caspase 3-like protease and Cdk activities are inhibited by Bcl-2, but upstream ICE-like protease activity is not. The kinetics of induction of death (A), cytoplasmic YVAD-specific ICE-like (B) and DEVD-specific caspase 3-like (C) activities, and nuclear H1-specific Cdk activity (D) upon shift to the nonpermissive temperature were monitored in DE cells and DE/Bcl-2 cells. Cysteine protease activities were not detectable in nuclear extracts, and only basal levels of H1 kinase activity were present in cytoplasmic extracts of dying cells. The data presented are derived from one experiment in which a single set of extracts was used for all activity determinations. These data are representative of three separate experiments. In other experiments, elevated DEVD-specific caspase activity was not detectable at time points before 3 h of temperature shift.

FIG. 2

Glucocorticoid-mediated cell death of T cells and associated caspase 3-like protease and Cdk activities are inhibited by Bcl-2, but upstream ICE-like protease activity is not. The kinetics of induction of death (A), cytoplasmic YVAD-specific ICE-like (B) and DEVD-specific caspase 3-like (C) activities, and nuclear H1-specific Cdk activity (D) were monitored after treatment with 10⁻⁶ M dexamethasone in DO11.10 cells and in representative clones of DO11.10 transfected with bcl-2 and crmA. The behaviors of DO11.10 cells transfected with p35 and with CrmA were identical; data for the DO11.10/p35 transfectants is omitted for clarity of presentation. Note that histone kinase activity in panel D was measured with histone H1 substrate peptide (see Materials and Methods). Comparable kinetics of appearance of kinase activity, although with lower apparent specific activity, were observed with biotinylated H1 substrate (see Fig. 6 for a comparison). The data presented are derived from one experiment in which a single set of extracts was used for all activity determinations. These data are representative of three separate experiments.

FIG. 3

Characterization in vitro of cell death-associated cysteine protease activities. YVAD-specific ICE-like (A) and DEVD-specific caspase 3-like (B) activities were assayed in the presence of the protease inhibitors NEM and PMSF and the specific aldehyde-derivatized tetrapetide inhibitors YVAD-CHO and DEVD-CHO.

FIG. 4

The appearance of class III caspase activity and cell death is dependent on upstream class I activity. The ability of aldehyde-derivatized tetrapeptide protease inhibitors to block in vivo DEVD-specific class III caspase activity (A) and cell death (B) induced by glucocorticoid treatment was monitored in DO11.10 cells. The derivatized DEVD tetrapeptide itself (left) or the derivatized YVAD tetrapeptide specific for class I proteases (right) was added at the indicated concentrations simultaneously with or 1 h before the addition of 10⁻⁶ M dexamethasone. DEVD-specific activity was assayed 12 h after glucocorticoid addition. Cell death was quantified by measurement of trypan blue inclusion 24 h after hormone addition.

FIG. 5

Immunoblot analysis of cell death-associated nuclear cyclin A-dependent kinases. Cyclin A (A) and Cdk (B) components were identified by immunoblot analysis. Cytoplasmic and nuclear extracts were prepared from DE and DE/Bcl-2 cells (upper panels) incubated at the permissive temperature (34°C; −) and at the restrictive temperature (39.5°C; +) for 4 h and from DO11.10 cells and representative clones of DO11.10 transfected with bcl-2 (DO/Bcl-2) and crmA (DO/CrmA) (lower panels) that were untreated (−) or after treatment with 10⁻⁶ M dexamethasone for 6 h (+). Analysis of a nuclear extract from DO11.10 cells treated for 12 h with 2 μg of aphidicolin per ml (Aph.) is included for comparison in panel A. The densitometric quantitation of nuclear cyclin A expression is given in Table 2.

FIG. 6

Cell death-associated histone H1 kinase activities represent cyclin A-dependent Cdks. (A) Death kinase activity was analyzed by immunodepletion analysis. Nuclear extracts from untreated DO11.10 cells or cells treated for 12 h with 10⁻⁶ M dexamethasone were analyzed for histone kinase activity following depletion with antibody specific for the indicated cyclin (αcyc) or Cdk (αCdk 2) or with irrelevant anti-Myc (αMyc) antibody. (B) Nuclear extracts from DE cells incubated at the restrictive temperature for 6 h and from DO11.10 cells treated with dexamethasone (Dex.) as above, as well as viable control (Ctrl.) cells, were treated with p9^cksHs2 agarose to adsorb nuclear Cdk activity. Adsorbed and unadsorbed Cdk activities from 1 μg of nuclear extract were quantified. Note that for the experiments in panel A, H1 substrate peptide was used, while the activity in panel B was assayed with biotinylated H1. Comparison of DO11.10 activity in the two panels demonstrates the difference in apparent specific activity obtained with the different substrates.