Bax-mediated cell death by the Gax homeoprotein requires mitogen activation but is independent of cell cycle activity

Abstract

Tissues with the highest rates of proliferation typically exhibit the highest frequencies of apoptosis, but the mechanisms that coordinate these processes are largely unknown. The homeodomain protein Gax is downregulated when quiescent cells are stimulated to proliferate, and constitutive Gax expression inhibits cell proliferation in a p21 WAF/CIP -dependent manner. To understand how mitogen-induced proliferation influences the apoptotic process, we investigated the effects of deregulated Gax expression on cell viability. Forced Gax expression induced apoptosis in mitogen-activated cultures, but quiescent cultures were resistant to cell death. Though mitogen activation was required for apoptosis, neither the cdk inhibitor p21 WAF/CIP nor the tumor suppressor p53 was required for Gax-induced cell death. Arrest in G 1 or S phases of the cell cycle with chemical inhibitors also did not affect apoptosis, further suggesting that Gax-mediated cell death is independent of cell cycle activity. Forced Gax expression led to Bcl-2 down-regulation and Bax up-regulation in mitogen-activated, but not quiescent cultures. Mouse embryonic fibroblasts homozygous null for the Bax gene were refractive to Gax-induced apoptosis, demonstrating the functional significance of this regulation. These data suggest that the homeostatic balance between cell growth and death can be controlled by mitogen-dependent pathways that circumvent the cell cycle to alter Bcl-2 family protein expression.

Adenovirus-mediated delivery of the Gax transcription factor to rat carotid arteries inhibits smooth muscle proliferation and induces apoptosis

“…Here, we have shown that Gax exhibits dual actions in VSMCs of balloon-injured rat carotid arteries, promoting both cell cycle arrest and apoptosis, consistent with previous in vitro observations. 19,20 It is noteworthy that Gax is unable to induce apoptosis in quiescent VSMCs, apoptosis is observed only in proliferating cells, 20 the subpopulation that forms the stenotic lesion in injured vessels. 29 Such a lack of toxicity to quiescent VSMCs indicates that Ad-Gax should prove an advantageous therapeutic strategy for treating conditions characterized by VSMC hyperproliferation.…”

“…in a volume of 100 l of either AdGax 19,20 or Ad-␤-gal. 19,20,30 The solution was injected via a cannula inserted just proximal to the carotid bifurcation and the vessel was incubated for 20 min, after which the solution was withdrawn and the cannula removed. All transgenes are expressed under control of the cytomegalovirus promoter.…”

“…19 In addition, Gax-expressing cells undergo apoptotic cell death under conditions of prolonged mitogen-activation while quiescent cells are refractive to Gax-induced apoptosis. 20 When the effects of constitutive Gax expression were analyzed in vivo, neointimal hyperplasia was markedly attenuated in both rat carotid 19 and rabbit iliac arteries 21 following injury. Other investigators have reported similar results with adenovirus-mediated expression of cell cycle inhibitors or cytotoxic genes in the vessel wall.…”

IL-6 and Matrix Metalloproteinase-1 Are Regulated by the Cyclin-Dependent Kinase Inhibitor p21 in Synovial Fibroblasts

“…For cell cycle analysis and determination of subdiploid DNA content, cultures were harvested by trypsinization, fixed in 70% ethanol overnight, and stained with propidium iodide (Roche, Indianapolis, IN) as previously described (32). A gate was established to count 10,000 events for each sample.…”

Effect of percutaneous adenovirus-mediated Gax gene delivery to the arterial wall in double-injured atheromatous stented rabbit iliac arteries

“…20 However, homeodomain transcription factors are widely recognized to function as pleiotropic regulators and gax overexpression may also affect VSMC differentiation, migration or viability. 22,61,62 In contrast to the Ad-Gaxtreated arteries, no discernible difference was seen in the extent of neointima formation between the Ad-CMVluctreated and saline-treated arteries in the control group of animals. The ability of Gax overexpression to inhibit neointima formation compares favorably with previous recent results reported by other groups using adenovirusencoded genes for wild-type and mutant form of RB in rat and porcine models, 46,63 the herpes simplex virus thymidine kinase gene in rat, rabbit and porcine models, 26,27,55,56 p21 53 in the rat model and hirudin in the rat model.…”