Abstract
The effects of enforced expression of p53 on the sensitivity of p53 −/− human monocytic leukemia cells (U937) to apoptosis following exposure to the S-phase-specific antimetabolite 1-[β- d-arabinofuranosyl]cytosine (ara-C) were examined. Cells were stably transfected with a plasmid containing a chimeric DNA construct encoding a temperature-sensitive p53 variant (135 ala⇒val), which transactivates at 32° but is non-functional at 37°. A significant reduction in the S-phase population was observed in ptsp53 mutants incubated at 32°. Nevertheless, while vector controls did not exhibit differential sensitivity to ara-C at 32° versus 37°, temperature-sensitive p53 mutants displayed a significant increase in apoptosis at the permissive temperature. This was not accompanied by increased ara-CTP formation, DNA incorporation of [ 3 H ]ara-C, or altered expression of Bcl-2 or Bax. Enhanced sensitivity was associated with increased mitochondrial injury (e.g. cytochrome c release), caspase activation, and loss of clonogenic survival. Significantly, ptsp53 cells synchronized in S phase were markedly more sensitive to ara-C-mediated mitochondrial injury and apoptosis at 32°, indicating that wild-type p53 specifically enhances the susceptibility of this subpopulation to ara-C lethality. Consistent with these results, transient transfection of human wild-type p53 cDNA rendered parental U937 cells more sensitive to ara-C-mediated cell death. Collectively, these findings indicate that p53 expression renders S-phase U937 cells more susceptible to ara-C-mediated mitochondrial dysfunction, cytochrome c release, apoptosis, and loss of clonogenic survival without enhancing ara-C metabolism. Such findings raise the possibility that loss of functional p53 activity allows leukemia cells to circumvent ara-C lethality.
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