UBIQUITIN PATHWAY INVOLVEMENT IN HUMAN LYMPHOCYTE GAMMA-IRRADIATION-INDUCED APOPTOSIS

Apoptosis (the classical type of programmed cell death) can be triggered in many cell types by widely diverse stimuli. Gamma rays, at low doses, can induce apoptosis in vitro in interphase human lymphocytes. In this type of apoptosis induction, activated gene expression is necessary for the fulfillment of the death program. In this report, we present evidence for a relationship between ubiquitin gene expression or ubiquitination and gamma-irradiation-mediated apoptosis in normal circulating human lymphocytes. Using in vitro nuclear transcription assays (run-on), Northern (RNA) blot analysis, immunolocalization studies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after immunoprecipitation, we demonstrate that (i) the ubiquitin mRNA level is increased as a consequence of the activation of ubiquitin gene transcription 15 to 90 min after initiation of apoptosis; (ii) specifically in apoptotic cells, and not in all irradiated cells, nuclear proteins are highly ubiquitinated; and (iii) ubiquitin sequence-specific antisense oligonucleotide inhibition results in a decreased level of ubiquitinated nuclear proteins and considerably diminishes the proportion of cells exhibiting the apoptotic death pattern. Each of these results might be explained by different modifications occurring in irradiated cells. Their convergence strongly suggests that the ubiquitin gene is one of the genes with induced activity in the apoptotic death program and that ubiquitination of nuclear proteins might be involved in chromatin disorganization and oligonucleosomal fragmentation, which are among the key events occurring in apoptosis.