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

Apoptosis (the classical type of programmed cell death) can be trigger ed in many cell types by widely diverse stimuli. Gamma rays, at low do ses, can induce apoptosis in vitro in interphase human lymphocytes. In this type of apoptosis induction, activated gene expression is necess ary for the fulfillment of the death program. In this report, we prese nt evidence for a relationship between ubiquitin gene expression or ub iquitination and gamma-irradiation-mediated apoptosis in normal circul ating human lymphocytes. Using in vitro nuclear transcription assays ( run-on), Northern (RNA) blot analysis, immunolocalization studies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis af ter immunoprecipitation, we demonstrate that (i) the ubiquitin mRNA le vel is increased as a consequence of the activation of ubiquitin gene transcription 15 to 90 min after initiation of apoptosis; (ii) specifi cally in apoptotic cells, and not in all irradiated cells, nuclear pro teins are highly ubiquitinated; and (iii) ubiquitin sequence-specific antisense oligonucleotide inhibition results in a decreased level of u biquitinated nuclear proteins and considerably diminishes the proporti on of cells exhibiting the apoptotic death pattern. Each of these resu lts might be explained by different modifications occurring in irradia ted cells. Their convergence strongly suggests that the ubiquitin gene is one of the genes with induced activity in the apoptotic death prog ram and that ubiquitination of nuclear proteins might be involved in c hromatin disorganization and oligonucleosomal fragmentation, which are among the key events occurring in apoptosis.