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The ubiquitination of nuclear proteins activated in human lymphocytes undergoing radiation-induced apoptosis and the subsequent downstream proteasomal protein processing, shown to be involved in apoptotic death control, may be dependent on an amino-terminal sequence identity of ubiquitin target proteins, the “N-end rule” pathway. Here we report that this selective pathway controls radiation-induced apoptosis and that it is involved in the initiation of this type of cell death. Dipeptide competitors of protein ubiquitination/processing dependent solely on the basic amino-terminal residues (type I) efficiently inhibited the radiation-induced apoptotic death phenotype, indicating that only the substrates of ubiquitination with basic NH2-terminal amino acids are involved in apoptotic death control. This selective inhibition was followed by an early, overall but also target-specific inhibition of ubiquitination and by an activation and stabilization of poly(ADP-ribose) polymerase (PARP) that occurs through inhibition of ubiquitination of its cleaved form (85 kDa). Interestingly, caspases-3 and -7 were not activated following irradiation, further suggesting that PARP cleavage may be regulated by an N-end rule pathway in a caspase-independent manner. These results highly suggest involvement of this subset of the ubiquitin system in the apoptotic death control and in the specific regulation of PARP activity.

The ability of a “vital” dye, acridine orange (AO), to intercalate into the DNA of living cells was investigated by quantitative intensified fluorescence microscopy and digital imaging under various conditions of dye concentration, excitation light intensity, and ionic concentration. Our results demonstrate that the bulk of chromatin DNA is packed in a way that does not allow AO intercalation. At low dye concentrations and very low levels of light intensity, the only fluorescent structures observed inside the nucleus are the nucleoli. This nonpermissive state of the chromatin appears to be a characteristic feature of the nucleus in living cells. AO intercalation into DNA can be mediated by raising the nuclear Na + concentration. This was achieved here by using a cation carrier, monensin, a procedure which permits the avoidance of cell permeabilization. Furthermore, we show that the discharge of lysosomal enzymes in the living cell, via a targeted photodynamic reaction which occurs at high levels of light intensity, can also release the constraints which impede dye intercalation into nuclear DNA. In conclusion, studies carried out under conditions where intercalative dyes such as AO are able to stain the nuclear DNA have to be interpreted with caution and do not provide direct evidence on the structural state of native chromatin. The molecular origin of the absence of AO intercalation in chromatin of the living cell is discussed with regard to the restrained uncoiling of the double helix which is required for dye intercalation.