CHROMATIN CONDENSATION DURING APOPTOSIS IS ACCOMPANIED BY DEGRADATIONOF LAMIN A+B, WITHOUT ENHANCED ACTIVATION OF CDC2 KINASE

Chromatin condensation paralleled by DNA fragmentation is one of the m ost important criteria which are used to identify apoptotic cells. How ever, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respe ct it is known that in mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar pr ocess is happening in apoptotic cells. The experiments described in th is study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum con ditions or mitosis. Solubilization of lamin A+B was analyzed by immuno blotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization . In apoptotic cells, a small amount of lamin is solubilized before th e onset of apoptosis, thereafter, chromatin condensation is accompanie d by degradation of lamin A+B to a 46-kD fragment. Analysis of cellula r lysates by probing blots with anti-PSTAIR followed by anti-phosphoty rosine showed that in contrast to mitosis, dephosphorylation on tyrosi ne residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activa tion of p34(cdc2) as determined in the histone H1 kinase assay. Coindu ction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase . The sudden breakdown of chromatin structure may be the result of det achment of the chromatin loops from their anchorage at the nuclear mat rix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 k bp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during a poptosis appears to be due to a rapid proteolysis of nuclear matrix pr oteins which does not involve the p34(cdc2) kinase.