The aim of our study was to explore the antiproliferative and pro-apoptotic action of
roscovitine (ROSC) on human
breast cancer MCF-7 cells. We examined the effect of ROSC on cell proliferation, cell cycle progression, nucleolar morphology, posttranslational modifications of
histones as well as on induction of apoptosis. The effects of ROSC on the argyrophilic nucleolar organizer regions (
AgNORs) and nucleolar
RNA of MCF-7 cells were marked: ROSC treatment changed the pattern of
AgNORs in a time-dependent manner. The disintegration of nucleoli manifested by increasing number of nucleolar fragments already began at 6 hr posttreatment. This was accompanied by a redistribution of the
nucleolin from the nucleolus beginning after 6 hr and preceded a decrease of
histone acetylation and phosphorylation. Inhibition of
DNA synthesis and accumulation of G(2)/M-arrested cells starting 6 hr posttreatment coincided with a strong increase of the p53 level and with an appearance of a few cells committed to undergo apoptosis. However, all these changes preceded the main wave of apoptosis, which occurred after 24 hr ROSC treatment as assessed by determination of the frequency of
Annexin binding, activation of
caspases as well as of DNA fragmentation. Onset of PARP-1 cleavage detected by immunoblotting and by immunohistochemistry 6 hr or 9 hr posttreatment, respectively, preceded for a few hours the DNA fragmentation detected in situ by TUNEL assay. Reconstitution of MCF-7 cells with
caspase-3 did not change the kinetics of ROSC-induced apoptosis. Our results show that disintegration of nucleoli is an early marker of ROSC-induced changes. Cell cycle arrest precedes the main wave of apoptosis.