Wilhelm bernhard workshop on the cell nucleus

DELAYED ENTRY TO QUIESCENCE BY p21^CDKN1A-NULL HUMAN FIBROBLASTS: A POSSIBLE INVOLVEMENT IN THE DISASSEMBLY OF PRE-REPLICATION COMPLEX

Stivala L.A., Perucca P., Cazzalini O. ¹, E. Prosperi ², Madine M., Laskey R.A.L. <sup>3

1</sup>Dipartimento di Medicina sperimentale, Sezione di Patologia generale and ²Istituto di Genetica Molecolare del CNR, Sezione di Istochimica e Citometria, Pavia, Italy; ³MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge, UK.
The cyclin-dependent kinase inhibitor p21^CDKN1A is involved in several cellular processes including cell cycle arrest induced by DNA damage, cell differentiation and senescence. In contrast, the role of this protein in the cell cycle exit to quiescence is more controversial. In order to investigate a possible role of p21 in inducing quiescent cell growth arrest, we have considered the assembly of the pre-replication complex as a possible step targeted by p21. To this end, we have used human embryonic p21-null fibroblasts (obtained by homologous recombination) with extended lifespan induced by expression of the catalytic subunit of human telomerase (htert). Cells were driven to quiescence both by contact inhibition and serum deprivation (0.5%) and residual DNA replicative capacity was assessed by BrdU incorporation. Cellular levels of p21 were determined by immunofluorescence. The results showed that in p21^+/+ parental fibroblasts but not in p21^-/- cells, p21 protein levels as well as the percentage of positive nuclei, were significantly increased after 3 days of serum starvation, and remained high at least up to 7 days. Increased levels of p27^kip1 protein were found in both cell lines, whereas p16 was not significantly modified. Analysis of the residual DNA replicative activity indicated that p21-null fibroblasts retained significant levels of BrdU incorporation, up to at least 10-18 days after serum deprivation, as compared to complete no incorporation observed after 7-9 days in the parental p21^+/+ cells. These findings were confirmed by the capacity to perform DNA replication in vitro, exhibited by nuclei isolated from serum-starved p21^-/- cells, as compared to parental p21^+/+ fibroblasts. Western blot analysis of the pre-replication complex during serum starvation, showed that chromatin-bound MCM2 protein was displaced with faster kinetics in p21^+/+ than in p21^-/- cells. These results suggest that p21 may be involved in the pathway that drive the cells to quiescence.

LIGHT AND ELECTRON MICROSCOPIC STUDY OF REPLICATION SITES IN HELA CELLS DURING S-PHASE

Koberna K., Pliss A., Siegel A.J., Malinsky J., Ligasova A., Dudnik O. Berezney R., and Raska I.

1^st Faculty of Medicine, Charles University and Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Department of Biological Sciences, State University of New York at Buffalo, USA
Replication sites (RSs) in synchronized HeLa cells have been studied with the help of light microscopy (LM) and electron microscopy (EM) detection of labeled nascent DNA. In agreement with previous studies, distinct patterns of replication are observed on the LM level during S-phase. The replication begins in non-numerous small fluorescence foci scattered throughout nucleoplasm, but the number of RSs becomes very high within several minutes. At later stages of S-phase the RSs are less numerous and often larger. Towards the very end of S-phase, the number as well as the size of RSs gradually decreases. The EM analysis of the early replicated chromatin identifies the replication pattern with the small RSs with a maximum size 240 nm, which we refer to as replication units (RUs). RUs of about the same size are clustered into larger domains during later stages of S-phase. These domains correspond to the large RSs observed on the LM level during both the mid and late periods of S-phase. Importantly, the stereological evaluation of the images indicates that the number of RUs is similar in the early andmid S-phase cells (with exception of very early S-phase cells) and declines towards the end of S-phase. Only individual RUs are observed at the very end of S-phase. About 1100 RUs are seen in the early and mid S-phase cells. The analysis of the overall fluorescence replication signal shows, however, an increase in the fluorescence replication signal during the transition from the early to mid S-phase cells. We cannot comment on the number of active replicons in early and mid S-phase cells. However, based on the analysis of the labeled and stretched DNA fibers from the early and mid S-phase cells, the elevation of the replication signal definitely reflects the higher speed of replication forks in the mid S-phase cells.

This work was supported by grants 304/01/0729, 304/03/1121, IAA5039103, EU CE ICA1-CT-200070028, AV0Z5039906, MSM111100003 and NIH GM2392.