Cell Biology International 1999, Vol. 23, No. 5, 323–334 Article No. cbir.1999.0362, available online at http://www.idealibrary.com on p53 DOES NOT CONTROL THE SPINDLE ASSEMBLY CELL CYCLE CHECKPOINT BUT MEDIATES G1 ARREST IN RESPONSE TO DISRUPTION OF MICROTUBULE SYSTEM ANNA A. SABLINA 1 , LARISSA S. AGAPOVA 1 , PETER M. CHUMAKOV 2 and BORIS P. KOPNIN 1 * 1 Institute of Cancerogenesis, Cancer Research Center, and 2 Engelhardt Institute of M olecular Biology, R ussian A cademy of S ciences, M oscow, R ussia Received 19 October 1998; accepted 9 February 1999 p53 plays a critical role as a tumour-suppressor in restricting the proliferation of damaged cells, thus preventing formation of genetically altered cell clones. Its inactivation leads, in particular, to accumulation of polyploid and aneuploid cells. To elucidate the role of p53 in control of chromosome number, we analysed its participation in the cell cycle checkpoints controlling: (1) spindle assembly; and (2) G1-to-S transitions in cells with disintegrated microtubule cytoskel- eton. Treatment with 8–10 ng/ml of colcemid causing no visible destruction of the spindle leads to arrest of metaphase-to-anaphase transition in both p53-positive and p53-negative murine fibroblasts, as well as in p53-positive REF52 cells and their counterparts (where the p53 function was inactivated by transduction of dominant-negative p53 fragment). Furthermore, p53-positive and p53-defective rodent and human cells showed no significant difference in kinetics of metaphase-to-interphase transitions in cultures treated with high colcemid doses preventing spindle formation. These data argue against the hypothesis that p53 is a key component of the spindle-assembly checkpoint. However, p53 mediates activation of the G1 checkpoint in response to depolymerization of microtubules in interphase cells. Treatment of synchronized G0/G1 cells with colcemid causes arrest of G1-to-S transition. Inactivation of the p53 function by transduction of dominant-negative p53 fragment abolishes the G1 checkpoint that prevents entry into S phase of cells with disrupted microtubules. Transduction of kinase-defective dominant-negative c-raf mutant or application of PD 098059, a specific inhibitor of MEK1, also abrogates the G1 cell cycle arrest in cells with disintegrated microtubule system. It seems that Raf-MAP-kinase signalling pathways are responsible for p53 activation induced by depolymerization of microtubules.  1999 Academic Press K : p53; microtubules; cell cycle checkpoints INTRODUCTION The tumour suppressor p53 is considered as a protein playing a key role in restricting of prolifer- ation of abnormal cells. It is activated in response to a variety of cellular stresses, including DNA damage, hypoxia, metabolic changes, microtubule disruption, viral infection and activated oncogenes, and is believed to be an integrator of these changes (for review see Hartwell and Kastan, 1994; Chernova et al., 1995; Cox and Lane, 1995; Jacks and Weinberg, 1996; Levine, 1997). The activation of p53 is due to accumulation and conformational changes of the protein (Fritsche et al., 1993; Kastan et al., 1991; Mosner et al., 1995; M eek et al., 1997). Substantial evidence has been com- piled showing that multi-site phosphorylation/ dephosphorylation of p53 is a major mechanism by which the p53 conformation or turnover rate can be altered (Zhang et al., 1994; Ko and Prives, 1996; Chernov and Stark, 1997). Indeed, p53 serves as a substrate for a number of protein *To whom correspondence should be addressed: Laboratory of Cytogenetics, Institute of Cancerogenesis, Cancer Research Center, Kashirskoye shosse 24, Moscow, 115478, Russia. 1065–6995/99/050323+ 12 $30.00/0  1999 Academic Press