ACTIVATION OF P53-MEDIATED CELL-CYCLE CHECKPOINT IN RESPONSE TO MICRONUCLEI FORMATION

Inactivation of p53 tumor-suppressor leads to genetic instability and, in particular, to accumulation of cells with abnormal numbers of chro mosomes. In order to better define the role of p53 function in maintai ning genome integrity we investigated the involvement of p53 in the co ntrol of proliferation of micronucleated cells resulting from abnormal chromosome segregation. Using cell lines expressing temperature-sensi tive (ts) p53 or containing p53 genetic suppressor element (p53-GSE) m e showed that inhibition of p53 function increases the frequency of ce lls with micronuclei. Immunofluorescence study revealed that in REF52 cell cultures with both spontaneous and colcemid-induced micronuclei t he proportion of p53-positive cells is considerably higher among micro nucleated variants as compared with their mononuclear counterparts. An alysis of 12(1)ConA cells expressing the beta-galactosidase reporter g ene under the control of a p53-responsive promoter showed activation o f p53-regulated transcription in the cells with micronuclei, Important ly, the percentage of cells manifesting specific p53 activity in colce mid-treated cultures increased with an augmentation of the number of m icronuclei in the cell. Activation of p53 in micronucleated cells was accompanied by a decrease in their ability to enter S-phase as was det ermined by comparative analysis of 5-bromodeoxyuridine (5-BrdU) incorp oration by the cells with micronuclei and their mononuclear counterpar ts. Inhibition of p53 function in the cells with tetracycline-regulate d p53 gene expression, as well as in the cells expressing ts-p53 or p5 3-GSE, abolished cell cycle arrest in micronucleated cells, These resu lts along with the data showing no increase in the frequency of chromo some breaks in REF52 cells after colcemid treatment suggest the existe nce of p53-mediated cell cycle checkpoint(s) preventing proliferation of micronucleated cells derived as a result of abnormal chromosome seg regation during mitosis.