ESCHERICHIA-COLI CYTOLETHAL DISTENDING TOXIN BLOCKS THE HELA-CELL CYCLE AT THE G(2) M TRANSITION BY PREVENTING CDC2 PROTEIN-KINASE DEPHOSPHORYLATION AND ACTIVATION/

Cytolethal distending toxins (CDT) constitute an emerging heterogeneou s family of bacterial toxins whose common biological property is to in hibit the proliferation of cells in culture by blocking their cycle at G(2)/M phase. In this study, we investigated the molecular mechanisms underlying the block caused by CDT from Escherichia coli on synchroni zed HeLa cell cultures, To this end, we studied specifically the behav ior of the two subunits of the complex that determines entry into mito sis, i.e., cyclin B1, the regulatory unit, and cdc2 protein kinase, th e catalytic unit. We thus demonstrate that CDT causes cell accumulatio n in G(2) and not in M, that it does not slow the progression of cells through S phase, and that it does not affect the normal increase of c yclin B1 from late S to G(2). On the other hand, we show that CDT inhi bits the kinase activity of cdc2 by preventing its dephosphorylation, an event which, in normal cells, triggers mitosis, This inhibitory act ivity was demonstrated for the three partially related CDTs so far des cribed for E. coli. Moreover, we provide evidence that cells exposed t o CDT during G(2) and M phases are blocked only at the subsequent G(2) phase, This observation means that the toxin triggers a mechanism of cell arrest that is initiated in S phase and therefore possibly relate d to the DNA damage checkpoint system.