UNCOUPLING OF S-PHASE AND MITOSIS BY RECOMBINANT CYTOTOXIC NECROTIZING FACTOR-2 (CNF2)

Cytotoxic necrotizing factor 2 (CNF2) is an exotoxin identified from v irulent clinical isolates of Escherichia coil. It has been characteriz ed in adherent cell lines as an inducer of cellular death, hyperploidy (multinucleation), and cytoskeletal reorganization, The molecular mec hanism of these actions is unclear, Two cellular mechanisms can be hyp othesized to explain the DNA content increase (hyperploidy) induced by the toxin, The first is that the toxin interferes with cytoplasmic di vision without interfering with normal nuclear cycling, such that DNA is replicated in the absence of cell division. The second is that the toxin drives the nuclear machinery to replicate the DNA multiple times within one cell cycle, without interfering with cytoplasmic division, In order to investigate these phenomena, we have constructed a recomb inant CNF2 gene that expresses a toxin with both an epitope tag and a polyhistidine tag. Extracts made from E. coli that express this gene h ave a high multinucleating activity that colocalizes with the recombin ant 115-kDa protein. To distinguish between these hypotheses, we used recombinant CNF2 and several growth conditions (time, partial differen tiation, and stage of growth) to establish a relationship between cell ular divisions and generation of hyperploidy. It was also determined t hat the toxin had no effect upon in vitro DNA replication using a Xeno pus egg extract system. In aggregate, these data are consistent with t he hypothesis that CNF2 is affecting cytoplasmic division and thereby removing the requirement for a completed mitosis before the initiation of another S-phase, These data are discussed in relation to the gener ation of polyploid cells during megakaryopoeisis and the generation of aneuploid cells during tumorigenesis. (C) 1997 Academic Press.