TY - JOUR
T1 - Uncoupling of S-phase and mitosis by recombinant cytotoxic necrotizing factor 2 (CNF2)
AU - Denko, Nicholas
AU - Langland, Rachel
AU - Barton, Michelle
AU - Lieberman, Michael A.
N1 - Funding Information:
1This work was supported in part by a grant from the National Institutes of Health (HL51555) to M.A.L. 2Current address: Department of Radiation Oncology, Stanford School of Medicine, Stanford University, Stanford, CA 94305. 3To whom correspondence and reprint requests should be addressed. Fax: (513) 558-8474. E-mail: lieberma@ucbeh.san.uc.edu.
PY - 1997/7/10
Y1 - 1997/7/10
N2 - Cytotoxic necrotizing factor 2 (CNF2) is an exotoxin identified from virulent clinical isolates of Escherichia coli. It has been characterized in adherent cell lines as an inducer of cellular death, hyperploidy (multinucleation), and cytoskeletal reorganization. The molecular mechanism of these actions is unclear. Two cellular mechanisms can be hypothesized to explain the DNA content increase (hyperploidy) induced by the toxin. The first is that the toxin interferes with cytoplasmic division 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 recombinant CNF2 gene that expresses a toxin with both an epitope tag and a polyhistidine tag. Extracts made from E. coli that express this gene have a high multinucleating activity that colocalizes with the recombinant 115-kDa protein. To distinguish between these hypotheses, we used recombinant CNF2 and several growth conditions (time, partial differentiation, and stage of growth) to establish a relationship between cellular divisions and generation of hyperploidy. It was also determined that the toxin had no effect upon in vitro DNA replication using a Xenopus egg extract system. In aggregate, these data are consistent with the 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 generation of polyploid cells during megakaryopoeisis and the generation of aneuploid cells during tumorigenesis.
AB - Cytotoxic necrotizing factor 2 (CNF2) is an exotoxin identified from virulent clinical isolates of Escherichia coli. It has been characterized in adherent cell lines as an inducer of cellular death, hyperploidy (multinucleation), and cytoskeletal reorganization. The molecular mechanism of these actions is unclear. Two cellular mechanisms can be hypothesized to explain the DNA content increase (hyperploidy) induced by the toxin. The first is that the toxin interferes with cytoplasmic division 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 recombinant CNF2 gene that expresses a toxin with both an epitope tag and a polyhistidine tag. Extracts made from E. coli that express this gene have a high multinucleating activity that colocalizes with the recombinant 115-kDa protein. To distinguish between these hypotheses, we used recombinant CNF2 and several growth conditions (time, partial differentiation, and stage of growth) to establish a relationship between cellular divisions and generation of hyperploidy. It was also determined that the toxin had no effect upon in vitro DNA replication using a Xenopus egg extract system. In aggregate, these data are consistent with the 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 generation of polyploid cells during megakaryopoeisis and the generation of aneuploid cells during tumorigenesis.
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U2 - 10.1006/excr.1997.3595
DO - 10.1006/excr.1997.3595
M3 - Article
C2 - 9223378
AN - SCOPUS:0031563151
SN - 0014-4827
VL - 234
SP - 132
EP - 138
JO - Experimental Cell Research
JF - Experimental Cell Research
IS - 1
ER -