Chapter 18 : Cytolethal Distending Toxin

Effects of CDT treatment on growing cells. (A) Phase-contrast microscopy and (B) cell cycle distribution of Chinese hamster ovary cells treated with Ec-CDT-I. Microscopic examination reveals the dramatic increase in cell size following 48 h of CDT treatment. Control cells were treated with a cell extract from a non-CDT-producing E. coli strain. Cell cycle distribution analysis by flow cytometry reveals that CDT cells are completely blocked at the G2/M boundary whereas control cells exhibit a normal distribution for cycling cells.

T7 expression and exclusive radio-labeling of CDT polypeptides. The Ec-CDTII genes were separated by cloning and expressed under a T7 promoter in the presence of rifampicin and 35S-methionine/cysteine. Total cell extract and supernatant fractions were then prepared and analyzed by SDS-PAGE and autoradiography. The expressed forms of Ec-CDT-II migrated with molecular sizes of approximately 32, 30, and 20 kDa. These results coincide with the calculated molecular sizes for CDT. As expected, CdtA, CdtB, and CdtC also appeared in the supernatant fraction of the cell culture.

Homology of CdtB to mammalian type I DNase. (A) PSI-BLAST alignment of Ec-CDT-II was performed through the National Center for Biotechnology Information BLAST site (http://www.ncbi.nlm.nih.gov/BLAST/). Iterations were repeated until the sequences converged. Shaded residues correspond to catalytic or metal ion-binding residues. The underlined sequence represents the phosphohydrolases-related motif found in all CdtBs, mammalian type I endonucleases, and sphingomyelinases (B). The CdtBs aligned are designated as shown in Table 1 . Hum. DNaseI, human DNase I; B.c. SMase, Bacillus cereus sphingomyelinase; S.a. SMase, Staphylococcus aureus sphingomyelinase.

Mitotic checkpoint pathway following DNA damage. DNAdamage following exposure to IR, UV light, or genotoxic chemical is sensed by either ATM or the ATM-related kinase ATR. Once activated, ATM and ATR phosphorylate several substrates that affect growth arrest. Chk2 (Cds1) is phosphorylated by ATM, which in turn phosphorylates the Cdc25 phosphatase on Ser-216. Phosphorylation of Cdc25 on Ser-216 creates a binding site for a 14-3-3 protein that binds to the phosphatase and escorts it to the cytoplasm. Cdc25 is required by the cell to mediate the transition between G2 and M phase by activating Cdc2. Cdc2 is dephosphorylated (activated) by Cdc25 in a cycle-regulated manner to allow formation of the active Cdc2/cyclin B complex, thus directing the onset of mitosis. The persistent phosphorylation of Cdc2 following CDT treatment is a result of the Chk2-dependent inactivation of Cdc25 ( 13 ). ATM activation also is responsible for stabilization of the tumor suppressor p53. Both direct phosphorylation of p53 and phosphorylation of MDM2 (a protein that normally escorts p53 to the cytoplasm for degradation) result in a dramatic elevation and activation of p53. Activation of p53 in turn induces expression of p21, a cyclin-dependent kinase inhibitor that blocks activation of Cdc2, thus enforcing the G2 arrest. In addition, p53 activation results in apoptosis in some cell types (see text and suggested reading for additional background).

Model for CDT action. According to the current understanding of CDT action, CDT appears to bind to a receptor on the surface of sensitive cells, enters the cell by receptor-mediated endocytosis, and traffics through the cell through early and late endosome, and following involvement of the Golgi, CdtB enters the nucleus. Nuclear CdtB is then responsible for imparting a damaging effect on chromosomal DNA, resulting in growth arrest either by the mitotic checkpoint cascade or DNA damage-mediated apoptosis.