PMC

A, structure of the chicken ICAD gene and the targeting strategy using a targeting vector with a resistance cassette flanked by ICAD 5′ and 3′ noncoding genomic regions. B, Southern blot analysis of genomic DNA prepared from the following: ICAD^+/+ (lanes 1 and 2); ICAD^+/-* (ICAD heterozygote with the puro/tk marker, lanes 3 and 4); ICAD^+/- (ICAD heterozygote with puro/tk marker excised, lanes 5 and 6); and ICAD^-/- (lanes 7 and 8). Genomic DNA (5 μg) was digested by BglII. The radioactive 5′-probe was located outside of the targeting vector. The figure shows data from two independent ICAD ^-/-- clones (lanes 7a and 7b) and two independent ICAD^-/-/CAD^-/- clones (lanes 8a and 8b).

Mitotic DNA damage occurs downstream of caspase-dependent, partial cleavage of ICAD. (A) Immunoprecipitation (IP) of ICAD followed by blotting for cleaved ICAD (c ICAD) at 24 h K5I showed less cleavage than 24 h 1 μM STS but more than UV treatment (see Materials and Methods). Ten percent input blot: ICAD shows total ICAD levels. (B) Mock, untreated (M) and G2-synchronized (synch) cells, and cells released into K5I showed decreased ICAD from 8 to 48 h and increased c ICAD from 16 h, correlating well with late mitotic arrest/slippage (p-H3). Dynein is the loading control. (C) Immunoprecipitation of ICAD followed by blotting for c ICAD showed partial cleavage of ICAD at 24 h K5I treatment is blocked by zVAD-fmk or expression of noncleavable ICAD (clone A). All blots in each panel are from the same gels and/or samples. (D) Noncleavable ICAD-expressing clone A and B showed the same background levels of γH2A.X as MCF7 CELLS. G2-synchronized clone A and B released into K5I for 16 h showed severely inhibited γH2A.X compared with MCF7 cells. Average integrated fluorescence intensity (± SE). RFU, relative fluorescence units. No K5I: MCF7, n = 31; clone A, n = 12; clone B, n = 16. 16 h K5I: MCF7, n = 210; clone A, n = 166; clone B, n = 142; two experiments each condition. *, p < 0.05 vs. MCF7 + 16 h K5I.

WT and ICAD^−/− mice received DMH injections once a week for 8 weeks. Mice were sacrificed 24 weeks later. (A) Diameter of tumors was assessed in the different experimental groups; n = 12. *, Difference from DMH-treated WT mice, p<0.01. (B) A gross examination of a large tumor detected in DMH-treated ICAD^−/− mice. (C) H&E-stained colonic tissue with a large tumor from a DMH-treated ICAD^−/− mouse; note the large necrotic cores (black arrows) and vascularization (yellow arrows). (D) PCNA expression in colon tumors from DMH-treated ICAD^−/− mice as assessed by IHC. (E) H&E-stained section with a tumor from a DMH-treated ICAD^−/− mouse with inflammatory cells (arrows) in the vicinity of the tumor. (F) MCP-1 and COX2 expression in colon tumors from DMH-treated ICAD^−/− mice as assessed by IHC; the right panel is an IgG control.

Electrophoresis of PCR products with primers for IS256 and icaD. Lane 1, 100 bp molecular weight marker; lane 2, 371-bp band from icaD positive control; lane 3 and lane 4, 371-bp bands (icaD) from S. haemolyticus strains; lane 5, 1102-bp band (IS256) and 371-band (icaD) from S. haemolyticus strain; lane 6, 371-bp band (icaD) from S. haemolyticus strain; lane 7, 1102-bp band (IS256) and 371-band (icaD) from S. haemolyticus strain; lane 8, 371-band (icaD) from S. haemolyticus strain.