The results presented here suggest that decatenation G2
checkpoint function is more accurately quantified by measuring the rate of mitotic entry, rather than measuring the mitotic index at a single time point. The mitotic index assay relies upon two events to detect decatenation G2
checkpoint function: G2
arrest and mitotic exit. Given that topo IIα contributes to a decatenation reaction at centromeres that suppresses a MAD2-dependent metaphase checkpoint, 13, 22
treatment with ICRF-193 can induce a metaphase delay that blocks mitotic exit. The mitotic entry rate assay quantifies the rate of G2
/M progression, and is not influenced by the ICRF-193-induced metaphase arrest or mitotic exit rates. By applying the improved mitotic entry rate assay, it was clear that ATR and CHEK1 are not required for decatenation G2
checkpoint function, whereas depletion or truncation of ATM significantly attenuated the checkpoint response in NHDFs and lymphoblasts, respectively.
The prior study of ATR function in the decatenation G2
checkpoint utilized an SV40-transformed human fibroblast line with inducible expression of kinase-inactive ATR.18
SV40-transformed human fibroblast lines typically display highly aneuploid genomes due to a phase of severe telomere crisis before establishment of immortality. 49
DNA damage checkpoints acting in G1
, S, and G2
may be defective in SV40-transformed and large T antigen-transformed lines. 50, 51
It is possible that the over-expression of kinase-inactive ATR and incubation with ICRF-193 in the SV40-transformed line disrupted centromeric decatenation and induced a metaphase arrest.
The complicated biology of SV40-transformed immortal human cell lines with aneuploid genomes requires that caution should be applied when interpreting features of cell cycle regulation in such immortalized lines. The telomerase-expressing diploid human fibroblast lines were developed to overcome this problem. Transduction of hTERT to immortalize human fibroblasts generates a cell culture model with extended-lifespan, stable diploid genomes, and retention of all DNA damage checkpoint functions at levels equivalent to those measured in telomerase-negative, parental fibroblast strains.30, 35
Severe depletion of ATR in three diploid normal human fibroblast lines had no effect on ICRF-193-induced G2
arrest ( and data not shown). As depletion of protein expression does not always produce the same phenotype as drug-induced over-expression of kinase-inactive mutants, 52
it is conceivable that over-expression of the kinase-inactive ATR allele produced biochemical alterations within SV40-transformed cells that affected mitotic entry or exit after treatment with ICRF-193.
The prior analysis in DT40 cells showed that 1 μM ICRF-193 reduced the rate of accumulation of mitotic cells during incubation with nocodazole by 15% relative to control but, in cells with genetic depletion of CHEK1, ICRF-193 had no effect on mitotic accumulation.43
The DT40 cells are known to display defects in DNA damage G1
checkpoint function, 53
and their modest response to ICRF-193 suggests they may express a defective decatenation G2
checkpoint. In diploid human fibroblast lines, 0.5 μM ICRF-193 reduced the rate of mitotic entry by 75% relative to control () and 4 μM produced a 97-99% inhibition. Fibroblasts depleted of CHEK1 responded to 4 μM ICRF-193 with a 97% reduction in the mitotic entry rate, indicating that CHEK1 was not required for decatenation G2
checkpoint function. Equivalent depletion of CHEK1 in normal fibroblasts produced a severe attenuation of the intra-S checkpoint response to UV irradiation, 54
demonstrating that the degree of depletion of CHEK1 protein was sufficient to inhibit DNA damage checkpoint signaling.
The prior studies of ATM and decatenation G2
checkpoint function used either lymphoblastoid lines with the mitotic index assay, 18
or an SV40-transformed AT fibroblast line with the mitotic entry rate assay. 40
The telomerase-expressing AT fibroblast lines display canonical defects in DNA damage checkpoint functions, hypersensitivity to ionizing radiation, 36
and significantly less mitotic inhibition and G2
delay than normal fibroblast lines when treated with ICRF-193 (). In addition, AT lymphoblastoid and ATM-depleted NHF1hTERTs displayed similar defects in decatenation G2
checkpoint function ( and ). Taken together, the biological data presented here strongly support the hypothesis that ATM contributes to decatenation G2
A few studies have questioned the existence of the decatenation G2
checkpoint. One study showed that high concentrations of ICRF-193 caused Indian muntjac cells first to delay in an early phase of mitosis called antephase and then to fall back into a G2
ICRF-193-treated muntjac cells also expressed γH2AX as a putative marker of DNA dsbs, 55
although recent studies suggest that γH2AX may not be specific for DNA dsbs. 56, 57
Another study examined DNA damage response markers in diploid human fibroblasts and HeLa cells after treatment with ICRF-193. 46
Elements of ATM-dependent DNA damage response were recognized, including activation of CHEK2 and expression of γH2AX. The demonstration of γH2AX in ICRF-193-treated cells has not been reproducible, and HeLa cells examined by the mitotic entry rate assay display an attenuated decatenation G2
checkpoint (Bower and Kaufmann, unpublished data). In addition, Skoufias et al.
found that while both IR and etoposide induced γH2AX in HeLa cells as expected, ICRF-193 did not,13
and neither Nakagawa et al.
nor Luo et al.
were able to detect γH2AX in human cancer lines after treatment with ICRF-193. 28, 47
The ultrasensitive method of alkaline elution chromatography did not detect DNA damage in ICRF-193-treated diploid human fibroblasts, 17
and we have been unable to detect appreciable activation of γH2AX in ICRF-193-treated NHDFs by flow cytometry or immunofluorescence (Bower and Kaufmann, in preparation). However, cells that evaded the decatenation G2
checkpoint after treatment with ICRF-193 displayed chromosomal aberrations, 26
which may be recognized as DNA dsbs upon mitotic exit and return to interphase.
The analysis of DNA damage response biomarkers reported here indicated that ATM signaling was induced by ICRF-193. Remarkably, treatment with ICRF-193 induced an ATM-dependent phosphorylation of p53 and CHEK2, but not H2AX. This result is reminiscent of recent reports showing that the topo II catalytic inhibitors chloroquine and ciprofloxacin can activate ATM without phosphorylation of H2AX. 33, 34
Thus, it appears that catalytic inhibitors of topo II may induce a conformational change in chromatin that activates ATM. This method of activation of ATM differs from the response to DNA dsbs, as γH2AX is not induced.
It has been recently demonstrated that Ser1524 in topo IIα is phosphorylated in HT1080 fibrosarcoma cells, and this phosphorylation is required for ICRF-193-induced G2
Cells in which endogenous topo IIα was replaced with ectopic topo IIα containing a non-phosphorylatable alanine at this position failed to arrest in G2
when treated with ICRF-193, and entered mitosis with severely entangled sister chromatids. The phospho-Ser1524 in topo IIα was bound by the BRCT repeat of the checkpoint mediator protein MDC1 and this binding was stimulated by inhibition of topo IIα with ICRF-193. Furthermore, depletion of MDC1 attenuated the ICRF-193-induced G2
arrest. It is interesting to note that MDC1 is phosphorylated by ATM and serves to recruit ATM to γH2AX on chromatin at the sites of DNA dsbs. 58
As ICRF-193 does not induce γH2AX, MDC1 may recruit ATM to catalytically inactive topo IIα, upon exposure to ICRF-193, chloroquine, and/or ciprofloxacin.