The human decatenation checkpoint

Abstract

Chromatid catenation is actively monitored in human cells, with progression from G(2) to mitosis being inhibited when chromatids are insufficiently decatenated. Mitotic delay was quantified in normal and checkpoint-deficient human cells during treatment with ICRF-193, a topoisomerase II catalytic inhibitor that prevents chromatid decatenation without producing topoisomerase-associated DNA strand breaks. Ataxia telangiectasia (A-T) cells, defective in DNA damage checkpoints, showed normal mitotic delay when treated with ICRF-193. The mitotic delay in response to ICRF-193 was ablated in human fibroblasts expressing an ataxia telangiectasia mutated- and rad3-related (ATR) kinase-inactive ATR allele (ATR(ki)). BRCA1-mutant HCC1937 cells also displayed a defect in ICRF-193-induced mitotic delay, which was corrected by expression of wild-type BRCA1. Phosphorylations of hCds1 or Chk1 and inhibition of Cdk1 kinase activity, which are elements of checkpoints associated with DNA damage or replication, did not occur during ICRF-193-induced mitotic delay. Over-expression of cyclin B1 containing a dominant nuclear localization signal, and inhibition of Crm1-mediated nuclear export, reversed ICRF-193-induced mitotic delay. In combination, these results imply that ATR and BRCA1 enforce the decatenation G(2) checkpoint, which may act to exclude cyclin B1/Cdk1 complexes from the nucleus. Moreover, induction of ATR(ki) produced a 10-fold increase in chromosomal aberrations, further emphasizing the vital role for ATR in genetic stability.

Figure 1

Sustained chromatid catenation induces mitotic delay in normal and A-T cells. (A) Normal and A-T lymphoblasts were sham-treated, irradiated with 1 Gy of γ-rays (IR), incubated in 2 μM ICRF-193, or incubated with DMSO solvent. Two hours after treatment or addition of drug, mitotic index was quantified as described in Materials and Methods. The results for IR and ICRF-193 are expressed as a percentage of the untreated control (mean + SD; n = 3–4 for all but GM03332C, where n = 2). (B) The mean values obtained in A were expressed as the average percentage of the untreated control for normal and A-T cells (mean + SD, n = 3). *, The mean inhibition of mitosis in A-T cells after IR was significantly less than in normal cells (Student's t test, P < 0.05).

Figure 2

ATR and BRCA1 are required for ICRF-193-induced mitotic delay. (A) Uninduced or induced GM847 cells were irradiated with 1.0 Gy of γ-rays (IR) or incubated with 2 μM ICRF-193 (I). Sham-treated and DMSO controls are denoted S and D, respectively. After 2 h, cells were harvested and Western immunoblot analysis was performed. (B) Uninduced (open bars) and induced GM847 (closed bars) cells were treated as described above. After 2 h, cells were assayed for mitosis. The results are expressed as the mean percentage of the untreated control (+ SD, for ICRF-193, n = 3; for IR, n = 2). *, The mean inhibition of mitosis in uninduced ICRF-193-treated cells was significantly different from that in induced ICRF-193-treated cells (Student's t test, P < 0.005). (C and D) Metaphase spreads of chromosomes from ATR^ki-induced GM847 cells. Arrows point to various chromosomal aberrations. (E) HCC1937 cells expressing GFP or BRCA1wt were irradiated with 1.0 Gy of γ-rays or incubated with 2 μM ICRF-193. After 2 h, cells were assayed for mitosis. The results are expressed as the mean percentage of the untreated control (+ SD, for ICRF-193, n = 3; for IR, n = 2). *, The mean inhibition of mitosis in GFP- expressing cells was significantly different from that in BRCA1wt-expressing cells (Student's t test, P < 0.005).

Figure 3

ICRF-193 does not induce phosphorylation of hCds1 or hChk1. (A) Normal and A-T lymphoblasts were sham-treated (S) or irradiated with 5 Gy of γ-rays (IR), incubated with DMSO solvent (D), incubated with 2 μM ICRF-193 (I), or retained untreated in the incubator (Inc). One hour after treatment or addition of drug, cells were harvested. (B) Uninduced or induced GM847 cells were harvested 2 h after sham treatment or irradiation with 1.0 or 5.0 Gy. Western immunoblot analysis was performed for expression of hCds1. (C) Uninduced or induced GM847 cells were harvested 2 h after sham treatment, irradiation with 50 J/m² UVC, or incubation with 2 mM HU or 2 μM ICRF-193 (I). IP-Western or Western blot analysis was performed for expression of ATR, Chk1, and Ser-345- or -317-phosphorylated Chk1.

Figure 4

Nuclear cyclin B1 abrogates ICRF-193-induced mitotic delay. (A) Human cell lines (normal and A-T lymphoblasts and fibroblasts, HeLa) were irradiated or incubated with ICRF-193 or DMSO. Two hours after treatment or addition of drug, cells were harvested and assayed for mitotic index and Cdk1 kinase activity. The results are expressed as a percentage of the untreated control. There was not a significant correlation between the reduction in mitotic index and Cdk1 kinase activity after incubation with ICRF-193 (R² = 0.03, P > 0.5). (B) Normal and A-T lymphoblastoid cell lines were treated with and without 6 ng/ml of LMB for 2 h, after which cells were fixed onto glass slides and immunofluorescence microscopy was performed. Representative images are shown for GM03189D (A-T). (C) Normal (GM03714A, GM01815, and GM03657A) and A-T lymphoblasts (GM09582, GM03189D, and GM03332C) were pretreated with and without 6 ng/ml of LMB and then were sham-treated, irradiated with 1 Gy of γ-rays, or incubated with 2 μM ICRF-193 or DMSO. Two hours after treatment or addition of drug, mitotic index was quantified. The results are expressed as the average percentage of control mitotic index in normal or A-T cells. (mean + SD, n = 3 for IR and n = 5 for ICRF-193). *, The mean inhibition of mitosis in LMB-treated cells was significantly different from the mean inhibition of mitosis in IR- or ICRF-193-treated normal and A-T cells (Student's t test, P < 0.05). (D) Log-phase NHF1hTERT cells were infected with adenovirus containing tetracycline transactivator (TTA) alone or coinfected with TTA and wt cyclin B1 (WTB) or cyclin B1 containing a dominant nuclear localization sequence (NB1). Twelve hours after infection, cells were harvested for cyclin B1 Western blot analysis, fixed for mitotic index determination, or incubated with DMSO or 2 μM ICRF-193 in the presence of 100 ng/ml of colcemid. After 6 h, cells were fixed and mitotic index was determined. The results are expressed as the percentage of mitotic cells at time of addition of DMSO or ICRF-193 (T0) and after 6 h of incubation in colcemid and ICRF-193 or DMSO. The results depicted were reproduced in two additional experiments.

Figure 5

(A) Mitotic entry checkpoints. (B) Model for ATR-dependent G₂ checkpoint function.