Hypersensitivity of Ku-Deficient Cells toward the DNA Topoisomerase II Inhibitor ICRF-193 Suggests a Novel Role for Ku Antigen during the G₂and M Phases of the Cell Cycle

Abstract

Ku antigen is a heterodimer, comprised of 86-and 70-kDa subunits, which binds preferentially to free DNA ends. Ku is associated with a catalytic subunit of 450 kDa in the DNA-dependent protein kinase (DNA-PK), which plays a crucial role in DNA double-strand break (DSB) repair and V(D)J recombination of immunoglobulin and T-cell receptor genes. We now demonstrate that Ku86 (86-kDa subunit)-deficient Chinese hamster cell lines are hypersensitive to ICRF-193, a DNA topoisomerase II inhibitor that does not produce DSB in DNA. Mutant cells were blocked in G 2 at drug doses which had no effect on wild-type cells. Moreover, bypass of this G 2 block by caffeine revealed defective chromosome condensation in Ku86-deficient cells. The hypersensitivity of Ku86-deficient cells toward ICRF-193 was not due to impaired in vitro decatenation activity or altered levels of DNA topoisomerase II␣ or -␤. Rather, wild-type sensitivity was restored by transfection of a Ku86 expression plasmid into mutant cells. In contrast to cells deficient in the Ku86 subunit of DNA-PK, cells deficient in the catalytic subunit of the enzyme neither accumulated in G 2 /M nor displayed defective chromosome condensation at lower doses of ICRF-193 compared to wild-type cells. Our data suggests a novel role for Ku antigen in the G 2 and M phases of the cell cycle, a role that is not related to its role in DNA-PK-dependent DNA repair.Ku is an abundant nuclear protein originally identified as an autoantigen recognized by sera from patients with autoimmune diseases (47). The Ku antigen is a heterodimer comprised of 86-and 70-kDa subunits (Ku86 and Ku70) which interacts preferentially with free DNA ends or particular DNA structures such as hairpins or gaps (46)(47)(48). Ku is associated with a catalytic subunit of 450 kDa (DNA-PK CS ), forming the DNA-dependent protein kinase (DNA-PK). It is presumed that the Ku antigen tethers the serine-threonine kinase activity of this enzyme to DNA ends, allowing phosphorylation of nearby substrates (22,28). Accordingly, in vitro phosphorylation of a large number of proteins by DNA-PK is stimulated in the presence of double-strand breaks (DSB) (12, 43; reviewed in reference 3). A series of genetic and biochemical data have converged to establish the crucial role of DNA-PK in DSB repair as well as in V(D)J recombination of immunoglobulin and T-cell receptor genes (reviewed in references 35, 69, and 75). Defective DSB repair and V(D)J recombination in X-raysensitive rodent cell lines of the complementation group for XRCC5 were rescued by transfection of the human Ku86 cDNA (9, 57, 59), and mutations that are responsible for the X-ray sensitivity of these cells were mapped to the Ku86 gene, demonstrating unambiguously that Ku86 is the product of XRCC5 (25). An additional role for DNA-PK in DNA replication has been suggested by its ability to phosphorylate replication protein A (10, 52). Furthermore, a role in transcription is indicated by the ability of DNA-PK to inhibit RNA polymerase I activity (39, 41), as well as to modu...

The bacterial cytolethal distending toxin (CDT) triggers a G2 cell cycle checkpoint in mammalian cells without preliminary induction of DNA strand breaks

“…The higher resistance of etoposideinduced block to ca eine alleviation is possibly mechanistically linked to the irreversible nature of this block, as compared to the transient nature of Xray-induced block (Poon et al, 1997a). In etoposidetreated cells, ca eine resistance may be related to the inability of cells to undergo normal chromosome condensation, an event that required the participation of DNA topoisomerase IIa (Munoz et al, 1998). In CDT-arrested cells the nature of this ca eineinsensitive component should be investigated further.…”

“…Another ca eine-sensitive checkpoint control speci®cally induced in S phase has also been described: the so-called topoisomerase IIdependent G2 checkpoint (Downes et al, 1994). This checkpoint is activated by inhibitors of topoisomerase II such as ICRF-193, which inhibits chromatid decatenation without damaging DNA (Kaufman and Kies, 1998;Munoz et al, 1998). Like CDT, ICRF-193 does not seem to interfere with cell progression through S phase (Ishida et al, 1994).…”

Hypersensitivity of Nonhomologous DNA End-joining Mutants to VP-16 and ICRF-193

“…Earlier work by Muñ oz et al (47) suggested a novel role for Ku at G 2 /M to explain their observation that Ku86-deficient cells were hypersensitive to ICRF-193. This was, however, based on the assumption that the drug is a pure catalytic inhibitor of topo II.…”

Degradation of ATM-Independent Decatenation Checkpoint Function in Human Cells is Secondary to Inactivation of p53 and Correlated with Chromosomal Destabilization

“…Moreover, cytogenetic analysis of ICRF-193-treated E6-expressing cells demonstrated the presence of improperly condensed, entangled chromatids in metaphase spreads (not shown) as commonly observed in ICRF-193-treated mammalian cells. 15,70,75 Evidently, ICRF-193 reached its nuclear target, topoisomerase II, and blocked chromatid decatenation in the E6-expressing cells at high PDL.…”

“…73 However, mammalian cells exposed to a lower concentration of ICRF-193 prior to mitosis arrest growth in G2. 14,15,74,75 The fact that caffeine could override ICRF-193-induced, but not IR-induced, G2 delay in the DM87 muntjac cell line suggested that IR and ICRF-193 trigger G2 checkpoint response using different signaling pathways. 15 The studies reported here were designed to characterize genetic requirements for the decatenation checkpoint in human cells and determine whether conditions that lead to inactivation of DNA damage G2 checkpoint function also affect the decatenation checkpoint.…”