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Identification of Decatenation G₂ Checkpoint Impairment Independently of DNA Damage G₂ Checkpoint in Human Lung Cancer Cell Lines

¹ Division of Molecular Oncology, Aichi Cancer Center Research Institute, Nagoya, and ² Division of Respiratory Medicine, Department of Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan

	ABSTRACT

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It has been suggested that attenuation of the decatenation G₂ checkpoint function, which ensures sufficient chromatid decatenation by topoisomerase II before entering into mitosis, may contribute to the acquisition of genetic instability in cancer cells. To date, however, very little information is available on this type of checkpoint defect in human cancers. In this study, we report for the first time that a proportion of human lung cancer cell lines did not properly arrest before entering mitosis in the presence of a catalytic, circular cramp-forming topoisomerase II inhibitor ICRF-193, whereas the decatenation G₂ checkpoint impairment was present independently of the impaired DNA damage G₂ checkpoint. In addition, the presence of decatenation G₂ checkpoint dysfunction was found to be associated with diminished activation of ataxia-telangiectasia mutated in response to ICRF-193, suggesting the potential involvement of an upstream pathway sensing incompletely catenated chromatids. Interestingly, hypersensitivity to ICRF-193 was observed in cell lines with decatenation G₂ checkpoint impairment and negligible activation of ataxia-telangiectasia mutated. These findings suggest the possible involvement of decatenation G₂ checkpoint impairment in the development of human lung cancers, as well as the potential clinical implication of selective killing of lung cancer cells with such defects by this type of topoisomerase II inhibitor.

	INTRODUCTION

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Lung cancer currently claims more lives than any other cancers, placing it as the leading cause of cancer death in economically well-developed countries (1 , 2) . Lung cancer cells frequently exhibit complex chromosomal abnormalities, including multiple numerical (i.e., aneuploidy) and structural alterations including deletion, translocation, homogeneously staining region, and double minutes (3) . Cell cycle checkpoints are a monitoring system that arrests cells at a particular phase of the cell cycle in response to a lack of appropriate conditions for progression, thereby maintaining genetic stability and ensuring a smooth progression through the cell cycle (4) . Recent molecular and cellular biological studies have revealed that cell cycle checkpoints are often impaired in cancer cells, leading to the failure of normal growth control and the generation of genetic instability in cancer cells (5 , 6) . We previously reported on the pervasive presence of numerical chromosomal instability phenotype (7) and the frequent impairment of the mitotic spindle checkpoint in human lung cancer cell lines (8) . We have also shown that DNA damage G₂ checkpoint is often impaired in small cell lung cancer in a histological type-specific manner (9) .

It has been proposed that cells monitor the status of intertwined daughter chromatids after DNA replication and actively delay mitosis until chromatids are sufficiently decatenated by topoisomerase II (10) . Because complete chromatid decatenation is required for accurate chromatid segregation, it has been suggested that attenuation of decatenation G₂ checkpoint function contributes to the acquisition of genetic instability in cancer cells (11) . The bisdioxopiperazine, ICRF-193, sequesters topoisomerase II in a closed-clamp conformation without causing DNA damage and leads to the activation of decatenation G₂ checkpoint, resulting in the blockade of topoisomerase II at a point in its catalytic cycle after strand passage and re-ligation but before release of the passed DNA and ATP hydrolysis (12 , 13) . With regard to the underlying mechanisms of decatenation G₂ checkpoint, Deming et al. (11) previously reported that in contrast to DNA damage checkpoint, decatenation G₂ checkpoint activation relies on ataxia telangiectasia and Rad3-related (ATR) activity and nuclear exclusion of cyclin B1 instead of ataxia-telangiectasia mutated (ATM)-dependent down-regulation of cdc2/cyclin B1 activity. They also showed that decatenation G₂ checkpoint was not properly activated in a BRCA1-null cell line and that reconstitution with wt-BRCA1 restored ICRF-193-induced G₂ arrest (11) . To date, however, little is known about the potential involvement of decatenation G₂ checkpoint impairment in human cancers, and virtually no data are available regarding its relation to the pathogenesis of lung cancers.

In this study, we examined a panel of human lung cancer cell lines and found for the first time that decatenation G₂ checkpoint is impaired in a proportion of human lung cancer cell lines independently of the presence of the impairment DNA damage G₂ checkpoint. In addition, initial attempts were made to elucidate the molecular mechanism of the impairment of decatenation G₂ checkpoint in lung cancers.

	MATERIALS AND METHODS

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Cell Lines and Culture.
ACC-LC-48, ACC-LC-49, ACC-LC-172, and ACC-LC-176 were established in our laboratory, whereas NCI-H460 and A549 were purchased from the American Type Culture Collection (Manassas, VA). QG56 and PC-10 were generously provided by Dr. Yoshihiro Hayata (Tokyo Medical University, Tokyo, Japan). This panel of lung cancer cell lines included three small cell lung cancers (ACC-LC-48, ACC-LC-49, and ACC-LC-172), an adenocarcinoma (A549), three squamous cell carcinoma (ACC-LC-176, QG56, and PC-10), and a large cell carcinoma (NCI-H460) cell lines. Normal human bronchial epithelial cells were purchased from Sanko Junyaku (Tokyo, Japan). These cell lines were tested with the TaKaRa PCR Mycoplasma detection kit (Takara Bio, Inc., Shiga, Japan) and were shown to be free of Mycoplasma contamination. Normal human bronchial epithelial cells were cultured essentially according to the manufacturer’s instructions. Other cell lines were cultured in RPMI 1640 supplemented with 5% fetal bovine serum (Invitrogen-Life Technologies, Inc., Grand Island, NY), 100 units/ml penicillin, and 100 µg/ml streptomycin.

Cell Cycle Synchronization.
All synchronization was performed using optimized conditions for each cell line via the double thymidine/aphidicolin sequential block, except for PC-10 via single thymidine block. In brief, 24-h treatment with 2 mM thymidine was used to arrest exponentially proliferating cells in the early S-phase, except for ACC-LC-172, in which 36-h treatment was carried out. Cells were then released from the arrest by three washes in PBS and grown in fresh medium supplemented with 1 µM deoxycytidine for the following durations: 10 h in A549 and ACC-LC-176; 12 h in NCI-H460; 14 h in QG56 and ACC-LC-48; and 18 h in ACC-LC-49 and 20 h in ACC-LC-172. Five µM aphidicolin were added in all of the cell lines, except for QG56 (2 µM), followed by additional incubation for 14 h in A549, NCI-H460, QG56, and ACC-LC-176, 16 h in ACC-LC-48, and for 24 h in ACC-LC-49 and ACC-LC-172. Cells were washed three times with PBS and placed into normal medium. At this point, 85% of the cells were found to be at the G₁-S border by propidium iodide-flow cytometry analysis. The cells were then grown without any constraints for the following periods of time, which were predetermined in each cell line to attain optimal synchronization in the G₂ phase: 6 h in A549 and NCI-H460; 7 h in QG56 and ACC-LC-176; 8 h in ACC-LC-48 and PC-10; 10 h in ACC-LC-172; and 12 h in ACC-LC-49. At this point, the following percentages of cells were found to be in the G₂ phase of the cycle as evidenced by 4N DNA content with very few mitotic figures: 77% in A549; 55% in NCI-H460; 83% in QG56; 74% in ACC-LC-49; 87% in ACC-LC-172; 70% in ACC-LC-48; 64% in ACC-LC-176; and 72% in PC10.

Antibodies.
The following antibodies were purchased from their respective companies: anti-cdc2 (sc-54), anti-cyclin B1 (GNS1), anti-ATR/FRP1 (N-19), and anti-BRCA1 (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA); anti--H2AX (rabbit) and anti-Plk1 (Upstate Biotechnology, Lake Placid, NY); anti-ATM p-S1981 (Rockland, Gilbertsville, PA); anti-ATM (Ab-3) (Calbiochem, La Jolla, CA); anti--tubulin (Sigma, St. Louis, MO); and anti-WRN (BD Transduction Laboratories, San Diego, CA). Anti-topoisomerase II (clone 1C5) and anti-53BP1 antibodies were generous gifts from Dr. Ryoji Ishida (Aichi Cancer Center Research Institute) and Dr. Thanos D. Halazonetis (Department of Molecular Genetics, The Wistar Institute, Philadelphia, PA), respectively.

Measurement of Mitotic Indices.
Mitotic delay was examined by quantifying the mitotic index in replicate cultures of treated and control cells. To measure responses to ICRF-193 or MST-16, cells were treated with solvent alone or with 2 µM ICRF-193 (a generous gift from Dr. Ryoji Ishida) or with 100 µM MST-16 (Zenyaku Kogyo, Tokyo, Japan), whereas 1 Gy of irradiation using Hitachi MBR-1520R (Hitachi, Tokyo, Japan) was conducted to examine DNA damage-induced mitotic delay in G₂. Sham-treated controls were subjected to the same movements into and out of incubators as treated cells. Cells were harvested by trypsinization, followed by fixation with 4% formalin in PBS for 30 min. Cells were then attached to glass slides by centrifugation using an Auto Smear CF-12D (Sakura, Tokyo, Japan) and stained with 0.1 µg/ml 4',6-diamidino-2-phenylindole. To measure the mitotic index, at least 2000 cells were counted for each slide under a fluorescence microscope as described previously (14) .

Western Blot Analysis.
An equal amount of total cell lysate solubilized in Laemmli’s sample buffer was electrophoresed on SDS-polyacrylamide gels and transferred to Immobilon-P filters (Millipore Corp., Bedford, MA). The filters were first incubated with a primary antibody and then with horseradish peroxidase-conjugated secondary antibodies [antimouse IgG and antirabbit IgG, Cell Signaling Technology (Beverly, MA); antigoat IgG MBL (Nagoya, Japan)]. For visualization, an enhanced chemiluminescence system (Amersham, Buckinghamshire, United Kingdom) was used.

Assay for Clamp Formation between Topoisomerase II and DNA.
Assay for clamp formation between topoisomerase II and DNA was performed essentially as described by Nishida et al. (15) . In brief, cells were incubated with ICRF-193 for 2 h, lysed in ice-cold lysis buffer [50 mM Tris-HCl (pH 7.4), 350 mM NaCl, 0.1% NP40, 5 mM EDTA, 50 mM NaF, and 1x complete protease inhibitor mixture (Roche, Mannheim, Germany)] containing ICRF-193 while standing for 1 h on ice, and then centrifuged at 15,000 rpm for 15 min at 4°C. The supernatants were then separated on a SDS-PAGE and blotted with the anti-topoisomerase II antibody.

Immunoprecipitation and in Vitro Kinase Assay.
Unsynchronized cells were exposed to solvent alone, 2 µM ICRF-193, or 1 Gy irradiation for 2 h. Cells were then harvested and lysed through sonication in NETN buffer [20 mM Tris (pH 8.0), 400 mM NaCl, 1 mM EDTA, 0.5% NP40, 10 mM NaF, 1 mM Na₃VO₄, 10 mM glycerophosphate, and 1x complete protease inhibitor mixture] as described by Smits et al. (16) . Lysates were centrifuged at 15,000 rpm at 4°C. Immunoprecipitation was carried out by incubating 500 µg of whole cell lysates with 2 µg of the anti-cyclinB1 antibody and 20 µl of protein G-Sepharose beads (1:1) for 1 h at 4°C. Beads were washed three times with NETN buffer and twice with kinase buffer [20 mM Tris (pH 7.5), 5 mM MgCl₂, 2.5 mM MnCl₂, and 1 mM DTT]. Half of the immunoprecipitates were incubated in kinase buffer supplemented with 50 µM ATP, 2.5 µCi [-³²P]ATP (Amersham), and 10 µg of histone H1 (Roche) for 30 min at 30°C. The kinase reaction was stopped by adding SDS sample buffer and boiling the samples for 5 min. Samples were separated on 12.5% polyacrylamide gel, and the phosphorylated histone H1 was visualized by autoradiography. The remaining immunoprecipitates were separated on a SDS-PAGE and blotted with the anti-cdc2 antibody.

Immunocytochemistry.
Cells grown on coverslips were either mock treated or exposed to 2 µM ICRF-193 for 1 h as described above. Cells were fixed in 1% paraformaldehyde for 15 min followed by treatment with 0.2% Triton X-100 on ice for 20 min. Coverslips were incubated with rabbit polyclonal anti--H2AX antibody and mouse monoclonal anti-53BP1 antibody overnight at 4°C, followed by 1-h incubation with an Alexa 488-conjugated goat antirabbit and Alexa 568-conjugated antimouse secondary antibody (Molecular Probes, Eugene, OR). Cell nuclei were then stained with 4',6-diamidino-2-phenylindole as described above.

Mutational Analysis.
PCR amplification using random-primed first-strand cDNAs was performed with the aid of the following oligonucleotide primers, and the PCR products were subjected to direct sequencing with an ABI3100 (Perkin-Elmer, Foster City, CA) DNA sequencer and a Dye Terminator Cycle Sequencing kit (Perkin-Elmer). The primer pairs used for amplification of the WRN gene were as follows: F1 (sense; 5'-TGGATCTTCTCGGGTTTTCTT-3') and R1 (antisense; 5'-TGGCAACATCTGTCAACTCC-3'); F2 (sense; 5'-GGAGTGGCCACCATTATACA-3') and R2 (antisense; 5'-TCCGTGGGTTTTCCAATTTA-3'); F3 (sense;5'-ATCCGCTGTAGCAATTGGAG-3') and R3 (sense; 5'-GACATCAAACAAGCTCTTTCCA-3'); F4 (sense; 5'-GAACTGAGGCCCAGCAATAA-3') and R4 (antisense; 5'-GGAAGACCCAGATTTCTTTCC-3'); F5 (sense; 5'-CTGAGCTGAGCATTTATCTCCCAATGA-3') and R5 (antisense; 5'-GCTGGGATGTTGGACATTTT-3'); F6 (sense; 5'-ACTTTGGCCATTCCAGTTTT-3') and R6 (antisense; 5'-TGGTCGATCAAAACCAGTACA-3'); F7 (sense; 5'-AGGTAACATGGGCCTGCTC-3') and R7 (antisense; 5'-ACTTGGCGAATGTCAGCTTT-3'); F8 (sense; 5'-TGGGAATTTGAAGGTCCAAC-3') and R8 (antisense; 5'-TCCATGGAATAGCAATGATCC-3'); F9 (sense; 5'-GGGCTCCTGCAGACATTAAC-3') and R9 (antisense; 5'-GCTTGAAGGATGAGGCTCTG-3'); F10 (sense; 5'-GCGTCTTGCCGATCAATATC-3') and R10 (antisense; 5'-TTTTGGCCATATCCACCAGT-3'); F11 (sense; 5'-TGGTACAGTCACCAGAAAAAGC-3') and R11 (antisense; 5'-TTCTGAACCTCTGGAGTCAGG-3'); F12 (sense; 5'-CAGAAGACGAGTCTGGTAGCAA-3') and R12 (antisense; 5'-ACCTCCCCTTTTCGTTTTGT-3'); and F13 (sense; 5'-CACATGGCAATTGAGATCCTT-3') and R13 (antisense; 5'-TCAGAGCACATAACATTTTCCAA-3'). PCR amplification was carried out in the presence (F1-R1, F2-R2, F4-R4, F6-R6, F7-R7, F8-R8, and F13-R13) or the absence (F3-R3, F5-R5, F9-R9, F10-R10, F11-R11, and F12-R12) of 10% glycerol and consisted of 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min (F1-R1, F3-R3, F4-R4, F5-R5, F7-R7, F8-R8, F9-R9, F10-R10, and F11-R11) or at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min (F2-R2, F6-R6, F12-R12, and F13-R13).

Cell Proliferation Assay.
ICRF-193-induced cytotoxicity was determined by modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Briefly, cells were plated in 96-well plates and treated with various concentrations of ICRF-193 in a final concentration of 0.2% DMSO. After 48 h, the number of cells was measured with a colorimetric assay reagent, TetraColor One (Seikagaku, Tokyo, Japan), according to the manufacturer’s protocol. Five replicate wells were measured for each drug concentration. The IC₅₀ values were calculated as the drug concentration that killed 50% of the cells.

	RESULTS

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Identification of Impaired G₂ Arrest in Response to ICRF-193 in Human Lung Cancer Cell Lines.
Eight human lung cancer cell lines were examined to investigate whether decatenation G₂ checkpoint is perturbed in this common adult cancer, resulting in the identification of two distinct types of response. Five cell lines showed significant inhibition of entry into mitosis by the treatment with ICRF-193, a finding consistent with the presence of proficient decatenation G₂ checkpoint (Fig. 1A) . In marked contrast, ACC-LC-48 and ACC-LC-176 showed virtually no or only negligible delay and inhibition of the cell cycle progression in the presence of ICRF-193, whereas ICRF-193-induced inhibition at G₂ was similarly observed in PC-10 in a slightly less pronounced manner (Fig. 1B) . We note that the assay for protein-closed clamp formation indicated that nonextractable, DNA-bound topoisomerase II was formed at similar levels in all cell lines examined (data not shown). In addition, another bisdioxopiperazine agent, MST-16, clearly reduced the rate of entry into mitosis, although to a slightly less pronounced extent (data shown for ACC-LC-48 and ACC-LC-172 shown in Fig. 1C and data not shown for QG-56 and ACC-LC-49), which might be due to its lower potency as a topoisomerase II inhibitor than ICRF-193 (12) . These findings provided the first direct evidence for the presence of impaired decatenation G₂ checkpoint in a proportion of human lung cancer cell lines.

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Impaired decatenation G2 checkpoint function in lung cancer cell lines. Cells were synchronized to the G2 phase of the cell cycle and then incubated with solvent alone or with 2 µM ICRF-193. A, significant inhibition of the entry into mitosis in the presence of 2 µM ICRF-193 in five of eight lung cancer cell lines, a finding consistent with the presence of proficient decatenation G2 checkpoint. B, significantly perturbed G2 arrest in response to ICRF-193 treatment in three of the eight lung cancer cell lines, indicating impaired decatenation G2 checkpoint function. C, significant abrogation of G2 arrest in response to MST-16 treatment in ACC-LC-48 in contrast to inhibition of mitotic entry in ACC-LC-172. bars, ±SD.

View larger version (63K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. In vitro kinase assay of the cdc2/cyclin B1 kinase. A, significantly diminished histone H1 kinase activity in response to ICRF-193 treatment in A549 and NCI-H460 but not in QG56, ACC-LC-172, and ACC-LC-49, all of which had intact decatenation G2 checkpoint. B, absence of reduction in the cdc2/cycB1 kinase activity in ACC-LC-48, ACC-LC-176, and PC-10, all with impaired decatenation G2 checkpoint.

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Representative results of formation of -H2AX and 53BP1 nuclear foci in response to ICRF-193 treatment. Colocalized clear nuclear foci of -H2AX and 53BP1 were observed only in A549 and NCI-H460 (data not shown).

View larger version (63K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Induction of ataxia-telangiectasia mutated (ATM) activation by ICRF-193 as reflected by its autophosphorylation. A, significant induction of ATM autophosphorylation in A549 and NCI-H460, a finding consistent with the occurrence of DNA DSBs. B, significant induction of ATM autophosphorylation in decatenation G2 checkpoint proficient cell lines. C, markedly impaired ATM activation by ICRF-193 treatment in decatenation G2 checkpoint impaired cell lines, especially in ACC-LC-48 and ACC-LC-176. The Western blot analysis shown in A, B, and C was performed in parallel, and the exposure time was exactly the same.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. A, sensitivity to ICRF-193 measured by colorimetric cell proliferation assay after 48 h of continuous treatment. bars, ±SD. Note that ACC-LC-176 and ACC-LC-48 with clearly impaired ataxia-telangiectasia mutated activation and deficient decatenation G2 checkpoint were hypersensitive to ICRF-193. B, representative results of flow cytometric analysis of cells treated with 2 µM ICRF-193 for 48 h. C, apoptosis induced by 48 h of continuous treatment with 2 µM ICRF-193 in a panel of human lung cancer cell lines. Bars indicate increase in the apoptotic cells in ICRF-193-treated cells. NHBE, normal human bronchial epithelial cells.

	DISCUSSION

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It is widely accepted that checkpoint impairment may contribute to the carcinogenesis and progression of human cancers. We previously reported on the frequent occurrence of DNA damage G₂ checkpoint impairment (9) , as well as of mitotic checkpoint dysfunction in human lung cancers (8 , 15) in addition to G₁ checkpoint impairment, because of frequent p53 inactivation (21) . In the study reported here, we have provided for the first time direct evidence that decatenation G₂ checkpoint is impaired in a proportion of human lung cancer cell lines, adding decatenation G₂ checkpoint as another checkpoint mechanism perturbed in lung cancers. Although chromosome instability is very common in lung cancer cells (3) , an intriguing link between inactivation of decatenation G₂ checkpoint function and acquisition of chromosomal abnormality has been suggested. Deming et al. (22) reported that chromatid discohesion and subsequent constrictions and tangles were caused by overriding ICRF-193-induced G₂ arrest with the use of an ATM/ATR inhibitor caffeine, which resulted in the eventual chromatid breaks and exchanges. Franchitto et al. (20) also suggested that inactivation of decatenation G₂ checkpoint could contribute to chromosomal instability in conjunction with other failures.

To date, however, very little information is available on this type of checkpoint defect in human cancer cells. We note that during the preparation of this article, Doherty et al. (23) reported the identification of decatenation G₂ checkpoint impairment by analyzing five bladder cancer cell lines. Although future studies of other types of human cancers are needed, it is possible that decatenation G₂ checkpoint impairment may be a common event in various types of human cancers, playing a role in the processes of carcinogenesis and/or progression by inducing a chromosome instability phenotype.

This study also clearly shows that impairment of decatenation G₂ checkpoint is not concordantly present with the perturbation of DNA damage G₂ checkpoint in lung cancer cell lines, suggesting that the signaling pathways, which activate these two checkpoints, are at least in part distinct, although this does not preclude the possibility that these checkpoints share a common molecule(s) in their signaling pathways. In this study, we were able to show that three lung cancer cell lines, namely QG56, ACC-LC-49, and ACC-LC-172, retained their ability to properly arrest at G₂ in response to ICRF-193 without causing appreciable DNA damage-induced reactions such as those observed by 1 Gy of irradiation in DNA damage G₂ checkpoint-proficient cell lines. In addition, we did not observe phosphorylation of Chk1 at Ser³⁴⁵ in ICRF-193-treated QG56 (unpublished observation), which is consistent with the result of a previous study (11) , supporting the notion that the observed delays in entry into mitosis in response to ICRF-193 was not caused by the imposition of replication stress. These findings are consistent with the previously held notion that ICRF-193 is a purely catalytic inhibitor of topoisomerase II, acting in the form of a closed protein clamp without inducing DNA damage (10 , 13 , 24 , 25) . However, we also observed the rather unexpected occurrence that ICRF-193-induced DNA DSBs and DNA damage checkpoint activation in two cell lines, i.e., A549 and NCI-H460. DNA DSBs observed in A549 and NCI-H460 are not attributable merely to the deficiency of decatenation G₂ checkpoint function because ACC-LC-48, ACC-LC-176, and PC-10 did not form nuclear foci of -H2AX and 53BP1 without proper G₂ arrest in response to ICRF-193 treatment. Our results suggest that ICRF-193 induces DNA DSBs in a certain cellular context by an as yet unknown underlying mechanism. In this connection, a similarly contradictory finding was recently reported by Hajii et al. (26) using the comet assay and pulsed-field gel electrophoresis analysis in cultured V79 and irs-2 Chinese hamster lung fibroblasts. These observations indicate that future studies of decatenation checkpoint need to pay careful attention to inadvertent activation of DNA damage checkpoint by a said catalytic, non-DNA-damaging topoisomerase II inhibitor such as ICRF-193.

As for a candidate molecule responsible for such a defect, it is notable that normal lymphoblast and fibroblast cells of patients with Werner syndrome have been shown to be unable to arrest before entering into mitosis when incubated with the bisdioxopiperazine ICRF-187, an agent with properties similar to ICRF-193, and that the introduction of wild-type WRN significantly restored ICRF-187induced G₂ arrest (20) . Thus, the WRN gene appears to be an excellent candidate for a genetic/epigenetic lesion responsible for decatenation G₂ checkpoint impairment in lung cancers. However, our extensive search for mutations in the WRN gene did not disclose the presence, and Western blot analysis did not indicate a readily detectable association between decatenation G₂ checkpoint impairment and expression levels of WRN. It has also been suggested that ATR, BRCA1, and Plk1 are involved in the decatenation G₂ checkpoint response (11 , 22) , but we did not find any clear association between decatenation G₂ checkpoint impairment and the expression levels of these molecules in the panel of cell lines used in this study. Therefore, additional studies are clearly needed to elucidate the underlying mechanisms responsible for the impairment of decatenation G₂ checkpoint in human lung cancer cell lines.

In this connection, the findings of our study suggests a possibility of a potential link between decatenation G₂ checkpoint impairment and diminished ATM activation in response to ICRF-193. Because it has been shown that ATM can be activated by hypotonic swelling or by treatment with chloroquine or trichostatin in the absence of a detectable DNA strand break (19) , it is possible that activation of ATM in the absence of DNA DSBs may reflect changes in chromatin structure such as incompletely catenated chromatids because of the ICRF-193 treatment. It has been shown that ICRF-193-induced G₂ arrest is ATM independent and relies on ATR activity by analyzing ataxia-telangiectasia cells and human fibroblast overexpressing a kinase-inactive ATR allele (11) . The ATM activation by the ICRF-193 treatment observed in our study may accordingly be a mere reflection of activation of a molecule(s) upstream to ATM. It remains possible, however, that ATM might cooperate with ATR and redundantly play a functional, yet dispensable, role in the implementation of decatenation G₂ checkpoint. Nevertheless, our findings strongly suggest that in a proportion of human lung cancer cell lines, the process of sensing incompletely catenated chromatids, which also triggers ATM activation in lung cancer cell lines as well as in primary normal human bronchial epithelial cells (data not shown), is impaired.

Another interesting point of this study is a potential association between decatenation G₂ checkpoint impairment and hypersensitivity to ICRF-193, although the small number of cell lines examined did not allow us to draw a definite conclusion. Both ACC-LC-48 and ACC-LC-176, which exhibited markedly impaired decatenation G₂ checkpoint and significantly diminished ATM activation, exhibited hypersensitivity to ICRF-193. This notion that ICRF-193 may selectively render decatenation G₂ checkpoint-defective cancer cells vulnerable to the induction of cell death is of great clinical interest because we may be able to take advantage of the presence of this association to confer cancer-specific killing activity with a catalytic circular cramp-forming topoisomerase II inhibitors. In this regard, catalytic topoisomerase inhibitors such as aclarubicin and sobuzoxane are being used as antineoplastic agents (27 , 28) , and novel agents can be expected to emerge soon.

We note that our results are also in line with those of a recently published study, which reported that continuous inhibition of topoisomerase II by ICRF-187 with activities similar to ICRF-193 induced apoptotic cell death in the WRN-defective normal cells of the Werner syndrome cases and that introduction of wild-type WRN cDNA restored decatenation checkpoint as well as resistance to ICRF-187-induced apoptosis (20) . Furthermore, they showed that normal cells, which could escape the decatenation G₂ checkpoint because of caffeine treatment and were treated continuously with ICRF-187, underwent apoptotic cell death only to a lesser extent. In this connection, we also observed that caffeine abrogated decatenation G₂ checkpoint in lung cancer cell lines with proficient decatenation G₂ checkpoint but did not significantly sensitize decatenation G₂ checkpoint proficient cell lines to ICRF-193 (data not shown). These findings suggest that hypersensitivity to ICRF-193 is not the result of solely and directly from entering into mitosis with insufficiently catenated chromatids but rather from the combination of these effects with failure of an additional surveillance pathway(s). Additional studies are obviously necessary to clarify the molecular basis of hypersensitivity to catalytic topoisomerase II inhibitors, which may ultimately lead to the development of an attractive strategy for lung cancer treatment, i.e., selective killing of targeted cancer cells without causing prominent toxicity in normal cells.

In conclusion, our study clearly shows that decatenation G₂ checkpoint is impaired in a proportion of lung cancer cell lines in association with diminished ATM activation and also potentially with hypersensitivity to ICRF-193. The observations reported here warrant further study aimed not only at a better understanding of the underlying mechanisms and biological consequences of decatenation G₂ checkpoint impairment in lung cancers but also the development of novel targeted therapeutic approaches for this fatal cancer.

	ACKNOWLEDGMENTS

 
We thank Dr. Ryoji Ishida for providing ICRF-193 and anti-topoisomerase II antibodies, as well as for helpful suggestions, and Dr. Thanos D. Halazonetis for the anti-53BP1 antibody.

	FOOTNOTES

 
Grant support: Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, Japan and a Grant-in-Aid for the Second Term Comprehensive Ten-Year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: A. Masuda is currently at Cellseed, Inc., Shinjuku, Shinjuku-ku, Tokyo, Japan.

Requests for reprints: Takashi Takahashi, Division of Molecular Oncology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan. Phone/Fax: 81-52-764-2983; E-mail: tak{at}aichi-cc.jp

Received 3/11/04. Revised 5/10/04. Accepted 5/13/04.

	REFERENCES

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