The DNA damage response to non-replicating adeno-associated virus: Centriole overduplication and mitotic catastrophe independent of the spindle checkpoint

Adeno-associated virus (AAV) type 2 or UV-inactivated AAV (UV-AAV2) infection provokes a DNA damage response that leads to cell cycle arrest at the G2/M border. p53-deficient cells cannot sustain the G2 arrest, enter prolonged impaired mitosis, and die. Here, we studied how non-replicating AAV2 kills p53-deficient osteosarcoma cells. We found that the virus uncouples centriole duplication from the cell cycle, inducing centrosome overamplification that is dependent on Chk1, ATR and CDK kinases, and on G2 arrest. Interference with spindle checkpoint components Mad2 and BubR1 revealed unexpectedly that mitotic catastrophe occurs independently of spindle checkpoint function. We conclude that, in the p53-deficient cells, UV-AAV2 triggers mitotic catastrophe associated with a dramatic Chk1-dependent overduplication of centrioles and the consequent formation of multiple spindle poles in mitosis. As AAV2 acts through cellular damage response pathways, the results provide information on the role of Chk1 in mitotic catastrophe after DNA damage signaling in general.     

p53 deficiency and defective mitotic checkpoint in proliferating T lymphocytes increase chromosomal instability through aberrant exit from mitotic arrest

During the proliferation of T cells for successful immune responses against pathogens, the fine regulation of cell cycle is important to the maintenance of T cell homeostasis and the prevention of lymphoproliferative disorders. However, it remains to be elucidated how the cell cycle is controlled at the mitotic phase in proliferating T cells. Here, we show that during the proliferation of primary T cells, the disruption of the mitotic spindle leads to cell-cycle arrest at mitosis and that prolonged mitotic arrest results in not only apoptosis but also the form of chromosomal instability observed in human cancers. It is interesting that in response to spindle damage, the phosphorylation of BubR1, a mitotic checkpoint kinase, was significantly induced in proliferating T cells, and the expression of the dominant-negative mutant of BubR1 compromised mitotic arrest and subsequent apoptosis and thus led to the augmentation of polyploidy formation. We also show that in response to prolonged spindle damage, the expression of p53 but not of p73 was significantly induced. In addition, following sustained mitotic arrest, p53-deficient T cells were found to be more susceptible to polyploidy formation than the wild type. These results suggest that during flourishing immune response, mitotic checkpoint and p53 play important roles in the prevention of chromosomal instability and in the maintenance of the genomic integrity of proliferating T cells.     

The C-terminus of Bfa1p in budding yeast is essential to induce mitotic arrest in response to diverse checkpoint-activating signals

During mitosis, genomic integrity is maintained by the proper coordination of anaphase entry and mitotic exit through mitotic checkpoints. In budding yeast, exit from mitosis is triggered by the activation of the small GTPase Tem1p. Bfa1p in association with Bub2p negatively regulates Tem1p in response to spindle damage, spindle misorientation, and DNA damage, resulting in cell cycle arrest. To delineate the Bfa1p domains that respond to distinct checkpoint signals, we constructed 13 Bfa1 deletion mutants. The C-terminal 184 amino acids of Bfa1p (Bfa1-D8(391-574)) contained the entire capacity of Bfa1p to generate mitotic arrest in response to spindle damage, spindle misorientation, and DNA damage. This domain was also enough to interact with the mitotic exit network proteins Tem1p, Bub2p, and Cdc5p, and to localize to the spindle pole body (SPB). Over-expression of Bfa1-D8(391-574) induced late anaphase arrest as efficient as the full-length Bfa1p in a Bub2p-dependent manner. In contrast, the N-terminal portion of Bfa1p (Bfa1-D16(1-376)) could not localize to SPB and did not block mitotic exit in response to diverse checkpoint signals. Bfa1-D16(1-376) interacted with Tem1p but not with Bub2p and its over-expression partially arrested cells in mitosis in the absence of Bub2p. By random mutagenesis of Bfa1-D8(391-574) with hydroxylamine, we isolated a point mutant of D8, D8(E438K), which interacts with both Tem1p and Bub2p but cannot respond to checkpoint signals. This mutant also showed reduced efficiency in the localization to SPB. Taken together, our study demonstrated that various checkpoint signals are transmitted to the C-terminal domain of Bfa1 (Bfa1-D8(391-574)) and that Bfa1p localization to SPB is necessary but not sufficient to regulate mitotic exit in response to various checkpoint signals.     

Meiotic DNA replication checkpoint control in fission yeast

In eukaryotes, the DNA replication checkpoint prevents entry into mitosis when DNA replication is incomplete and is crucial for maintaining genomic integrity. Much less is known about equivalent controls that operate during meiosis. Here, we show that a DNA replication checkpoint control operates during meiosis in fission yeast. The mitotic checkpoint Rad genes and the Cds1 protein kinase are required for the DNA replication checkpoint during meiosis, with Cds1 playing a more prominent role than it does during mitosis. When DNA replication is blocked, the checkpoint maintains Cdc2 tyrosine 15 phosphorylation keeping Cdc2 protein kinase activity low and preventing onset of meiosis I. Additionally, there is a second checkpoint acting during meiosis that is revealed if cells are prevented from maintaining Cdc2 tyrosine 15 phosphorylation when DNA replication is blocked. Such cells arrest with high Cdc2 protein kinase activity and separated spindle pole bodies, an arrest state similar to that observed in mitotic budding yeast cells when DNA replication is incomplete. This second checkpoint is meiosis specific and may reflect processes occurring only during meiosis such as increased recombination rates, an extended duration of nuclear division, or homolog chromosome pairing.     

A positive feedback loop between the p53 and Lats2 tumor suppressors prevents tetraploidization

Damage to the mitotic spindle and centrosome dysfunction can lead to cancer. To prevent this, cells trigger a succession of checkpoint responses, where an initial mitotic delay is followed by slippage without cytokinesis, spawning tetraploid G1 cells that undergo a p53-dependent G1/S arrest. We describe the importance of Lats2 (Large Tumor Suppressor 2) in this checkpoint response. Lats2 binds Mdm2, inhibits its E3 ligase activity, and activates p53. Nocodazole, a microtubule poison that provokes centrosome/mitotic apparatus dysfunction, induces Lats2 translocation from centrosomes to the nucleus and p53 accumulation. In turn, p53 rapidly and selectively up-regulates Lats2 expression in G2/M cells, thereby defining a positive feedback loop. Abrogation of Lats2 promotes accumulation of polyploid cells upon exposure to nocodazole, which can be prevented by direct activation of p53. The Lats2-Mdm2-p53 axis thus constitutes a novel checkpoint pathway critical for the maintenance of proper chromosome number.     

The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint

The spindle checkpoint prevents cell cycle progression in cells that have mitotic spindle defects. Although several spindle defects activate the spindle checkpoint, the exact nature of the primary signal is unknown. We have found that the budding yeast member of the Aurora protein kinase family, Ipl1p, is required to maintain a subset of spindle checkpoint arrests. Ipl1p is required to maintain the spindle checkpoint that is induced by overexpression of the protein kinase Mps1. Inactivating Ipl1p allows cells overexpressing Mps1p to escape from mitosis and segregate their chromosomes normally. Therefore, the requirement for Ipl1p in the spindle checkpoint is not a consequence of kinetochore and/or spindle defects. The requirement for Ipl1p distinguishes two different activators of the spindle checkpoint: Ipl1p function is required for the delay triggered by chromosomes whose kinetochores are not under tension, but is not required for arrest induced by spindle depolymerization. Ipl1p localizes at or near kinetochores during mitosis, and we propose that Ipl1p is required to monitor tension at the kinetochore.     

Pds1 and Esp1 control both anaphase and mitotic exit in normal cells and after DNA damage

The separation of sister chromatids in anaphase is followed by spindle disassembly and cytokinesis. These events are governed by the anaphase-promoting complex (APC), which triggers the ubiquitin-dependent proteolysis of key regulatory proteins: anaphase requires the destruction of the anaphase inhibitor Pds1, whereas mitotic exit requires the destruction of mitotic cyclins and the inactivation of Cdk1. We find that Pds1 is not only an inhibitor of anaphase, but also blocks cyclin destruction and mitotic exit by a mechanism independent of its effects on sister chromatid separation. Pds1 is also required for the mitotic arrest and inhibition of cyclin destruction that occurs after DNA damage. Even in anaphase cells, where Pds1 levels are normally low, DNA damage stabilizes Pds1 and prevents cyclin destruction and mitotic exit. Pds1 blocks cyclin destruction by inhibiting its binding partner Esp1. Mutations in ESP1 delay cyclin destruction; overexpression of ESP1 causes premature cyclin destruction in cells arrested in metaphase by spindle defects and in cells arrested in metaphase and anaphase by DNA damage. The effects of Esp1 are dependent on Cdc20 (an activating subunit of the APC) and on several additional proteins (Cdc5, Cdc14, Cdc15, Tem1) that form a regulatory network governing mitotic exit. We speculate that the inhibition of cyclin destruction by Pds1 may contribute to the ordering of late mitotic events by ensuring that mitotic exit is delayed until after anaphase is initiated. In addition, the stabilization of Pds1 after DNA damage provides a mechanism to delay both anaphase and mitotic exit while DNA repair occurs.     

Induction of hRAD9 is required for G2/M checkpoint signal transduction in gastric cancer cells.

DNA damage triggers the activation of checkpoints that delay cell cycle progression to allow for DNA repair. Loss of G2 checkpoints provides a growth advantage for tumor cells undergoing aberrant mitosis. However, the precise mechanisms of G2 checkpoints acting in gastric cancer are unknown. Here, we analyzed the G2 checkpoint function in two gastric cancer cells, MKN-28 cells containing a mutant p53 gene and MKN-45 cells which have wild-type p53. Two agents damaging DNA, camptothecin (CPT) or ultraviolet light (UV), were utilized to trigger a G2 phase cell cycle checkpoint response in these tumor cells. Both CPT and UV inhibited the growth of MKN-45 cells, whereas they did not affect the growth of MKN-28 cells. CPT induced cell cycle arrest at the G2/M phase and enhanced the expression of human RAD9 (hRAD9) in MKN-45 cells. In addition, hRAD9 showed perinuclear staining and similar localization with Bcl-2 in MKN-45 cells but not in MKN-28 cells after having applied CPT or UV light. These results suggest that besides p53 activity, the induction of hRAD9 is required for G2/M checkpoint signal transduction in gastric cancer cells.     

The dominant-negative effect of p53 mutants and p21 induction in tetraploid G1 arrest depends on the type of p53 mutation and the nature of the stimulus

Many p53 mutant proteins possess a dominant-negative activity that is under the control of several factors, namely p53 mutations and the cell type. The goals of our study were to determine the following: (1) the dominant-negative effect of different p53 mutations in response to mitotic spindle inhibitors, and (2) if this dominant-negative activity is dependent on the nature of the stimulus. We therefore examined the cellular response of the near-diploid LoVo colon carcinoma cell line possessing two wild-type TP53 alleles and three other clones transfected with different TP53 mutants (p53-273H, p53-175H, and p53-143A) to treatments with different mitotic spindle inhibitors. Flow cytometric studies and analysis of retinoblastoma protein (pRb) dephosphorylation and 5-bromo-2'-deoxyuridine incorporation by immunocytochemistry revealed a tetraploid G1 arrest of the wild-type LoVo clone and the p53-273H mutant clone after exposure to mitotic spindle inhibitors, preventing tetraploid cells from entering into an additional S phase. On the other hand, the p53-175H and p53-143A mutant clones re-enter S phase with no apparent arrest. Therefore, our results confirm that p53 mutant dominant-negative activity and the tetraploid G1 arrest in response to mitotic spindle inhibitor treatment depend on the type of p53 mutation, involve p21 induction, and require pRb dephosphorylation. Moreover, when we compare our results with those obtained by other investigators after ionizing radiation exposure using the same cell lines, we identify the nature of the stimulus as a new factor that determines the dominant-negative effect of p53 mutants.     

Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics

Medulloblastoma (MB) is the most common pediatric CNS malignancy. We identify EAG2 as an overexpressed potassium channel in MBs across different molecular and histological subgroups. EAG2 knockdown not only impairs MB cell growth in vitro, but also reduces tumor burden in vivo and enhances survival in xenograft studies. Mechanistically, we demonstrate that EAG2 protein is confined intracellularly during interphase but is enriched in the plasma membrane during late G2 phase and mitosis. Disruption of EAG2 expression results in G2 arrest and mitotic catastrophe associated with failure of premitotic cytoplasmic condensation. While the tumor suppression function of EAG2 knockdown is independent of p53 activation, DNA damage checkpoint activation, or changes in the AKT pathway, this defective cell volume control is specifically associated with hyperactivation of the p38 MAPK pathway. Inhibition of the p38 pathway significantly rescues the growth defect and G2 arrest. Strikingly, ectopic membrane expression of EAG2 in cells at interphase results in cell volume reduction and mitotic-like morphology. Our study establishes the functional significance of EAG2 in promoting MB tumor progression via regulating cell volume dynamics, the perturbation of which activates the tumor suppressor p38 MAPK pathway, and provides clinical relevance for targeting this ion channel in human MBs.     

Mitosis in vertebrates: the G2/M and M/A transitions and their associated checkpoints

In this review, I stress the importance of direct data and accurate terminology when formulating and communicating conclusions on how the G2/M and metaphase/anaphase transitions are regulated. I argue that entry into mitosis (i.e., the G2/M transition) is guarded by several checkpoint control pathways that lose their ability to delay or stop further cell cycle progression once the cell becomes committed to divide, which in vertebrates occurs in the late stages of chromosome condensation. After this commitment, progress through mitosis is then mediated by a single Mad/Bub-based checkpoint that delays chromatid separation, and exit from mitosis (i.e., completion of the cell cycle) in the presence of unattached kinetochores. When cells cannot satisfy the mitotic checkpoint, e.g., when in concentrations of spindle poisons that prohibit the stable attachment of all kinetochores, they are delayed in mitosis for many hours. In normal cells, the duration of this delay depends on the organism and ranges from ∼4 h in rodents to ∼22 h in humans. Recent live cell studies reveal that under this condition, many cancer cells (including HeLa and U2OS) die in mitosis by apoptosis within ∼24 h, which implies that biochemical studies on cancer cell populations harvested in mitosis after a prolonged mitotic arrest are contaminated with dead or dying cells.     

Nutritional folate-deficiency in Chinese hamster ovary cells. Chromosomal abnormalities associated with perturbations in nucleic acid precursors

Folate deficiency is known to induce chromosomal abnormalities. We used a nutritionally folate-deficient Chinese hamster ovary (CHO) cell culture system to examine modulation of chromosome damage by purine or pyrimidine supplementation. The cells were cultured in folate-deficient (Fol-) medium or Fol- medium supplemented with thymidine (dT) or hypoxanthine (Hx) until population growth arrest. The cultures were then switched to complete medium, permitting the cells to begin cell division. Cell-cycle progression was followed by flow cytometry to identify the first mitosis, when samples for analysis were collected. The mitotic index, frequency of chromosomal aberrations in mitotic cells, and relative distribution of different types of aberrations were determined. Cells grown in Fol- medium supplemented with Hx entered the G2/M phase of the cell cycle at 14 hours after media change as compared with 16 hours for Fol- cultures or 24 hours for Fol- cultures supplemented with dT. Cells cultured in Fol- medium alone or supplemented with dT showed similar frequencies of damage, averaging 20-22%, as compared with 2% for control cultures. In contrast, cells grown in Hx-supplemented medium exhibited a lower frequency of damaged mitoses (15%), as well as a reduction in certain types of abnormalities. This latter finding is surprising in light of our previous work showing that the presence of Hx during folate deficiency produced a more severe perturbation of phenotype (cellular enlargement) and growth control (S-phase delay and cell killing) than did dT supplementation or the absence of both nucleotide precursors.     

Variable levels of chromosomal instability and mitotic spindle checkpoint defects in breast cancer

Cytogenetic analyses have revealed that many aneuploid breast cancers have cell-to-cell variations of chromosome copy numbers, suggesting that these neoplasms have instability of chromosome numbers. To directly test for possible chromosomal instability in this disease, we used fluorescent in situ hybridization to monitor copy numbers of multiple chromosomes in cultures of replicating breast cancer-derived cell lines and nonmalignant breast epithelial cells. While most (7 of 9) breast cancer cell lines tested are highly unstable with regard to chromosome copy numbers, others (2 of 9 cell lines) have a moderate level of instability that is higher than the "background" level of normal mammary epithelial cells and MCF-10A cells, but significantly less than that seen in the highly unstable breast cancer cell lines. To evaluate the potential role of a defective mitotic spindle checkpoint as a cause of this chromosomal instability, we used flow cytometry to monitor the response of cells to nocodazole-induced mitotic spindle damage. All cell lines with high levels of chromosomal instability have defective mitotic spindle checkpoints, whereas the cell lines with moderate levels of chromosomal instability (and the stable normal mammary cells and MCF10A cells) arrest in G(2) when challenged with nocodazole. Notably, the extent of mitotic spindle checkpoint deficiency and chromosome numerical instability in these cells is unrelated to the presence or absence of p53 mutations. Our results provide direct evidence for chromosomal instability in breast cancer and show that this instability occurs at variable levels among cells from different cancers, perhaps reflecting different functional classes of chromosomal instability. High levels of chromosomal instability are likely related to defective mitotic checkpoints but not to p53 mutations.     

Targeting Mitosis for Anti-Cancer Therapy

Basic research that has focused on achieving a mechanistic understanding of mitosis has provided unprecedented molecular and biochemical insights into this highly complex phase of the cell cycle. The discovery process has uncovered an ever-expanding list of novel proteins that orchestrate and coordinate spindle formation and chromosome dynamics during mitosis. That many of these proteins appear to function solely in mitosis makes them ideal targets for the development of mitosis-specific cancer drugs. The clinical successes seen with anti-microtubule drugs such as taxanes and the vinca alkaloids have also encouraged the development of drugs that specifically target mitosis. Drugs that selectively inhibit mitotic kinesins involved in spindle and kinetochore functions, as well as kinases that regulate these activities, are currently in various stages of clinical trials. Our increased understanding of mitosis has also revealed that this process is targeted by inhibitors of farnesyl transferase, histone deacetylase, and Hsp90. Although these drugs were originally designed to block cell proliferation by inhibiting signaling pathways and altering gene expression, it is clear now that these drugs can also directly interfere with the mitotic process. The increased attention to mitosis as a chemotherapeutic target has also raised an important issue regarding the cellular determinants that specify drug sensitivity. One likely contribution is the mitotic checkpoint, a failsafe mechanism that delays mitotic exit so that cells whose chromosomes are not properly attached to the spindle have extra time to correct their errors. As the biochemical activity of the mitotic checkpoint is finite, cells cannot indefinitely sustain the delay, as in cases where cells are treated with anti-mitotic drugs. When the mitotic checkpoint activity is eventually lost, cells will exit mitosis and become aneuploid. While many of the aneuploid cells may die because of massive chromosome imbalance, survivors that continue to proliferate will no doubt be selected. This is clearly an undesirable outcome, thus efforts to obtain fundamental insights into why some cells that arrest in mitosis die without exiting mitosis will be exceedingly important in enhancing our understanding of the drug sensitivity of cancer cells.     

p53-dependent apoptosis is essential for the antitumor effect of paclitaxel response to DNA damage in papillary thyroid carcinoma

A functional p53 protein plays an important role in killing tumor cells. Previous studies showed that chemotherapeutic drug, paclitaxel (PTX), showed anti-tumor activity through inducing G2/M arrest and apoptosis by targeting microtubules in tumor cells. However, PTX was not sensitive to p53-inactivated papillary thyroid carcinoma (PTC) cells by inducing G2/M arrest only. Recombinant adenovirus-p53 (rAd-p53) was used to increase the level of p53, which significantly increased the sensitivity of PTC cells to PTX by inducing S arrest, G2/M arrest and apoptosis. To discuss the anti-tumor mechanism of rAd-p53 + PTX and found p53 activation was necessary for anti-tumor effect of PTX in PTC cells. There was high level of p53 in rAd-p53-treated PTC cells. rAd-p53 + PTX increased the level of p21, p-ATM and γ-H2AX and decreased the level of Cyclin D1/E1, suggesting p53 activated p21 which negatively regulated cyclins to induce S arrest response to DNA damage in PTC cells. rAd-p53 + PTX increased the levels of cleaved-PARP-1, cleaved -Caspase 3, and BAX and decreased the level of BCL-XL, suggesting p53 regulates the expression of BAX/BCL-XL to mediate DNA damage-induced apoptosis in PTC cells. Furthermore, rAd-p53 + PTX showed significant tumor inhibition in TPC-1 xenograft model, with an inhibitory rate of 79.39%. TUNEL assay showed rAd-p53 + PTX induced notable apoptosis in tumor tissues. rAd-p53 showed good sensitization of PTX in vitro and in vivo through inducing DNA damage induced-apoptosis indicated p53-dependent apoptosis was essential for the antitumor effect of PTX in PTC.     

Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53

Chk2/hcds1, the human homolog of the Saccharomyces cerevisiae RAD53/SPK1 and Schizosaccharomyces pombe cds1 DNA damage checkpoint genes, encodes a protein kinase that is post-translationally modified after DNA damage. Like its yeast homologs, the Chk2/hCds1 protein phosphorylates Cdc25C in vitro, suggesting that it arrests cells in G(2) in response to DNA damage. We expressed Chk2/hCds1 in human cells and analyzed their cell cycle profile. Wild-type, but not catalytically inactive, Chk2/hCds1 led to G(1) arrest after DNA damage. The arrest was inhibited by cotransfection of a dominant-negative p53 mutant, indicating that Chk2/hCds1 acted upstream of p53. In vitro, Chk2/hCds1 phosphorylated p53 on Ser-20 and dissociated preformed complexes of p53 with Mdm2, a protein that targets p53 for degradation. In vivo, ectopic expression of wild-type Chk2/hCds1 led to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative Chk2/hCds1 mutant abrogated both phosphorylation of p53 on Ser-20 and p53 stabilization. Thus, in response to DNA damage, Chk2/hCds1 stabilizes the p53 tumor suppressor protein leading to cell cycle arrest in G(1).     

Identification of a new APC/C recognition domain,the A box,which is required for the Cdh1-dependent destruction of the kinase Aurora-A during mitotic exit

The mitotic kinase Aurora A (Aur-A) is required for formation of a bipolar mitotic spindle and accurate chromosome segregation. In somatic cells, Aur-A protein and kinase activity levels peak during mitosis, and Aur-A is degraded during mitotic exit. Here, we investigated how Aur-A protein and kinase activity levels are regulated, taking advantage of the rapid synchronous cell division cycles of Xenopus eggs and cell-free systems derived from them. Aur-A kinase activity oscillates in the early embryonic cell cycles, just as in somatic cells, but Aur-A protein levels are constant, indicating that regulated activation and inactivation, instead of periodic proteolysis, is the dominant mode of Aur-A regulation in these cell cycles. Cdh1, the APC/C activator that targets many mitotic proteins for ubiquitin-dependent proteolysis during late mitosis and G1 in somatic cells, is missing in Xenopus eggs and early embryos. We find that addition of Cdh1 to egg extracts undergoing M phase exit is sufficient to induce rapid degradation of Aur-A. Aur-A contains both of the two known APC/C recognition signals, (1) a C-terminal D box similar to those required for ubiquitin-dependent destruction of cyclin B and several other mitotic proteins, and (2) an N-terminal KEN box similar to that found on cdc20, which is ubiquitinated in response to APC/C(Cdh1). The D box is required for Cdh1-induced destruction of Aur-A but the KEN box is not. Destruction also requires a short region in the N terminus, which contains a newly identified recognition signal, the A box. The A box is conserved in vertebrate Aur-As and contains serine 53, which is phosphorylated during M phase. Mutation of serine 53 to aspartic acid, which can mimic the effect of phosphorylation, completely blocks Cdh1-dependent destruction of Aur-A. These results suggest that dephosphorylation of serine 53 during mitotic exit could control the timing of Aur-A destruction, allowing recognition of both the A box and D box by Cdh1-activated APC/C.     

Bromodeoxyuridine induces p53-dependent and -independent cell cycle arrests in human gastric carcinoma cell lines.

OBJECTIVES: This study was designed to clarify the effects of bromodeoxyuridine (BrdU) on cell cycle progression and induction of apoptosis, and to demonstrate the role of P53 in these processes. METHODS: We continuously exposed four human gastric carcinoma cell lines with different P53 status (P53 wild-type AGS and MKN-45, P53-mutated MKN-28 and P53-deleted KATO-III) to BrdU in asynchronous and synchronous culture conditions, and analyzed DNA histograms of apoptotic and nonapoptotic cells determined by static DNA cytofluorometry. RESULTS: Continuous exposure to 20 microM BrdU after synchronization with hydroxyurea resulted in S phase delay and G1 arrest in MKN-45 and an increase of apoptosis in the first S/G(2) phase in AGS and MKN-45. In the second S phase, a delay of 3-6 h was observed in all the four cell lines. In asynchronous cultures, continuous exposures to 20 and 200 microM BrdU for 72 h or more caused growth suppression with G(1) and G(2) arrests, respectively, in all the cell lines. CONCLUSIONS: These data suggested that the BrdU-induced growth suppression of the cell lines examined was mainly caused by cell cycle arrest rather than cell death, and that the cell cycle arrests in the first S and G(1) phases (elicited by BrdU in the single DNA strand) and those in the second S, G(2) and G(1) phases (elicited by BrdU in the double DNA strands) were mediated by p53-dependent and -independent pathways, respectively.