A Topoisomerase II-Dependent Checkpoint in G2-Phase Plant Cells Can Be Bypassed by Ectopic Expression of Mitotic Cyclin B2

DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclin-dependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.

Key Words:

Plant cyclin B2, Topoisomerase II, ICRF-193, G2 checkpoint, Microtubules     

Degradation of ATM-independent decatenation checkpoint function in human cells is secondary to inactivation of p53 and correlated with chromosomal destabilization

DNA topoisomerase II is required in the cell cycle to decatenate intertwined daughter chromatids prior to mitosis. To study the mechanisms that cells use to accomplish timely chromatid decatenation, the activity of a catenation-responsive checkpoint was monitored in human skin fibroblasts with inherited or acquired defects in the DNA damage G2 checkpoint. G2 delay was quantified shortly after a brief incubation with ICRF-193, which blocks the ability of topoisomerase II to decatenate chromatids, or treatment with ionizing radiation (IR), which damages DNA. Both treatments induced G2 delay in normal human fibroblasts. Ataxia telangiectasia fibroblasts with defective G2 checkpoint response to IR displayed normal G2 delay after treatment with ICRF-193, demonstrating that ATM kinase was not required for signaling when chromatid decatenation was blocked. The G2 delay induced by ICRF-193 was reversed by caffeine, indicating that active checkpoint signaling was involved. ICRF-193-induced G2 delay also was independent of p53 function, being evident in cells expressing HPV16E6 to inactivate p53. However, as fibroblasts expressing HPV16E6 aged in culture, they lost the ability to delay entry to mitosis, both after DNA damage and when decatenation was blocked. This age-related loss of G2 delay in response to ICRF-193 and IR in E6-expressing cells was blocked by induction of telomerase. Expression of telomerase also prevented chromosomal destabilization in aging E6-expressing cells. These observations lead to a new model of genetic instability, in which attenuation of G2 decatenatory checkpoint function permits cells to enter mitosis with insufficiently decatenated chromatids, leading to aneuploidy and polyploidy.     

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

DNA topoisomerase II is required in the cell cycle to decatenate intertwined daughter chromatids prior to mitosis. To study the mechanisms that cells use to accomplish timely chromatid decatenation, the activity of a catenation-responsive checkpoint was monitored in human skin fibroblasts with inherited or acquired defects in the DNA damage G2 checkpoint. G2 delay was quantified shortly after a brief incubation with ICRF-193, which blocks the ability of topoisomerase II to decatenate chromatids, or treatment with ionizing radiation (IR), which damages DNA. Both treatments induced G2 delay in normal human fibroblasts. Ataxia telangiectasia fibroblasts with defective G2 checkpoint response to IR displayed normal G2 delay after treatment with ICRF-193, demonstrating that ATM kinase was not required for signaling when chromatid decatenation was blocked. The G2 delay induced by ICRF-193 was reversed by caffeine, indicating that active checkpoint signaling was involved. ICRF-193-induced G2 delay also was independent of p53 function, being evident in cells expressing HPV16E6 to inactivate p53. However, as fibroblasts expressing HPV16E6 aged in culture, they lost the ability to delay entry to mitosis, both after DNA damage and when decatenation was blocked. This age-related loss of G2 delay in response to ICRF-193 and IR in E6-expressing cells was blocked by induction of telomerase. Expression of telomerase also prevented chromosomal destabilization in aging E6-expressing cells. These observations lead to a new model of genetic instability, in which attenuation of G2 decatenatory checkpoint function permits cells to enter mitosis with insufficiently decatenated chromatids, leading to aneuploidy and polyploidy.

Key Words:

Checkpoints, DNA damage, Decatenation, Topoisomerase II, ICRF-193, Radiation     

A topoisomerase II-dependent checkpoint in G2-phase plant cells can be bypassed by ectopic expression of mitotic cyclin B2

DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclindependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.     

Revised genetic requirements for the decatenation G2 checkpoint: The role of ATM

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in Chk2, but no appreciable phosphorylation of Ser139-H2AX or Ser345-Chk1. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage.     

G1 tetraploidy checkpoint and the suppression of tumorigenesis

Checkpoints suppress improper cell cycle progression to ensure that cells maintain the integrity of their genome. During mitosis, a metaphase checkpoint requires the integration of all chromosomes into a metaphase array in the mitotic spindle prior to mitotic exit. Still, mitotic errors occur in mammalian cells with a relatively high frequency. Metaphase represents the last point of control in mitosis. Once the cell commits to anaphase there are no checkpoints to sense segregation defects. In this context, we will explore our recent finding that non-transformed mammalian cells have a checkpoint that acts subsequent to mitotic errors to block the proliferation of cells that have entered G1 with tetraploid status. This arrest is dependent upon both p53 and pRb, and may represent an important function of both p53 and pRb as tumor suppressors. Further, we discuss the possibility that this mechanism may similarly impose G1 arrest in cells that become aneuploid through errors in mitosis.     

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.     

ATR enforces the topoisomerase II-dependent G2 checkpoint through inhibition of Plk1 kinase

An ATR-dependent G(2) checkpoint responds to inhibition of topoisomerase II and delays entry into mitosis by sustaining nuclear exclusion of cyclin B1-Cdk1 complexes. Here we report that induction of this checkpoint with ICRF-193, a topoisomerase II catalytic inhibitor that does not cause DNA damage, was associated with an ATR-dependent inhibition of polo-like kinase 1 (Plk1) kinase activity and a decrease in cyclin B1 phosphorylation. Expression of constitutively active Plk1 but not wild type Plk1 reversed ICRF-193-induced mitotic delay in HeLa cells, suggesting that Plk1 kinase activity is important for the checkpoint response to ICRF-193. G(2)/M synchronized normal human fibroblasts, when treated with ICRF-193, showed a decrease in cyclin B1 phosphorylation and Plk1 kinase activity despite high cyclin B1-Cdk1 kinase activity. G(2) fibroblasts that were treated with caffeine to override the checkpoint response to ICRF-193 displayed a high incidence of chromosomal aberrations. Taken together, these results suggest that ATR-dependent inhibition of Plk1 kinase activity may be one mechanism to regulate cyclin B1 phosphorylation and sustain nuclear exclusion during the G(2) checkpoint response to topoisomerase II inhibition. Moreover, the results demonstrate an important role for the topoisomerase II-dependent G(2) checkpoint in the preservation of human genomic stability.     

Mitotic death: a mechanism of survival? A review

Mitotic death is a delayed response of p53 mutant tumours that are resistant to genotoxic damage. Questions surround why this response is so delayed and how its mechanisms serve a survival function. After uncoupling apoptosis from G1 and S phase arrests and adapting these checkpoints, p53 mutated tumour cells arrive at the G2 compartment where decisions regarding survival and death are made. Missed or insufficient DNA repair in G1 and S phases after severe genotoxic damage results in cells arriving in G2 with an accumulation of point mutations and chromosome breaks. Double strand breaks can be repaired by homologous recombination during G2 arrest. However, cells with excessive chromosome lesions either directly bypass the G2/M checkpoint, starting endocycles from G2 arrest, or are subsequently detected by the spindle checkpoint and present with the features of mitotic death. These complex features include apoptosis from metaphase and mitosis restitution, the latter of which can also facilitate transient endocycles, producing endopolyploid cells. The ability of cells to initiate endocycles during G2 arrest and mitosis restitution most likely reflects their similar molecular environments, with down-regulated mitosis promoting factor activity. Resulting endocycling cells have the ability to repair damaged DNA, and although mostly reproductively dead, in some cases give rise to mitotic progeny. We conclude that the features of mitotic death do not simply represent aberrations of dying cells but are indicative of a switch to amitotic modes of cell survival that may provide additional mechanisms of genotoxic resistance.     

Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase

Upon DNA damage, cell cycle progression is temporally blocked to avoid propagation of mutations. While transformed cells largely maintain the competence to recover from a cell cycle arrest, untransformed cells past the G1/S transition lose mitotic inducers, and thus the ability to resume cell division. This permanent cell cycle exit depends on p21, p53, and APC/C^Cdh1. However, when and how permanent cell cycle exit occurs remains unclear. Here, we have investigated the cell cycle response to DNA damage in single cells that express Cyclin B1 fused to eYFP at the endogenous locus. We find that upon DNA damage Cyclin B1-eYFP continues to accumulate up to a threshold level, which is reached only in G2 phase. Above this threshold, a p21 and p53-dependent nuclear translocation required for APC/C^Cdh1-mediated Cyclin B1-eYFP degradation is initiated. Thus, cell cycle exit is decoupled from activation of the DNA damage response in a manner that correlates to Cyclin B1 levels, suggesting that G2 activities directly feed into the decision for cell cycle exit. Once Cyclin B1-eYFP nuclear translocation occurs, checkpoint inhibition can no longer promote mitotic entry or re-expression of mitotic inducers, suggesting that nuclear translocation of Cyclin B1 marks the restriction point for permanent cell cycle exit in G2 phase.     

Cell cycle-dependent DNA damage signaling induced by ICRF-193 involves ATM, ATR, CHK2, and BRCA1

Topoisomerase II is essential for cell proliferation and survival and has been a target of various anticancer drugs. ICRF-193 has long been used as a catalytic inhibitor to study the function of topoisomerase II. Here, we show that ICRF-193 treatment induces DNA damage signaling. Treatment with ICRF-193 induced G2 arrest and DNA damage signaling involving gamma-H2AX foci formation and CHK2 phosphorylation. DNA damage by ICRF-193 was further demonstrated by formation of the nuclear foci of 53BP1, NBS1, BRCA1, MDC1, and FANCD2 and increased comet tail moment. The DNA damage signaling induced by ICRF-193 was mediated by ATM and ATR and was restricted to cells in specific cell cycle stages such as S, G2, and mitosis including late and early G1 phases. Downstream signaling of ATM and ATR involved the phosphorylation of CHK2 and BRCA1. Altogether, our results demonstrate that ICRF-193 induces DNA damage signaling in a cell cycle-dependent manner and suggest that topoisomerase II might be essential for the progression of the cell cycle at several stages including DNA decondensation.     

Flow cytometric analysis of ICRF-193 influence on cell passage through mitosis

Studying the effect of topoisomerase II (topo II) inhibitors on cell passage through mitosis seems to be important for understanding the role of this enzyme during chromosome condensation and segregation. A flow cytometric assay (Zenin et al., 2001) allowed to determine the mitotic index, and to discriminate between not only cells in G2 and M phases (including metaphase and anaphase cells), but also cells in pseudo-G1 with 4c DNA content. It is shown that topo II catalytic inhibitor ICRF-193 blocks G2-M transition in a lymphoblastoid cell line GM-130. Addition of caffeine to cells abrogated a block of their entering mitosis but not the inhibitor action. Cells entered mitosis, which was proven by the presence of chromosomes in the examined specimen, and, bypassing anaphase, appeared in pseudo-G1 with 4c DNA content. We have found that in the presence of ICRF-193 cells, GM-130 and Hep-2 lines, previously blocked by nocodazole when in mitosis and then washed, pass through metaphase, enter anaphase and leave it to pass to pseudo-G1 with the 4c DNA content. Thus, by inhibiting topo II activity ICRF-193 causes abnormal mitotic transition.     

p53-Independent Apoptosis and p53-Dependent Block of DNA Rereplication Following Mitotic Spindle Inhibition in Human Cells

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.     

Fission yeast Clp1p phosphatase regulates G2/M transition and coordination of cytokinesis with cell cycle progression.

Background: In Saccharomyces cerevisiae the mitotic-exit network (MEN) functions in anaphase to promote the release of the Cdc14p phosphatase from the nucleolus. This release causes mitotic exit via inactivation of the cyclin-dependent kinase (Cdk). Cdc14p-like proteins are highly conserved; however, it is unclear if these proteins regulate mitotic exit as in S. cerevisiae. In Schizosaccharomyces pombe a signaling pathway homologous to the MEN and termed the septation initiation network (SIN) is required not for mitotic exit, but for initiation of cytokinesis and for a cytokinesis checkpoint that inhibits further cell cycle progression until cytokinesis is complete.Results: We have identified the S. pombe Cdc14p homolog, Clp1p, and show that it is not required for mitotic exit but rather functions together with the SIN in coordinating cytokinesis with the nuclear-division cycle. As cells enter mitosis, Clp1p relocalizes from the nucleolus to the spindle and site of cell division. Clp1p exit from the nucleolus does not depend on the SIN, but the SIN is required for keeping Clp1p out of the nucleolus until completion of cytokinesis. Clp1p, in turn, may promote the activation of the SIN by antagonizing Cdk activity until cytokinesis is complete and thus ensuring that cytokinesis is completed prior to the initiation of the next cell cycle. In addition to its roles in anaphase, Clp1p regulates the G2/M transition since cells deleted for clp1 enter mitosis precociously and cells overexpressing Clp1p delay mitotic entry. Unlike Cdc14p, Clp1p appears to antagonize Cdk activity by preventing dephosphorylation of Cdc2p on tyrosine.Conclusions: S. pombe Clp1p affects cell cycle progression in a markedly different manner than its S. cerevisiae homolog, Cdc14p. This finding raises the possibility that related phosphatases in animal cells will prove to have important roles in coordinating the onset of cytokinesis with the events of mitosis.     

Mitotic catastrophe occurs in the absence of apoptosis in p53-null cells with a defective G1 checkpoint

Cell death occurring during mitosis, or mitotic catastrophe, often takes place in conjunction with apoptosis, but the conditions in which mitotic catastrophe may exhibit features of programmed cell death are still unclear. In the work presented here, we studied mitotic cell death by making use of a UV-inactivated parvovirus (adeno-associated virus; AAV) that has been shown to induce a DNA damage response and subsequent death of p53-defective cells in mitosis, without affecting the integrity of the host genome. Osteosarcoma cells (U2OSp53DD) that are deficient in p53 and lack the G1 cell cycle checkpoint respond to AAV infection through a transient G2 arrest. We found that the infected U2OSp53DD cells died through mitotic catastrophe with no signs of chromosome condensation or DNA fragmentation. Moreover, cell death was independent of caspases, apoptosis-inducing factor (AIF), autophagy and necroptosis. These findings were confirmed by time-lapse microscopy of cellular morphology following AAV infection. The assays used readily revealed apoptosis in other cell types when it was indeed occurring. Taken together the results indicate that in the absence of the G1 checkpoint, mitotic catastrophe occurs in these p53-null cells predominantly as a result of mechanical disruption induced by centrosome overduplication, and not as a consequence of a suicide signal.     

Critical role of the nucleolus in activation of the p53-dependent postmitotic checkpoint

Cells eventually exit from mitosis during sustained arrest at the spindle checkpoint, without sister chromatid separation and cytokinesis. The resulting tetraploid cells are arrested in the subsequent G1 phase in a p53-dependent manner by the regulatory function of the postmitotic G1 checkpoint. Here we report how the nucleolus plays a critical role in activation of the postmitotic G1 checkpoint. During mitosis, the nucleolus is disrupted and many nucleolar proteins are translocated from the nucleolus into the cytoplasm. Among the nucleolar factors, Myb-binding protein 1a (MYBBP1A) induces the acetylation and accumulation of p53 by enhancing the interaction between p300 and p53 during prolonged mitosis. MYBBP1A-dependent p53 activation is essential for the postmitotic G1 checkpoint. Thus, our results demonstrate a novel nucleolar function that monitors the prolongation of mitosis and converts its signal into activation of the checkpoint machinery.     

Chk1 inhibition in p53-deficient cell lines drives rapid chromosome fragmentation followed by caspase-independent cell death

Activation of Checkpoint kinase 1 (Chk1) following DNA damage mediates cell cycle arrest to prevent cells with damaged DNA from entering mitosis. Here we provide a high-resolution analysis of cells as they undergo S- and G₂-checkpoint bypass in response to Chk1 inhibition with the selective Chk1 inhibitor GNE-783. Within 4–8 h of Chk1 inhibition following gemcitabine induced DNA damage, cells with both sub-4N and 4N DNA content prematurely enter mitosis. Coincident with premature transition into mitosis, levels of DNA damage dramatically increase and chromosomes condense and attempt to align along the metaphase plate. Despite an attempt to congress at the metaphase plate, chromosomes rapidly fragment and lose connection to the spindle microtubules. Gemcitabine mediated DNA damage promotes the formation of Rad51 foci; however, while Chk1 inhibition does not disrupt Rad51 foci that are formed in response to gemcitabine, these foci are lost as cells progress into mitosis. Premature entry into mitosis requires the Aurora, Cdk1/2 and Plk1 kinases and even though caspase-2 and -3 are activated upon mitotic exit, they are not required for cell death. Interestingly, p53, but not p21, deficiency enables checkpoint bypass and chemo-potentiation. Finally, we uncover a differential role for the Wee-1 checkpoint kinase in response to DNA damage, as Wee-1, but not Chk1, plays a more prominent role in the maintenance of S- and G₂-checkpoints in p53 proficient cells.     

Mammalian cells lack checkpoints for tetraploidy,aberrant centrosome number,and cytokinesis failure

Background  

Mammalian cells have been reported to have a p53-dependent tetraploidy checkpoint that blocks cell cycle progression in G1 in response to failure of cell division. In most cases where the tetraploidy checkpoint has been observed cell division was perturbed by anti-cytoskeleton drug treatments. However, other evidence argues against the existence of a tetraploidy checkpoint. Cells that have failed to divide differ from normal cells in having two nuclei, two centrosomes, a decreased surface to volume ratio, and having undergone an abortive cytokinesis. We tested each of these to determine which, if any, cause a G1 cell cycle arrest.     

Activation of p38 MAP kinase by DNA double-strand breaks in V(D)J recombination induces a G2/M cell cycle checkpoint

Delay of cell cycle progression in response to double-strand DNA breaks (DSBs) is critical to allow time for DNA repair and prevent cellular transformation. Here, we show that the p38 mitogen-activated protein (MAP) kinase signaling pathway is activated in immature thymocytes along with TcRbeta gene V(D)J recombination. Active p38 MAP kinase promotes a G2/M cell cycle checkpoint through the phosphorylation and activation of p53 in these cells in vivo. Inactivation of p38 MAP kinase and p53 is required for DN3 thymocytes to exit the G2/M checkpoint, progress through mitosis and further differentiate. We propose that p38 MAP kinase is activated by V(D)J-mediated DSBs and induces a p53-mediated G2/M checkpoint to allow DNA repair and prevent cellular transformation.