Polyploidization increases the sensitivity to DNA-damaging agents in mammalian cells.

Polyploidization occurs during normal development as well as during tumorigenesis. In this study, we investigated if the responses to genotoxic stress in cancer cells are influenced by the ploidy. Prolonged treatment of Hep3B cells with the spindle inhibitor nocodazole resulted in mitotic slippage, followed by re-replication of the DNA to produce polyploids. Reintroduction of p53 restored the checkpoints and suppressed polyploidization. Remarkably, a stable tetraploidy cell line could be generated from Hep3B by a transient nocodazole treatment followed by a period of recovery. Using this novel tetraploid system, we found that tetraploidization increased the cell volume without significantly affecting the cell cycle. Although tetraploidization was accompanied by an increase in centrosome number, the majority of mitoses in the tetraploid cells remained bipolar. Polyploidization sensitized cells to genotoxic stress inflicted by ionizing radiation and topoisomerase inhibitors without affecting the sensitivity to spindle inhibitors. Accordingly, more gamma-H2AX foci were induced by radiation in tetraploids than in normal Hep3B cells. Likewise, primary tetraploid human fibroblasts displayed higher gamma-H2AX foci formation than diploid human fibroblasts. An implication for chemotherapy is that some cancer cells can be sensitized to genotoxic agents by a preceding step that induces polyploidization.

Ubiquitination of p53 at multiple sites in the DNA-binding domain.

The tumor suppressor p53 is negatively regulated by the ubiquitin ligase MDM2. The MDM2 recognition site is at the NH2-terminal region of p53, but the positions of the actual ubiquitination acceptor sites are less well defined. Lysine residues at the COOH-terminal region of p53 are implicated as sites for ubiquitination and other post-translational modifications. Unexpectedly, we found that substitution of the COOH-terminal lysine residues did not diminish MDM2-mediated ubiquitination. Ubiquitination was not abolished even after the entire COOH-terminal regulatory region was removed. Using a method involving in vitro proteolytic cleavage at specific sites after ubiquitination, we found that p53 was ubiquitinated at the NH2-terminal portion of the protein. The lysine residue within the transactivation domain is probably not essential for ubiquitination, as substitution with an arginine did not affect MDM2 binding or ubiquitination. In contrast, several conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination. Removal of the DNA-binding domain reduced ubiquitination and increased the stability of p53. These data provide evidence that in addition to the COOH-terminal residues, p53 may also be ubiquitinated at sites in the DNA-binding domain.

Stalled replication induces p53 accumulation through distinct mechanisms from DNA damage checkpoint pathways.

Stalled replication forks induce p53, which is required to maintain the replication checkpoint. In contrast to the well-established mechanisms of DNA damage-activated p53, the downstream effectors and upstream regulators of p53 during replication blockade remain to be deciphered. Hydroxyurea triggered accumulation of p53 through an increase in protein stability. The requirement of p53 accumulation for the replication checkpoint was not due to p21(CIP1/WAF1) as its down-regulation with short-hairpin RNA did not affect the checkpoint. Similar to DNA damage, stalled replication triggered the activation of the MRN-ataxia telangiectasia mutated (ATM)/ATM and Rad3-related-CHK1/CHK2 axis. Down-regulation of CHK1 or CHK2, however, reduced p53 basal expression but not the hydroxyurea-dependent induction. Moreover, p53 was still stabilized in ataxia telangiectasia cells or in cells treated with caffeine, suggesting that ATM was not a critical determinant. These data also suggest that the functions of ATM, CHK1, and CHK2 in the replication checkpoint were not through the p53-p21(CIP1/WAF1) pathway. In contrast, induction of p53 by hydroxyurea was defective in cells lacking NBS1 and BLM. In this connection, the impaired replication checkpoint in several other genetic disorders has little correlation with the ability to stabilize p53. These data highlighted the different mechanisms involved in the stabilization of p53 after DNA damage and stalled replication forks.

The relative contribution of CHK1 and CHK2 to Adriamycin-induced checkpoint.

Topoisomerase II poisons like Adriamycin (ADR, doxorubicin) are clinically important chemotherapeutic agents. Adriamycin-induced DNA damage checkpoint activates ATM and ATR, which could in turn inhibit the cell cycle engine through either CHK1 or CHK2. In this study, we characterized whether CHK1 or CHK2 is required for Adriamycin-induced checkpoint. We found that both CHK1 and CHK2 were phosphorylated after Adriamycin treatment. Several lines of evidence from dominant-negative mutants, short hairpin RNA (shRNA), and knockout cells indicated that CHK1, but not CHK2, is critical for Adriamycin-induced cell cycle arrest. Disruption of CHK1 function bypassed the checkpoint, as manifested by the increase in CDC25A, activation of CDC2, increase in histone H3 phosphorylation, and reduction in cell survival after Adriamycin treatment. In contrast, CHK2 is dispensable for Adriamycin-induced responses. Finally, we found that CHK1 was upregulated in primary hepatocellular carcinoma (HCC), albeit as an inactive form. The presence of a stockpile of dormant CHK1 in cancer cells may have important implications for treatments like topoisomerase II poisons. Collectively, the available data underscore the pivotal role of CHK1 in checkpoint responses to a variety of stresses.

Topoisomerase poisons differentially activate DNA damage checkpoints through ataxia-telangiectasia mutated-dependent and -independent mechanisms.

Camptothecin and Adriamycin are clinically important inhibitors for topoisomerase (Topo) I and Topo II, respectively. The ataxia-telangiectasia mutated (ATM) product is essential for ionizing radiation-induced DNA damage responses, but the role of ATM in Topo poisons-induced checkpoints remains unresolved. We found that distinct mechanisms are involved in the activation of different cell cycle checkpoints at different concentrations of Adriamycin and camptothecin. Adriamycin promotes the G(1) checkpoint through activation of the p53-p21(CIP1/WAF1) pathway and decrease of pRb phosphorylation. Phosphorylation of p53(Ser20) after Adriamycin treatment is ATM dependent, but is not required for the full activation of p53. The G(1) checkpoint is dependent on ATM at low doses but not at high doses of Adriamycin. In contrast, the Adriamycin-induced G(2) checkpoint is independent on ATM but sensitive to caffeine. Adriamycin inhibits histone H3(Ser10) phosphorylation through inhibitory phosphorylation of CDC2 at low doses and down-regulation of cyclin B1 at high doses. The camptothecin-induced intra-S checkpoint is partially dependent on ATM, and is associated with inhibitory phosphorylation of cyclin-dependent kinase 2 and reduction of BrdUrd incorporation after mid-S phase. Finally, apoptosis associated with high doses of Adriamycin or camptothecin is not influenced by the absence of ATM. These data indicate that the involvement of ATM following treatment with Topo poisons differs extensively with dosage and for different cell cycle checkpoints.

How many mutant p53 molecules are needed to inactivate a tetramer?

The tumor suppressor p53 is transcription factor composed of four identical subunits. The majority of the mutations in p53 are missense mutations that impair DNA binding. On the other hand, the p53-related p63 and p73 genes are rarely mutated, but many cell types express natural variants lacking the N-terminal transactivation domain (NDelta). Compelling evidence indicates that both the DNA binding-defective and NDelta mutants can impair the function of wild-type p53 in a dominant-negative manner. Interestingly, it is uncertain how many mutant subunit(s) a p53 tetramer can tolerate. In this study, we first made theoretical predictions based on the number of mutant p53 monomers needed to inactivate a tetramer and then tested how well the experimental data fit the predicted values. Surprisingly, these experiments reveal that DNA binding-defective p53 mutants (R249S and R273H) are very ineffective in impairing the transcriptional activity of p53: at least three mutants are required to inactivate a tetramer. In marked contrast, p53NDelta is a very potent inhibitor of p53: one NDelta subunit per tetramer is sufficient to abolish the transcriptional activity. DNA binding is not necessary for the NDelta proteins to inactivate p53. Similarly, NDelta variants of p63 and p73 are also powerful inhibitors of members of the p53 family. These results have important implications for our thinking about the mechanism of tumorigenesis involving missense p53 mutants or the N-terminally truncated isoforms.

Differential mode of regulation of the checkpoint kinases CHK1 and CHK2 by their regulatory domains.

CHK1 and CHK2 are key mediators that link the machineries that monitor DNA integrity to components of the cell cycle engine. Despite the similarity and potential redundancy in their functions, CHK1 and CHK2 are unrelated protein kinases, each having a distinctive regulatory domain. Here we compare how the regulatory domains of human CHK1 and CHK2 modulate the respective kinase activities. Recombinant CHK1 has only low basal activity when expressed in cultured cells. Surprisingly, disruption of the C-terminal regulatory domain activates CHK1 even in the absence of stress. Unlike the full-length protein, C-terminally truncated CHK1 displays autophosphorylation, phosphorylates CDC25C on Ser(216), and delays cell cycle progression. Intriguingly, enzymatic activity decreases when the entire regulatory domain is removed, suggesting that the regulatory domain contains both inhibitory and stimulatory elements. Conversely, the kinase domain suppresses Ser(345) phosphorylation, a major ATM/ATR phosphorylation site in the regulatory domain. In marked contrast, CHK2 expressed in either mammalian cells or in bacteria is already active as a kinase against itself and CDC25C and can delay cell cycle progression. Unlike CHK1, disruption of the regulatory domain of CHK2 abolishes its kinase activity. Moreover, the regulatory domain of CHK2, but not that of CHK1, can oligomerize. Finally, CHK1 but not CHK2 is phosphorylated during the spindle assembly checkpoint, which correlates with the inhibition of the kinase. The mitotic phosphorylation of CHK1 requires the regulatory domain, does not involve Ser(345), and is independent on ATM. Collectively, these data reveal the very different mode of regulation between CHK1 and CHK2.

ING1b decreases cell proliferation through p53-dependent and -independent mechanisms.

ING1b can stimulate cell cycle arrest, repair, senescence, and apoptosis. The actions of ING1b are attributed to its activation of the tumor suppressor p53. Here we investigate the more subtle effects of ING1b on the cell cycle and DNA damage responses in the absence of p53. To this end, we have generated isogenic cell lines that expressed ING1b and p53 either individually or in combination under the control of inducible promoters. A five- to 10-fold induction of ING1b over the endogenous protein in a p53-null H1299 background slightly impairs proliferation by increasing the doubling time by approximately 10%. Significantly, ectopic expression of ING1b enhanced the G(2)/M DNA damage checkpoint induced by adriamycin. We demonstrated that the DNA damage-induced cell death mediated by the cooperation between ING1b and p53 was more prominent than by the individual proteins alone. In adriamycin-treated cells, p53 was stabilized and induced the expression of p21(CIP1/WAF1), but the expression of ING1b was not affected. The exact targets of ING1b in the p53-null background are not known, but we demonstrated that the transcriptional activities of other members of the p53 family, p63alpha and p73alpha, could be activated by ING1b. These data indicate that ING1 has a subtle antiproliferative effect even in the absence of p53, and ING1b enhances the DNA damage responses through p53-dependent and -independent mechanisms.

DNA damage during the spindle-assembly checkpoint degrades CDC25A, inhibits cyclin-CDC2 complexes, and reverses cells to interphase.

Cell cycle checkpoints that monitor DNA damage and spindle assembly are essential for the maintenance of genetic integrity, and drugs that target these checkpoints are important chemotherapeutic agents. We have examined how cells respond to DNA damage while the spindle-assembly checkpoint is activated. Single cell electrophoresis and phosphorylation of histone H2AX indicated that several chemotherapeutic agents could induce DNA damage during mitotic block. DNA damage during mitotic block triggered CDC2 inactivation, histone H3 dephosphorylation, and chromosome decondensation. Cells did not progress into G1 but seemed to retract to a G2-like state containing 4N DNA content, with stabilized cyclin A and cyclin B1 binding to Thr14/Tyr15-phosphorylated CDC2. The loss of mitotic cells was not due to cell death because there was no discernible effect on caspase-3 activation, DNA fragmentation, or viability. Extensive DNA damage during mitotic block inactivated cyclin B1-CDC2 and prevented G1 entry when the block was removed. The mitotic DNA damage responses were independent of p53 and pRb, but they were dependent on ATM. CDC25A that accumulated during mitosis was rapidly destroyed after DNA damage in an ATM-dependent manner. Ectopic expression of CDC25A or nonphosphorylatable CDC2 effectively inhibited the dephosphorylation of histone H3 after DNA damage. Hence, although spindle disruption and DNA damage provide conflicting signals to regulate CDC2, the negative regulation by the DNA damage checkpoint could overcome the positive regulation by the spindle-assembly checkpoint.

Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints.

Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.

The candidate tumor suppressor ING1b can stabilize p53 by disrupting the regulation of p53 by MDM2.

ING1b is a candidate tumor suppressor that can stimulate the transcriptional activity of p53 and inhibit cell proliferation. The molecular basis of how ING1b activates p53 function remains unclear. Here we show that ING1b could stimulate the activity of p53 by increasing the level and stability of the p53 protein. The stabilization and activation of p53 by ING1b could be reversed by MDM2 in a dose-dependent manner. Conversely, ING1b could reverse the inhibition and degradation of p53 caused by MDM2 in a dose-dependent manner. Furthermore, ING1b and MDM2 bound to p53 in a mutually exclusive manner. In agreement with these observations, we found that similarly to MDM2, ING1b binds to the NH(2)-terminal region of p53. These data suggest a model in which ING1b disrupts the interaction between p53 and MDM2, leading to the stabilization of p53 and growth inhibition.

Cyclin F is degraded during G2-M by mechanisms fundamentally different from other cyclins.

Cyclin F, a cyclin that can form SCF complexes and bind to cyclin B, oscillates in the cell cycle with a pattern similar to cyclin A and cyclin B. Ectopic expression of cyclin F arrests the cell cycle in G(2)/M. How the level of cyclin F is regulated during the cell cycle is completely obscure. Here we show that, similar to cyclin A, cyclin F is degraded when the spindle assembly checkpoint is activated and accumulates when the DNA damage checkpoint is activated. Cyclin F is a very unstable protein throughout much of the cell cycle. Unlike other cyclins, degradation of cyclin F is independent of ubiquitination and proteasome-mediated pathways. Interestingly, proteolysis of cyclin F is likely to involve metalloproteases. Rapid destruction of cyclin F does not require the N-terminal F-box motif but requires the COOH-terminal PEST sequences. The PEST region alone is sufficient to interfere with the degradation of cyclin F and confer instability when fused to cyclin A. These data show that although cyclin F is degraded at similar time as the mitotic cyclins, the underlying mechanisms are entirely distinct.