The mammalian UV response: c-Jun induction is required for exit from p53-imposed growth arrest

The mammalian UV response results in rapid and dramatic induction of c-jun. Induction of a protooncogene, normally involved in mitogenic responses, by a genotoxic agent that causes growth arrest seems paradoxical. We now provide an explanation for the role of c-Jun in the UV response of mouse fibroblasts. c-Jun is necessary for cell-cycle reentry of UV-irradiated cells, but does not participate in the response to ionizing radiation. Cells lacking c-Jun undergo prolonged cell-cycle arrest, but resist apoptosis, whereas cells that express c-Jun constitutively do not arrest and undergo apoptosis. This function of c-Jun is exerted through negative regulation of p53 association with the p21 promoter. Cells lacking c-Jun exhibit prolonged p21 induction, whereas constitutive c-Jun inhibits UV-mediated p21 induction.     

A role of the mitotic spindle checkpoint in the cellular response to DNA replication stress

Replication stress is a frequent and early event during tumorigenesis. Whereas the cellular responses to a persistent block of replication fork progression have been extensively studied, relatively little is known about how cells respond to low-intensity replication stress. However, transient replication fork perturbations are likely to occur even more frequently in tumor cells than a permanent replication arrest. We report here that transient, low intensity replication stress leads to a rapid activation of the DNA replication checkpoint but to a significantly delayed apoptotic response in a small but significant number of cells. This late apoptotic response was independent of p53 and we found evidence for cell death during mitosis in a proportion of cells. To further explore the role of p53 in the response to replication stress, we analyzed mouse embryonic fibroblasts (MEFs) deficient of p53 in comparison to wild-type or p63- or p73-deficient MEFs. We detected a significant increase of apoptosis and morphological signs of failed mitosis such as multinucleation in p53-deficient MEFs following replication stress, but not in wild-type or p63- or p73-deficient cells. Multinucleated p53-deficient MEFs frequently retained cyclin B1 expression indicating a persistently activated mitotic spindle checkpoint. Collectively, our results suggest that the cellular response to replication stress involves the mitotic spindle checkpoint in a proportion of cells. These findings imply that the mitotic spindle checkpoint may act in concert with DNA damage and cell-cycle checkpoints as an early anti-tumor barrier and provide a possible explanation for its frequent relaxation in human cancer.     

Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest

Centrosomes organize the microtubule cytoskeleton for both interphase and mitotic functions. They are implicated in cell-cycle progression but the mechanism is unknown. Here, we show that depletion of 14 out of 15 centrosome proteins arrests human diploid cells in G1 with reduced Cdk2-cyclin A activity and that expression of a centrosome-disrupting dominant-negative construct gives similar results. Cell-cycle arrest is always accompanied by defects in centrosome structure and function (for example, duplication and primary cilia assembly). The arrest occurs from within G1, excluding contributions from mitosis and cytokinesis. The arrest requires p38, p53 and p21, and is preceded by p38-dependent activation and centrosomal recruitment of p53. p53-deficient cells fail to arrest, leading to centrosome and spindle dysfunction and aneuploidy. We propose that loss of centrosome integrity activates a checkpoint that inhibits G1-S progression. This model satisfies the definition of a checkpoint in having three elements: a perturbation that is sensed, a transducer (p53) and a receiver (p21).     

Puma and p21 represent cooperating checkpoints limiting self-renewal and chromosomal instability of somatic stem cells in response to telomere dysfunction

The tumour suppressor p53 activates Puma-dependent apoptosis and p21-dependent cell-cycle arrest in response to DNA damage. Deletion of p21 improved stem-cell function and organ maintenance in progeroid mice with dysfunctional telomeres, but the function of Puma has not been investigated in this context. Here we show that deletion of Puma improves stem- and progenitor-cell function, organ maintenance and lifespan of telomere-dysfunctional mice. Puma deletion impairs the clearance of stem and progenitor cells that have accumulated DNA damage as a consequence of critically short telomeres. However, further accumulation of DNA damage in these rescued progenitor cells leads to increasing activation of p21. RNA interference experiments show that upregulation of p21 limits proliferation and evolution of chromosomal imbalances of Puma-deficient stem and progenitor cells with dysfunctional telomeres. These results provide experimental evidence that p53-dependent apoptosis and cell-cycle arrest act in cooperating checkpoints limiting tissue maintenance and evolution of chromosomal instability at stem- and progenitor-cell levels in response to telomere dysfunction. Selective inhibition of Puma-dependent apoptosis can result in temporary improvements in maintenance of telomere-dysfunctional organs.     

Cell cycle checkpoints and their impact on anticancer therapeutic strategies

Cells contain numerous pathways designed to protect them from the genomic instability or toxicity that can result when their DNA is damaged. The p53 tumor suppressor is particularly important for regulating passage through G1 phase of the cell cycle, while other checkpoint regulators are important for arrest in S and G2 phase. Tumor cells often exhibit defects in these checkpoint proteins, which can lead to hypersensitivity; proteins in this class include ataxia-telangiectasia mutatated (ATM), Meiotic recanbination 11 (Mre11), Nijmegen breakage syndrome 1 (Nbs 1), breast cancer susceptibility genes 1 and 2 (BRCA1), and (BRCA2). Consequently, tumors should be assessed for these specific defects, and specific therapy prescribed that has high probability of inducing response. Tumors defective in p53 are frequently considered resistant to apoptosis, yet this defect also provides an opportunity for targeted therapy. When their DNA is damaged, p53-defective tumor cells preferentially arrest in S or G2 phase where they are susceptible to checkpoint inhibitors such as caffeine and UCN-01. These inhibitors preferentially abrogate cell cycle arrest in p53-defective cells, driving them through a lethal mitosis. Wild type p53 can prevent abrogation of arrest by elevating levels of p21(waf1) and decreasing levels of cyclins A and B. During tumorigenesis, tumor cells frequently loose checkpoint controls and this facilitates the development of the tumor. However, these defects also represent an Achilles heel that can be targeted to improve current therapeutic strategies.     

The Haemophilus ducreyi cytolethal distending toxin induces cell cycle arrest and apoptosis via the DNA damage checkpoint pathways

The cytolethal distending toxins (CDTs) induce cell cycle arrest by a mechanism still not well characterized. We demonstrate that the effect of the Haemophilus ducreyi CDT (HdCDT) is cell type-specific: B cell lines underwent apoptosis, epithelial cells and keratinocytes arrested exclusively in G(2), whereas normal fibroblasts arrested both in G(1) and G(2). We studied normal keratinocytes and fibroblasts, which are relevant for understanding the pathogenicity of H. ducreyi. The response to HdCDT resembles the checkpoint response activated by ionizing radiation. Both responses were characterized by an early induction of the p53 gene and the cyclin-dependent kinase inhibitor p21 in fibroblasts, and activation of the chk2 kinase in epithelial cells. In the Ataxia Telangiectasia-mutated gene (ATM)-deficient lymphoblastoid cell lines, intoxication was significantly delayed compared with ATM wild type cells, and was associated with a slower kinetic of p53 stabilization, suggesting that the early response to HdCDT is ATM-dependent. Activation of ATM-dependent pathways was further confirmed by the ability of caffeine to partially override the HdCDT-mediated cell cycle arrest. Our data shed new light on the mechanism of action of this novel family of bacterial toxins, limiting the target candidates to DNA or molecules directly involved in activation of checkpoint responses.     

Reoxygenation following hypoxia activates DNA-damage checkpoint signaling pathways that suppress cell-cycle progression in cultured human lymphocytes

Cellular responses to DNA damage after hypoxia and reoxygenation (H/R) were examined in human lymphocytes. Cultured lymphocytes exposed to H/R showed a lower cytokinesis block proliferation index and a higher frequency of micronuclei in comparison to control cells. Western blots showed that H/R exposure induced p53 expression; however, p21 and Bax expression did not increase, indicating that H/R did not affect p53 transactivational activity. Phosphorylation of p53 (Ser15), Chk1 (Ser345), and Chk2 (Thr68) was also observed, suggesting that H/R activates p53 through checkpoint signals. In addition, H/R exposure caused the phosphorylation and negative regulation of Cdc2 and Cdc25C, proteins that are involved in cell-cycle arrest at the G2/M checkpoint. The S-phase checkpoint, regulated by the ATM-p95/NBS1-SMC1 pathway, was also triggered in H/R-exposed lymphocytes. These results demonstrate that H/R exposure triggers checkpoint signaling and induces cell-cycle arrest in cultured human lymphocytes.     

Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent

Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G1/S checkpoint, ATM, and p53 signaling pathways in p53-wildtype cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G1/S checkpoint pathway in p53-wildtype lines and not in p53-mutant cells. These responses are coupled with G2/G1 checkpoint effectors p21CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G2 cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wildtype and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G2 arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wildtype and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell-cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.     

Effect of inactivating various components of the signal pathways of the tumor suppressor p53 on genomic stability

To evaluate the role of different p53-regulated signaling pathways in the control of genomic integrity, we studied the frequency of changes in chromosome number and structure of cells of the sublines of mouse primary embryonic fibroblasts with the "knocked-out" genes for proteins p53, p21WAF, pRb, and p19ARF. Protein p21WAF is transactivated by p53 and is responsible for the cell block in the G1 phase of the damaged cells; protein pRb is a target for p21WAF which controls the G1-S-phase transition; and p19ARF protein is responsible for p53 activation in cells with certain anomalies. Inactivation of either of the studied genes proved to increase significantly the frequency of changes in the karyotype. However, the resultant chromosome instability differed: the frequency of the chromosome breaks, both spontaneous and induced with ethylmethane sulfonate (EMS), was in cells with inactivated p53 and lowest in cells with inactivated pRb. These distinctions were not caused by a different effect of various gene inactivation on the cell cycle progression: in all sublines, the cell block in G1 was abolished and the checkpoint function in G2 remained normal. However, the induction of apoptosis in EMS-treated cells differed in the studied sublines. The lowest number of apoptotic nuclei were determined in p53-/- cultures, whereas the highest were in the Rb-/- cultures. It is apparent that the degree of genetic instability is determined by a combined effect of apoptosis and abnormal regulation of the cell-cycle checkpoints.     

Hzf Determines cell survival upon genotoxic stress by modulating p53 transactivation

A critical unresolved issue about the genotoxic stress response is how the resulting activation of the p53 tumor suppressor can lead either to cell-cycle arrest and DNA repair or to apoptosis. We show here that hematopoietic zinc finger (Hzf), a zinc-finger-containing p53 target gene, modulates p53 transactivation functions in an autoregulatory feedback loop. Hzf is induced by p53 and binds to its DNA-binding domain, resulting in preferential transactivation of proarrest p53 target genes over its proapoptotic target genes. Thus, p53 activation results in cell-cycle arrest in Hzf wild-type MEFs, while in Hzf(-/-) MEFs, apoptosis is induced. Exposure of Hzf null mice to ionizing radiation resulted in enhanced apoptosis in several organs, as compared to in wild-type mice. These findings provide novel insights into the regulation of p53 transactivation function and suggest that Hzf functions as a key player in regulating cell fate decisions in response to genotoxic stress.     

Characterization of adriamycin-induced G2 arrest and its abrogation by caffeine in FL-amnion cells with or without p53

We investigated the effect of Adriamycin on FL-amnion (FL) cells. After treatment with the drug, the cells arrested at G2, but we did not detect an increase in the p21 levels. We established a p53-deficient derivative of these cells, in which G2 arrest also occurred after treatment with Adriamycin, suggesting that the arrest we observed in these cells is independent of the p53 pathway. Low doses of Adriamycin (100-200 ng/ml) induced G2 arrest, while late S-phase arrest was observed at high doses (500-1000 ng/ml) in both FL and p53-deficient FL cells. Accumulation of cyclin B1 was detected only in cells arrested at G2, and not in those arrested at S phase, suggesting that the S-phase checkpoint functioned efficiently even in p53-deficient FL cells. In both cell lines, caffeine-induced activation of CDC2 kinase was detected only in cells arrested at G2 and CDC2 kinase-activated cells died exhibiting features of apoptosis. CDC2 kinase activation was inhibited by cycloheximide. Furthermore, cycloheximide inhibited activation of CDK2:cyclin A, which normally precedes CDC2 kinase activation in caffeine-treated cells. These results suggest that p53 and p21 do not have special roles in the S- and G2-phase checkpoints and that CDK2:cyclin A could be the target of the G2-phase DNA damage checkpoint.     

Different levels of p53 induced either apoptosis or cell cycle arrest in a doxycycline-regulated hepatocellular carcinoma cell line <Emphasis Type="Italic">in vitro</Emphasis>

Induction of p53 gene expression in cancer cells can lead to both cell cycle arrest and apoptosis. To clarify whether the level of p53 expression determines the apoptotic response of hepatocellullar carcinoma (HCC) cells, we assessed the effect of various levels of expression of p53 gene on a p53-deficient HCC cell line, Hep3B, utilizing a doxycycline (Dox)-regulated inducible p53 expression system. Our results showed that apoptosis was induced in HCC cells with high levels of p53 expression. However, lower level of p53 expression induced only cell cycle arrest but not apoptosis. Bax expression was up-regulated following high levels of p53 expression, while bcl-2 expression was not altered by the level of p53 expression. Moreover, p21 expression was observed in both high and low expression of p53. These results suggest the level of p53 expression could determine if the HCC cells would go into cell cycle arrest or apoptosis. Bax may participate, at least in part, in inducing p53-dependent apoptosis and the induction of p21 alone was able to cause cell cycle arrest but not apoptosis.     

SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.     

AMP-activated protein kinase induces a p53-dependent metabolic checkpoint

Replicative cell division is an energetically demanding process that can be executed only if cells have sufficient metabolic resources to support a doubling of cell mass. Here we show that proliferating mammalian cells have a cell-cycle checkpoint that responds to glucose availability. The glucose-dependent checkpoint occurs at the G(1)/S boundary and is regulated by AMP-activated protein kinase (AMPK). This cell-cycle arrest occurs despite continued amino acid availability and active mTOR. AMPK activation induces phosphorylation of p53 on serine 15, and this phosphorylation is required to initiate AMPK-dependent cell-cycle arrest. AMPK-induced p53 activation promotes cellular survival in response to glucose deprivation, and cells that have undergone a p53-dependent metabolic arrest can rapidly reenter the cell cycle upon glucose restoration. However, persistent activation of AMPK leads to accelerated p53-dependent cellular senescence. Thus, AMPK is a cell-intrinsic regulator of the cell cycle that coordinates cellular proliferation with carbon source availability.     

ECRG2 disruption leads to centrosome amplification and spindle checkpoint defects contributing chromosome instability

Cancer cells contain an abnormal number of chromosomes (aneuploidy), which is a prevalent form of genetic instability in human cancers. Abnormal amplification of centrosomes and defects of spindle assembly checkpoint are the major causes of chromosome instability in cancer cells. Here we present biochemical evidence to suggest a role of ECRG2, a novel tumor suppressor gene, in maintaining chromosome stability. ECRG2 localized to centrosomes during interphase and kinetochores during mitosis. Further analysis revealed that ECRG2 participates in centrosome amplification in a p53-dependent manner. Depletion of ECRG2 not only destabilized p53, down-regulated p21, and increased the cyclin E/CDK2 activity, thus initiating centrosome amplification, but also abolished the ability of p53 localize to centrosomes. Overexpression of ECRG2 restored the p53-dependent suppression of centrosome duplication. Furthermore, ECRG2-depleted cells show severely disrupted spindle phenotype but fail to maintain the mitotic arrest due to minimal BUBR1 protein levels. Taken together, our results indicate that ECRG2 is important for ensuring centrosome duplication, spindle assembly checkpoint, and accurate chromosome segregation, and its depletion may contribute to chromosome instability and aneuploidy in human cancers.     

BUB1 mediation of caspase-independent mitotic death determines cell fate

The spindle checkpoint that monitors kinetochore-microtubule attachment has been implicated in tumorigenesis; however, the relation between the spindle checkpoint and cell death remains obscure. In BUB1-deficient (but not MAD2-deficient) cells, conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel, or 17-AAG) induced DNA fragmentation during early mitosis. This mitotic cell death was independent of caspase activation; therefore, we named it caspase-independent mitotic death (CIMD). CIMD depends on p73, a homologue of p53, but not on p53. CIMD also depends on apoptosis-inducing factor and endonuclease G, which are effectors of caspase-independent cell death. Treatment with nocodazole, paclitaxel, or 17-AAG induced CIMD in cell lines derived from colon tumors with chromosome instability, but not in cells from colon tumors with microsatellite instability. This result was due to low BUB1 expression in the former cell lines. When BUB1 is completely depleted, aneuploidy rather than CIMD occurs. These results suggest that cells prone to substantial chromosome missegregation might be eliminated via CIMD.     

Alteration of the ATM gene occurs in gastric cancer cell lines and primary tumors associated with cellular response to DNA damage

Ataxia telangiectasia mutated (ATM) is the gene mutated in the genetic disorder ataxia telangiectasia (AT), the symptoms of which include sensitivity to radiation and an increased risk of cancer. ATM is a kinase involved in activating the appropriate damage-response pathway, leading to either cell-cycle arrest or apoptosis, and is therefore a key checkpoint molecule in regulating cell-cycle response to DNA damage and responsible for maintenance of genome integrity. However, little is known about the association of ATM mutations with human gastric cancer (HGC). In order to determine the mutation and mRNA expression changes of the ATM gene in HGC, we performed analyses by denaturing high-performance liquid chromatography (DHPLC), DNA sequencing and RT-PCR technique on 13 human gastric tumor cell lines and 30 cases of fresh tumor specimens matched normal tissue. We compared the potential effect of the ATM gene mutation and cell behavior including cell-cycle arrest and induction of apoptosis in the tumor cell lines MGC803 and BGC823 with and without ionizing radiation (IR) exposure. Our data show that frequent variations were observed at 10 exons and 2 cDNA fragments which covered 8 other exons of the ATM gene as 5 out of 13 on the cell lines (38.5%) and 2 out of 30 cases in the tissue specimens (6.7%). All point mutations were confirmed as base substitutions (5982T-C; 6620A-G; 8684G-G/A; 9389C-G) and deletions (1079delC) by use of DNA sequencing. Among the mutations, one was reported previously in breast cancer, the other five have not yet been reported. The expression of ATM was significantly lower in five cell lines (MGC803; MKN45; SGC7901; GES and SUN-1) than in two others (BGC823 and RF48). G2/M cell-cycle arrest and apoptosis were observed in ATM-deficient MGC803 cells challenged with IR. A transient up-regulation of p53 occurred 1h post-IR in BGC823 cells but not in MGC803 cells. Our findings suggest that ATM mutations might be a pathogenic factor for an increased risk of gastric cancer, and the dysfunction of ATM may lead to a hypersensitivity to ionizing radiation in gastric cancer cells, possibly by a p53-dependent pathway.     

Chk1 and p21 cooperate to prevent apoptosis during DNA replication fork stress

Cells respond to DNA replication stress by triggering cell cycle checkpoints, repair, or death. To understand the role of the DNA damage response pathways in determining whether cells survive replication stress or become committed to death, we examined the effect of loss of these pathways on cellular response to agents that slow or arrest DNA synthesis. We show that replication inhibitors such as excess thymidine, hydroxyurea, and camptothecin are normally poor inducers of apoptosis. However, these agents become potent inducers of death in S-phase cells upon small interfering RNA-mediated depletion of the checkpoint kinase Chk1. This death response is independent of p53 and Chk2. p21-deficient cells, on the other hand, produce a more robust apoptotic response upon Chk1 depletion. p21 is normally induced only late after thymidine treatment. In Chk1-depleted cells p21 induction occurs earlier and does not require p53. Thus, Chk1 plays a primary role in the protection of cells from death induced by replication fork stress, whereas p21 mediates through its role in regulating entry into S phase. These findings are of potential importance to cancer therapy because we demonstrate that the efficacy of clinically relevant agents can be enhanced by manipulation of these signaling pathways.