Nuclear Localization of p38 MAPK in Response to DNA Damage

p38 MAP kinase (MAPK) is activated in response to environmental stress, cytokines and DNA damage, and mediates death, cell differentiation and cell cycle checkpoints. The intracellular localization of p38 MAPK upon activation remains unclear, and may depend on the stimulus. We show here that activation of p38 MAPK by stimuli that induce DNA double strand breaks (DSBs), but not other stimuli, leads to its nuclear translocation. In addition, naturally occurring DSBs generated through V(D)J recombination in immature thymocytes also promote nuclear accumulation of p38 MAPK. Nuclear translocation of p38 MAPK does not require its catalytic activity, but is induced by a conformational change of p38 MAPK triggered by phosphorylation within the active site. The selective nuclear accumulation of p38 MAPK in response to DNA damage could be a mechanism to facilitate the phosphorylation of p38 MAPK nuclear targets for the induction of a G2/M cell cycle checkpoint and DNA repair.     

Understanding the limitations of radiation-induced cell cycle checkpoints

The DNA damage response pathways involve processes of double-strand break (DSB) repair and cell cycle checkpoint control to prevent or limit entry into S phase or mitosis in the presence of unrepaired damage. Checkpoints can function to permanently remove damaged cells from the actively proliferating population but can also halt the cell cycle temporarily to provide time for the repair of DSBs. Although efficient in their ability to limit genomic instability, checkpoints are not foolproof but carry inherent limitations. Recent work has demonstrated that the G1/S checkpoint is slowly activated and allows cells to enter S phase in the presence of unrepaired DSBs for about 4-6?h post irradiation. During this time, only a slowing but not abolition of S-phase entry is observed. The G2/M checkpoint, in contrast, is quickly activated but only responds to a level of 10-20 DSBs such that cells with a low number of DSBs do not initiate the checkpoint or terminate arrest before repair is complete. Here, we discuss the limitations of these checkpoints in the context of the current knowledge of the factors involved. We suggest that the time needed to fully activate G1/S arrest reflects the existence of a restriction point in G1-phase progression. This point has previously been defined as the point when mitogen starvation fails to prevent cells from entering S phase. However, cells that passed the restriction point can respond to DSBs, albeit with reduced efficiency.     

Deficiency in the response to DNA double-strand breaks in mouse early preimplantation embryos

DNA double-strand breaks (DSBs) are caused by various environmental stresses, such as ionizing radiation and DNA-damaging agents. When DSBs occur, cell cycle checkpoint mechanisms function to stop the cell cycle until all DSBs are repaired; the phosphorylation of H2AX plays an important role in this process. Mouse preimplantation-stage embryos are hypersensitive to ionizing radiation, and X-irradiated mouse zygotes are arrested at the G2 phase of the first cell cycle. To investigate the mechanisms responding to DNA damage at G2 in mouse preimplantation embryos, we examined G2/M checkpoint and DNA repair mechanisms in these embryos. Most of the one- and two-cell embryos in which DSBs had been induced by gamma-irradiation underwent a delay in cleavage and ceased development before the blastocyst stage. In these embryos, phosphorylated H2AX (gamma-H2AX) was not detected in the one- or two-cell stages by immunocytochemistry, although it was detected after the two-cell stage during preimplantation development. These results suggest that the G2/M checkpoint and DNA repair mechanisms have insufficient function in one- and two-cell embryos, causing hypersensitivity to gamma-irradiation. In addition, phosphorylated ataxia telangiectasia mutated protein and DNA protein kinase catalytic subunits, which phosphorylate H2AX, were detected in the embryos at one- and two-cell stages, as well as at other preimplantation stages, suggesting that the absence of gamma-H2AX in one- and two-cell embryos depends on some factor(s) other than these kinases.     

Sentinels of DNA integrity in stem cells: Safeguarding future generations of cells

Integrated into the somatic cell cycle are multi-faceted mechanisms to protect genomic fidelity from genotoxic threats occurring during cell division or cellular quiescence. How embryonic stem cells respond to an array of attacks on genomic integrity has been uncertain, particularly in light of embryonic-like rapid cell cycle phases versus adult cells and the lack of an effective G1/S checkpoint. Whether a DNA damage response is activated similarly to somatic cells or apoptotic pathways used to purge damaged cells are important questions, since the longevity of embryonic stem cells provides opportunities for accumulated mutations and a source for carcinogenic cells. In this issue, Chuyikin et al. investigate the timing and sensitivity of the DNA damage response pathway to double strand breaks (DSBs) in mouse embryonic stem cells (ESCs), validating its responsiveness and providing a comprehensive view of key signaling events.

DNA DSBs are potently mutagenic lesions incurring chromosome breaks, potential rearrangements, mutation and loss of information.¹ The cellular response is immediate, sensitive and persistent, occurring within 30 seconds of damage upon detection of as little as 8 DSBs per cell. The response can be fully active in 15 minutes and persist for hours. Repair is preferred and may elicit checkpoint delays to cell cycle progression, with extreme genotoxic conditions initiating apoptotic pathways. The majority of DSB proteins are activated by PI-3 like kinases, with the primary mammalian response to DSBs occurring via the ATM kinase that is able to respond directly to DSBs. Phosphorylated downstream targets include the uncommon histone, H2AX. This histone provides a cytological platform at DSB sites for the recruitment of DSB mediator and effector proteins such as MDC1 and NBS1. To this scaffold further DSB proteins are recruited, amplifying the signal. NBS1 is part of the MRN complex that includes MRE11 and Rad50 and mediates nuclear localization of the complex to the DNA for stabilizing chromatin ends. The nucleolytic processing of DNA ends by MRE11 resection triggers a second pathway modulated by ATR, that responds to RPA coated ssDNA. Chuyikin et al., used antibodies to phosphorylated ATM and H2AX (pATM, pH2AX) as sensitive temporal markers of DNA repair foci that form at DSBs and followed these events through the cell cycle.

In fast proliferating undifferentiated cells an increase in single strand DNA breaks (SSBs) is typically observed, attributed to ongoing DNA replication, and not generally considered mutagenic. Chuyikin et al. used sensitive comet assays along with pH2AX and pATM antibodies to confirm the presence of SSBs in mESCs and a low background of pH2AX positive/pATM absent poised foci. Upon γ-irradiation to induce DSBs, dramatic detection of DNA repair foci including both pH2AX and pATM occurs. FACs analysis indicated no cell cycle arrest at G1/S from γ-irradiation, although a slight delay at G2/M. Chuyikin et al. did find that mESCs have an active spindle assembly checkpoint allowing cells to be blocked at G2/M with nocodazole and then released synchronously through the cell cycle. The key to their detection of this checkpoint was a six hour treatment with drug, versus longer timepoints. Indeed Reider and Maiato² have shown that in mammalian cells, spindle assembly checkpoint duration is variable and need not be satisfied to be overridden by adaptation, slippage or leakage, quite unlike the tight cell cycle arrest observed in fungi. Therefore longer treatments with nocodazole to arrest mESCs at this stage would be expected to simply be ineffective and promote further polyploidy by attenuating the mitotic mechanism. The authors detailed analysis of induction of DNA repair foci in all cell cycle stages revealed that all stages generate foci, including metaphase chromosomes in mitosis, although foci were most prominent in G1, G2 phases. Thus the primary response by the ATM pathway in these cells is not limited by cell cycle phase.

The maintenance of genomic fidelity in ESCs may require more enhanced DNA repair³ as well as alternative mechanisms to DNA repair, such as increased apoptosis. Chuyikin et al. observed increased caspase activity triggered after γ-irradiation of mESCs, but found no significant increase in cell death. They also found that protein levels of p53, a downstream target of the ATM kinase that is important for the G1/S checkpoint as well as p53-dependent apoptosis, were comparable to fibroblast cells, however p53 lacked activating phosphorylation. Both of these observations help to explain an ineffective G1/S checkpoint and the need for p53-independent apoptosis.

Additional alternate mechanisms for maintaining genomic integrity ESCs have been reported and contribute. This includes a 100X reduction in mutations versus somatic cells and resistance to oxidative stress. Asymmetry mechanisms,⁴ that are a commonly used means of cellular signaling and polarity from yeast to man may also apply, as in the Cairns immortal strand hypothesis. In 1975 Cairns proposed that stem cells might minimize mutations to their genomes from DNA replication by asymmetric segregation of their DNA. Retention of parental strands in the stem cell and segregation of potential mutation carrying DNAs into non-stem cell or differentiating daughters could reduce the mutation potential.4 Such asymmetric sister chromatid strand segregation is still controversial despite having been observed during mitosis in several stem cell populations. Continued elegant studies, such at that by Chuyikin et al, that define which pathways are present and examine the crosstalk in pathways used to detect, signal, repair and protect genomic integrity will continue to provide exciting new systemic views into stem cells. Our therapeutic use of stem cells in the future including understanding of cellular differentiation and cancer depends on it.

ReferencesRiches LC, et al. Mutagenesis 2008; In press.Rieder CL, et al. Dev Cell 2004; 7:637-51.Maynard S, et al. Stem Cells 2008; In press. Doxsey S, et al. Annu Rev Cell Dev Biol 2005; 21:411-34.Cairns J. Genetics 2006; 174:1069-72.Chuykin I, et al. Cell Cycle 2008; 7:In this issue.     

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations¹. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in²). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones^3,4, forming foci within 5-15 minutes⁵. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair⁶. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins^7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs⁶. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis¹⁰.In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1''s behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs¹¹. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis¹². Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.     

Genetic dissection of vertebrate 53BP1: a major role in non-homologous end joining of DNA double strand breaks

53BP1 (p53 binding protein) is a BRCT domain-containing protein that is rapidly recruited to DNA double strand breaks (DSBs). To investigate the role of 53BP1 in the DNA damage response, we generated 53BP1(-/-) cells from the chicken DT40 cell line. As in mammalian cells, mutation of 53BP1 increased cellular sensitivity to ionizing radiation. Although depletion of 53BP1 resulted in checkpoint defects in mammalian cells, DT40 53BP1(-/-) cells had normal intra S phase and G2/M checkpoints. G1 specific radiosensitivity and a higher sensitivity to topoisomerase II suggested defective non-homologous end joining (NHEJ) defects in DT40 53BP1(-/-) cells. Genetic analyses confirm this suggestion as we have demonstrated an epistatic relationship between 53BP1 and the NHEJ genes, Ku70 and Artemis, but not with Rad54, a gene essential for repair of DSBs by homologous recombination. We conclude that the major role of 53BP1 in supporting survival of DT40 cells that have suffered DNA DSBs is in facilitating repair by NHEJ.     

Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle.

Mice homozygous for the scid (severe combined immune deficiency) mutation are defective in the repair of DNA double-strand breaks (DSBs) and are consequently very X-ray sensitive and defective in the lymphoid V(D)J recombination process. Recently, a strong candidate for the scid gene has been identified as the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex. Here, we show that the activity of the DNA-PK complex is regulated in a cell cycle-dependent manner, with peaks of activity found at the G1/early S phase and again at the G2 phase in wild-type cells. Interestingly, only the deficit of the G1/early S phase DNA-PK activity correlated with an increased hypersensitivity to X-irradiation and a DNA DSB repair deficit in synchronized scid pre-B cells. Finally, we demonstrate that the DNA-PK activity found at the G2 phase may be required for exit from a DNA damage-induced G2 checkpoint arrest. These observations suggest the presence of two pathways (DNA-PK-dependent and -independent) of illegitimate mammalian DNA DSB repair and two distinct roles (DNA DSB repair and G2 checkpoint traversal) for DNA-PK in the cellular response to ionizing radiation.     

New alternative phosphorylation sites on the cyclin dependent kinase 1/cyclin a complex in p53-deficient human cells treated with etoposide: possible association with etoposide-induced apoptosis

Cell cycle arrest is a major cellular response to DNA damage preceding the decision to repair or die. Many malignant cells have non-functional p53 rendering them more “aggressive” in nature. Arrest in p53-negative cells occurs at the G2M cell cycle checkpoint. Failure of DNA damaged cells to arrest at G2 results in entry into mitosis and potential death through aberrant mitosis and/or apoptosis. The pivotal kinase regulating the G2M checkpoint is Cdk1/cyclin B whose activity is controlled by phosphorylation. The p53-negative myeloid leukemia cell lines K562 and HL-60 were used to determine Cdk1 phosphorylation status during etoposide treatment. Cdk1 tyrosine 15 phosphorylation was associated with G2M arrest, but not with cell death. Cdk1 tyrosine 15 phosphorylation also led to suppression of nuclear cyclin B-associated Cdk1 kinase activity. However cell death, associated with broader tyrosine phosphorylation of Cdk1 was not attributed to tyrosine 15 alone. This broader phosphoryl isoform of Cdk1 was associated with cyclin A and not cyclin B. Alternative phosphorylations sites were predicted as tyrosines 4, 99 and 237 by computer analysis. No similar pattern was found on Cdk2. These findings suggest novel Cdk1 phosphorylation sites, which appear to be associated with p53-independent cell death following etoposide treatment.     

Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage

Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G(2)/M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G(2)/M to the G(1) phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G(2) arrest that is alleviated by activated Akt. Furthermore, the transition from the G(2)/M to the G(1) phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G(2)/M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G(2)/M cell cycle checkpoint and apoptosis in MMR-deficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.     

TAO kinases mediate activation of p38 in response to DNA damage

Thousand and one amino acid (TAO) kinases are Ste20p-related MAP kinase kinase kinases (MAP3Ks) that activate p38 MAPK. Here we show that the TAO kinases mediate the activation of p38 in response to various genotoxic stimuli. TAO kinases are activated acutely by ionizing radiation, ultraviolet radiation, and hydroxyurea. Full-length and truncated fragments of dominant negative TAOs inhibit the activation of p38 by DNA damage. Inhibition of TAO expression by siRNA also decreases p38 activation by these agents. Cells in which TAO kinases have been knocked down are less capable of engaging the DNA damage-induced G2/M checkpoint and display increased sensitivity to IR. The DNA damage kinase ataxia telangiectasia mutated (ATM) phosphorylates TAOs in vitro; radiation induces phosphorylation of TAO on a consensus site for phosphorylation by the ATM protein kinase in cells; and TAO and p38 activation is compromised in cells from a patient with ataxia telangiectasia that lack ATM. These findings indicate that TAO kinases are regulators of p38-mediated responses to DNA damage and are intermediates in the activation of p38 by ATM.     

A role for the Fanconi anemia C protein in maintaining the DNA damage-induced G2 checkpoint

Fanconi anemia (FA) is a complex, heterogeneous genetic disorder composed of at least 11 complementation groups. The FA proteins have recently been found to functionally interact with the cell cycle regulatory proteins ATM and BRCA1; however, the function of the FA proteins in cell cycle control remains incompletely understood. Here we show that the Fanconi anemia complementation group C protein (Fancc) is necessary for proper function of the DNA damage-induced G2/M checkpoint in vitro and in vivo. Despite apparently normal induction of the G2/M checkpoint after ionizing radiation, murine and human cells lacking functional FANCC did not maintain the G2 checkpoint as compared with wild-type cells. The increased rate of mitotic entry seen in Fancc-/-mouse embryo fibroblasts correlated with decreased inhibitory phosphorylation of cdc2 kinase on tyrosine 15. An increased inability to maintain the DNA damage-induced G2 checkpoint was observed in Fancc -/-; Trp53 -/-cells compared with Fancc -/-cells, indicating that Fancc and p53 cooperated to maintain the G2 checkpoint. In contrast, genetic disruption of both Fancc and Atm did not cooperate in the G2 checkpoint. These data indicate that Fancc and p53 in separate pathways converge to regulate the G2 checkpoint. Finally, fibroblasts lacking FANCD2 were found to have a G2 checkpoint phenotype similar to FANCC-deficient cells, indicating that FANCD2, which is activated by the FA complex, was also required to maintain the G2 checkpoint. Because a proper checkpoint function is critical for the maintenance of genomic stability and is intricately related to the function and integrity of the DNA repair process, these data have implications in understanding both the function of FA proteins and the mechanism of genomic instability in FA.     

Wortmannin增强细胞辐射敏感性的分子机制

 Ｗ ｏｒｔｍ ａｎｎｉｎ（ Ｗ Ｏ Ｒ Ｔ）是 Ｐ Ｉ３ Ｋ 家族激酶特异抑制剂,对 ｐ５３ 野生型及突变型细胞的辐射敏感性均有提高．为了阐明 Ｗ Ｏ Ｒ Ｔ 的辐射增敏机制,通过免疫沉淀及免疫印迹法分析了 Ｗ Ｏ Ｒ Ｔ对辐射引起的细胞 Ｇ２／ Ｍ 转换中关键分子 ｃｄｃ２ 酪氨酸脱磷酸化延迟的影响;通过流式细胞术分析了 Ｗ Ｏ Ｒ Ｔ 对辐射引起的细胞 Ｇ２ 期延迟、细胞凋亡的影响;经报告基因转染的方法分析了 Ｗ Ｏ Ｒ Ｔ对宿主细胞对辐射损伤报告基因修复的影响;发现 Ｗ Ｏ Ｒ Ｔ 可促进受照细胞 ｃｄｃ２ 酪氨酸脱磷酸化、减弱辐射引起的细胞 Ｇ２ 期延迟、增强细胞凋亡并抑制损伤 Ｄ Ｎ Ａ 修复．提示 Ｗ Ｏ Ｒ Ｔ 辐射增敏是通过干扰细胞 Ｇ２ 期检查点调控、抑制损伤 Ｄ Ｎ Ａ 修复和促进细胞凋亡等多种途径实现的．     

Role of the Saccharomyces cerevisiae Rad53 checkpoint kinase in signaling double-strand breaks during the meiotic cell cycle

DNA double-strand breaks (DSBs) can arise at unpredictable locations after DNA damage or in a programmed manner during meiosis. DNA damage checkpoint response to accidental DSBs during mitosis requires the Rad53 effector kinase, whereas the meiosis-specific Mek1 kinase, together with Red1 and Hop1, mediates the recombination checkpoint in response to programmed meiotic DSBs. Here we provide evidence that exogenous DSBs lead to Rad53 phosphorylation during the meiotic cell cycle, whereas programmed meiotic DSBs do not. However, the latter can trigger phosphorylation of a protein fusion between Rad53 and the Mec1-interacting protein Ddc2, suggesting that the inability of Rad53 to transduce the meiosis-specific DSB signals might be due to its failure to access the meiotic recombination sites. Rad53 phosphorylation/activation is elicited when unrepaired meiosis-specific DSBs escape the recombination checkpoint. This activation requires homologous chromosome segregation and delays the second meiotic division. Altogether, these data indicate that Rad53 prevents sister chromatid segregation in the presence of unrepaired programmed meiotic DSBs, thus providing a salvage mechanism ensuring genetic integrity in the gametes even in the absence of the recombination checkpoint.     

ATR-dependent activation of p38 MAP kinase is responsible for apoptotic cell death in cells depleted of Cdc7

Cdc7 is a serine/threonine kinase that plays essential roles in the initiation of eukaryotic DNA replication and checkpoint response. In previous studies, depletion of Cdc7 by small interfering RNA was shown to induce an abortive S phase that led to the cell cycle arrest in normal human fibroblasts and apoptotic cell death in various cancer cells. Here we report that stress-activated p38 MAP kinase was activated and responsible for apoptotic cell death in Cdc7-depleted HeLa cells. The activation of p38 MAP kinase in the Cdc7-depleted cells was shown to depend on ATR, a major sensor kinase for checkpoint or DNA damage responses. Only the p38 MAP kinase, and not the other stress-activated kinases such as JNK or ERK, was activated, and both caspase 8 and caspase 9 were activated for the induction of apoptosis. Activation of apoptosis in Cdc7-depleted cells was completely abolished in cells treated with small interfering RNA or an inhibitor of the p38 MAP kinase, suggesting that p38 MAP kinase activation was responsible for apoptotic cell death. Taken together, we suggest that the ATR-dependent activation of the p38 MAP kinase is a major signaling pathway that induces apoptotic cell death after depletion of Cdc7 in cancer cells.     

Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development

Cdc7 kinase plays an essential role in firing of replication origins by phosphorylating components of the replication complexes. Cdc7 kinase has also been implicated in S phase checkpoint signaling downstream of the ATR and Chk1 kinases. Inactivation of Cdc7 in yeast results in arrest of cell growth with 1C DNA content after completion of the ongoing DNA replication. In contrast, conditional inactivation of Cdc7 in undifferentiated mouse embryonic stem (ES) cells leads to growth arrest with rapid cessation of DNA synthesis, suggesting requirement of Cdc7 functions for continuation of ongoing DNA synthesis. Furthermore, loss of Cdc7 function induces recombinational repair (nuclear Rad51 foci) and G2/M checkpoint responses (inhibition of Cdc2 kinase). Eventually, p53 becomes highly activated and the cells undergo massive p53-dependent apoptosis. Thus, defective origin activation in mammalian cells can generate DNA replication checkpoint signals. Efficient removal of those cells in which replication has been perturbed, through cell death, may be beneficial to maintain the highest level of genetic integrity in totipotent stem cells. Partial, rather than total, loss of Cdc7 kinase expression results in retarded growth at both cellular and whole body levels, with especially profound impairment of germ cell development.     

The nuclear death domain protein p84N5 activates a G2/M cell cycle checkpoint prior to the onset of apoptosis

In contrast to extracellular signals, the mechanisms utilized to transduce nuclear apoptotic signals are not well understood. Characterizing these mechanisms is important for predicting how tumors will respond to genotoxic radiation or chemotherapy. The retinoblastoma (Rb) tumor suppressor protein can regulate apoptosis triggered by DNA damage through an unknown mechanism. The nuclear death domain-containing protein p84N5 can induce apoptosis that is inhibited by association with Rb. The pattern of caspase and NF-kappaB activation during p84N5-induced apoptosis is similar to p53-independent cellular responses to DNA damage. One hallmark of this response is the activation of a G(2)/M cell cycle checkpoint. In this report, we characterize the effects of p84N5 on the cell cycle. Expression of p84N5 induces changes in cell cycle distribution and kinetics that are consistent with the activation of a G(2)/M cell cycle checkpoint. Like the radiation-induced checkpoint, caffeine blocks p84N5-induced G(2)/M arrest but not subsequent apoptotic cell death. The p84N5-induced checkpoint is functional in ataxia telangiectasia-mutated kinase-deficient cells. We conclude that p84N5 induces an ataxia telangiectasia-mutated kinase (ATM)-independent, caffeine-sensitive G(2)/M cell cycle arrest prior to the onset of apoptosis. This conclusion is consistent with the hypotheses that p84N5 functions in an Rb-regulated cellular response that is similar to that triggered by DNA damage.     

Non-Classical P38 Map Kinase Functions: Cell Cycle Checkpoints and Survival

The p38 MAPK kinase pathway is activated in response to a wide range of cellular stress stimuli and cytokines. Our understanding of the important functions of p38 MAPK in the process of differentiation and cell death has grown considerably in the recent years and is now relatively established. Here we discuss the role of p38 MAPK in the mediation of cell cycle checkpoints and cell survival, processes that have received less attention. We describe how p38 MAPK regulates both the G2/M as well as a G1/S cell cycle checkpoint in response to cellular stress such as DNA damage. While p38 MAPK has classically been associated with the induction of apoptosis, we discuss that p38 MAPK can also mediate cell survival in specific situations, such as in response to DNA damage. It is important to recognize these less appreciated functions of p38 MAPK when considering the potential use of pharmacological inhibitors of p38 MAPK in therapeutic treatments for disease.