DNA Damage-induced Cell Cycle Research Articles

Tumor Treating Fields (TTFields) downregulate the Fanconi Anemia-BRCA pathway and increase the efficacy of chemotherapy in malignant pleural mesothelioma preclinical models

ObjectivesTumor Treating Fields (TTFields) are low intensity, intermediate frequency, alternating electric fields with antimitotic effects on cancerous cells. TTFields concomitant with pemetrexed and a platinum agent are approved in the US and EU as first line therapy for unresectable, locally advanced or metastatic malignant pleural mesothelioma (MPM). The goal of the current study was to characterize the mechanism of action of TTFields in MPM cell lines and animal models. MethodsHuman MPM cell lines MSTO-211H and NCI-H2052 were treated with TTFields to determine the frequency that elicits maximal cytotoxicity. The effect of TTFields on DNA damage and repair, and the cytotoxic effect of TTFields in combination with cisplatin and/or pemetrexed were examined. Efficacy of TTFields concomitant with cisplatin and pemetrexed was evaluated in orthotopic IL-45 and subcutaneous RN5 murine models. ResultsTTFields at a frequency of 150 kHz demonstrated the highest cytotoxicity to MPM cells. Application of 150 kHz TTFields resulted in increased formation of DNA double strand breaks, elevated expression of DNA damage induced cell cycle arrest proteins, and reduced expression of Fanconi Anemia (FA)-BRCA DNA repair pathway proteins. Co-treatment of TTFields with cisplatin or pemetrexed significantly increased treatment efficacy versus each modality alone, with additivity and synergy exhibited by the TTFields-pemetrexed and TTFields-cisplatin combinations, respectively. In animal models, tumor volume was significantly lower for the TTFields-cisplatin-pemetrexed combination compared to control, accompanied by increased DNA damage within the tumor. ConclusionThis research demonstrated that the efficacy of TTFields for the treatment of MPM is associated with reduced expression of FA-BRCA pathway proteins and increased DNA damage. This mechanism of action is consistent with the observed synergism for TTFields-cisplatin vs additivity for TTFields-pemetrexed, as cisplatin-induced DNA damage is repaired via the FA-BRCA pathway.

MicroRNA-16 feedback loop with p53 and Wip1 can regulate cell fate determination between apoptosis and senescence in DNA damage response.

Cell fate regulation is an open problem whose comprehension impacts several areas of the biosciences. DNA damage induces cell cycle checkpoints that activate the p53 pathway to regulate cell fate mechanisms such as apoptosis or senescence. Experiments with different cell types show that the p53 pathway regulates cell fate through a switch behavior in its dynamics. For low DNA damage the pathway presents an oscillatory pattern associated with intense DNA damage repair while for high damage there are no oscillations and either p53 concentration increases inducing apoptosis or the cell enters a senescence state. Apoptosis and senescence phenotypes seem to have compensatory functions in tissues and the microRNA 16–1 (miR-16) is involved in the regulation of the fate between both phenotypes in cancer cells. To investigate the regulation of cell fate we developed a logical model of the G1/S checkpoint in DNA damage response that takes into account different levels of damage and contemplates the influence of miR-16 through its positive feedback loop formed with p53 and Wip1. The model reproduces the observed cellular phenotypes in experiments: oscillatory (for low DNA damage) regulated by negative feedback loops involving mainly p53 and Mdm2 and apoptotic or senescent (for high DNA damage) regulated by the positive p53/Wip1/miR-16 feedback loop. We find good agreement between the level of DNA damage and the probability of the phenotype produced according to experiments. We also find that this positive feedback makes senescent and apoptotic phenotypes to be determined stochastically (bistable), however controlling the expression level of miR-16 allows the control of fate determination as observed experimentally.

Abstract IA24: Targeting Chk1

Abstract The majority of traditional anticancer drugs inhibit DNA synthesis either by directly damaging DNA or by inhibiting synthesis of deoxyribonucleotide precursors. DNA damage induces cell cycle arrest through activation of cell cycle checkpoints whose goal is to prevent further DNA synthesis or mitosis until the damage is repaired. These checkpoints have undergone intense investigation as potential therapeutic targets, and Chk1 inhibitors (Chk1i) have emerged as promising novel therapeutic agents (1). Chk1 was initially recognized as a regulator of the DNA damage-induced S and G2 checkpoints, and its inhibition forced S phase progression followed by mitotic catastrophe of arrested cells. Subsequently, it was found that the combination of Chk1i with antimetabolites (particularly gemcitabine and cytarabine) more effectively enhanced cell killing (2, 3), and these combinations entered clinical trials. Little clinical benefit has been reported to date, and development of several Chk1i has been terminated for toxicity; whether the toxicity was due to on-target effects remains to be established. It is worth noting one outlier response in which a patient receiving irinotecan and the Chk1i AZD7762 attained a durable response; the sensitivity was tracked to a mutation in RAD50. This suggests that subsets of patients may have therapeutic benefit from this combination (4). Intriguingly, a few cell lines are also highly sensitive to Chk1i as a single agent suggesting subsets of tumors may exist that are uniquely sensitive to these drugs (5). Development of AZD7762 and another Chk1i, MK-8776, were terminated, but clinical trials are continuing with two other Chk1i: LY2606368 and GDC-0425; and a Phase I trial with V158411 is planned. The sensitivity to Chk1i as a single agent results from inappropriate activation of CDK2 in S phase, which in turn depends on the differential activation of the upstream phosphatase CDC25A (5, 6). This results in rapid appearance of DNA double-strand breaks that are dependent on Mre11 and Mus81 nucleases (7). The ability of Chk1i to abrogate DNA damage-induced S phase arrest is commonly attributed to activation of CDC25A/CDK2, yet we find that abrogation of arrest occurs even when CDK2 is inhibited. In contrast, inhibition of CDC7 prevents S phase progression. Similarly, the sensitivity to ribonucleotide reductase inhibitors such as gemcitabine is independent of CDK2 but dependent on CDC7. In this case, the continued absence of dNTPs prevents S phase progression, but DNA helicases are activated by Chk1i creating excess single-strand DNA that exhausts the protective single-strand binding protein RPA. The unprotected DNA is then susceptible to nuclease activity. The mechanistic link between Chk1 and CDC7 remains to be established. Administering a combination of two drugs to a patient, for example gemcitabine and a Chk1i, requires knowledge of how both drugs work in a human. One critical problem with preclinical experiments is that they are too often performed with continuous incubation over many days, yet in a patient, the drugs are usually administered over a short period of time. In the case of gemcitabine, a brief incubation in cell culture, or a bolus administration to a mouse or patient, leads to rapid and irreversible inhibition of ribonucleotide reductase and persistent S phase arrest. To assess the optimum time of addition of Chk1i, we added MK-8776 for 6 h periods following a 6 h treatment with gemcitabine. The greatest growth inhibition in vitro occurred when the addition of MK-8776 was delayed to 18 h, and this correlated with the maximal accumulation of cells in S phase. This delayed schedule was also shown to be more effective than concurrent administration in a xenograft model (3). Despite the apparent efficacy of MK-8776 in combination with gemcitabine, experiments to assess the single agent activity of MK-8776 have not detected tumor growth inhibition. We propose several explanations: when used as a single agent, a higher concentration of MK-8776 is required to inhibit cell growth than when combined with gemcitabine; cells also appear to recover better following a 6-h incubation with MK-8776 as a single agent than a 6-h treatment in combination suggesting that longer target inhibition is required for single agent activity. These 6-h treatment periods were selected because they reflect the in vivo concentration and half-life of MK-8776 in patients. These disappointing effects may be circumvented with alternate Chk1i that achieve greater bioavailability for a longer period of time. In this regard, both GDC-0425 and LY2606368 exhibit longer plasma half-lives, and Vernalis has a lead compound that has exhibited single agent activity in a xenograft model (Andrew Massey, personal communication). In summary, while initial clinical trials have generally resulted in marginal therapeutic activity, there may be subsets of patients who may respond well, and Chk1i with prolonged bioavailability may have enhanced in vivo efficacy either alone or in combination.

Abstract A45: Neat1 is a p53-inducible lincRNA important for pancreatic cancer suppression

Abstract p53’s role as a critical tumor suppressor is well illustrated by the fact that it is found mutated in more than half of all human cancers. In addition, the highly penetrant cancer predisposition observed in Li-Fraumeni patients with germline mutations in p53 and in p53 null mice further highlight the importance of p53 in tumor suppression. p53 is a transcription factor, able to sense stress signals such as DNA damage and hyperproliferative signals, promoting in turn the expression of a network of target genes to induce anti-proliferative responses such as cell cycle arrest or apoptosis. Although studies of genes in the p53 network have largely focused on protein-coding genes, in recent years the role of p53-regulated non-coding (nc) RNAs in p53 biology has been increasingly recognized. To identify new p53-regulated ncRNAs that could contribute to tumor suppression, we leveraged chromatin immunoprecipitation (ChIP)-sequencing and RNA-sequencing (RNA-seq) data we generated previously in p53 wild-type and p53-/- mouse embryonic fibroblasts (MEFs). This analysis uncovered the lincRNA Neat1 as a p53 target gene. We showed that Neat1 is induced by different DNA damaging agents in a p53-dependent manner in different mouse and human cell types, including pancreatic ductal cells. Although we found that Neat1 is not involved in DNA damage-induced p53-dependent cell cycle arrest or apoptosis responses, we found that it does play a role in p53-dependent transformation suppression in both oncogene-expressing fibroblasts and pancreatic cancer cells. Furthermore, using oncogene-expressing fibroblasts, we showed that Neat1 loss promotes cancer in vivo in a subcutaneous xenograft model. Preliminary evidence suggests also that Neat1 loss promotes acinar to ductal metaplasia, bolstering its importance in pancreatic cancer suppression. These findings highlight the importance of Neat1 for p53 tumor suppressor function. Detailed analyses of Neat1’s mechanism of action will provide insights in its function as a tumor suppressor. Citation Format: Stephano S. Mello, Carolyn Sinow, Pawel K. Mazur, Hannes Vogel, John Rinn, Laura D. Attardi.{Authors}. Neat1 is a p53-inducible lincRNA important for pancreatic cancer suppression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A45.

RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage-induced cell senescence.

The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase-regulated nuclear-cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage-induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β-dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP-regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage.

Abstract A2-65: Integrating computational and experimental approaches to build predictive models of the SMG1 kinase network in glioblastoma stem cells

Abstract Glioblastoma multiforme (GBM) is the most common and most aggressive tumor of the central nervous system, with a mean survival of only 12-14 months post diagnosis and treatment. A major reason for the poor prognosis of GBM is attributed to a small population of refractory GBM stem cells (GSC) that have been shown to initiate and sustain tumor growth. Current evidence suggests that GSC preferentially reside in hypoxic niches in the tumor that contribute to maintenance of its stem cell properties such as self-renewal and resistance to therapy. In order to identify targets for achieving effective tumor remission, we have performed an RNAi kinome screen to identify specific kinases that enable the survival of GSCs in hypoxic microenvironment. Only 40% of the hits were common between the two cell lines screened, emphasizing heterogeneity of tumor response that necessitates personalized targeted therapy for GBM. The SMG1 kinase was identified in this screen as a gene that is preferentially inhibitory to one of our GSC lines under hypoxia (1% oxygen). SMG1 is a member of the phosphoinositide-like family of kinases that includes ATM, ATR and mTOR. SMG1 is a potential target for cancer therapy to counteract tumor cell growth and resistance to DNA damaging chemotherapeutic agents due to its role in nonsense mediated decay (NMD) and DNA damage response (DDR) pathways. Our results are therapeutically significant since we found additive growth inhibitory effect of SMG1 knockdown in GSC in the presence of DNA damaging drug Temozolomide (TMZ), the current standard of care for GBM. The Upf1 is an essential NMD factor that is phosphorylated by SMG1 and is also required for DNA damage induced cell cycle S phase progression. Interestingly, our results show that the growth inhibitory effect of SMG1 knockdown correlated with the induction of Upf1 in GS7-2 cells specifically under hypoxia. We assessed the role of downstream mediators in NMD pathway (ATF-4, MAP4K12, SMG7 and p-EIF2alpha) and DDR pathway (p53, p-p53 (Ser 15), p21, ATM, ATR, Chk1, p-Chk1, Chk2, p-Chk2 and MRN complex) to identify critical components of the pathways that contribute to the differential growth inhibition effect. We have used the Cell Collective modeling software platform (BMC Systems Biology, 6, 96) to model the SMG1 signaling network by integrating our data with results in the literature in order to simulate cellular growth and survival of GSC under hypoxia and TMZ induced DNA damage. Our model predicts that the preferential growth inhibitory role of SMG1 inhibition in GS7-2 cells under hypoxia specifically is due to the role of NMD pathway in regulating autophagy and this is currently being tested experimentally in multiple cell lines. The long-term goal of this study will be to develop therapeutic predictions for targeting SMG1 or other kinases from the screen for a given tumor and thereby develop personalized therapies for treating GBM. Citation Format: Sejuti Sengupta, Steven K. Young, Surbhi Goel-Bhattacharya, Brent H. Cochran. Integrating computational and experimental approaches to build predictive models of the SMG1 kinase network in glioblastoma stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-65.

DNA Damage Sensitivity Assays in Caenorhabditis elegans

C. elegans has served as a genetically tractable multicellular model system to examine DNA damage-induced genotoxic stress which threatens genome integrity. Importantly, the high degree of conservation shared between worms and humans offers the advantage that findings about DNA damage-induced cell cycle arrest/checkpoint response and DNA double-strand break repair in worms are applicable to human studies. Here, we describe simple DNA damage sensitivity assays to quantify the response of C. elegans to diverse types of DNA damaging agents. These assays have provided important insights into the mechanisms of function for factors such as ZTF-8 that are involved in DNA damage repair and response in the C. elegans germline. These DNA damage sensitivity assays rely on the straightforward readouts of either egg or larval lethality and involve the use of various DNA damaging agents. We use γ-irradiation (γ-IR), which produces DNA double-strand breaks (DSBs), camptothecin (CPT), which induces single-strand breaks, nitrogen mustard (HN2), which produces interstrand crosslinks (ICLs), hydroxyurea (HU), which results in replication fork arrest thus preventing DNA synthesis, and UV-C, which causes photoproducts (pyrimidine dimers). See Table 1. Comparisons between the relative sensitivity/resistance observed in, for example, mutants compared to wild type, for various DNA damaging agents allows for inferences regarding potential repair pathways being affected.

Abstract 1786: UV damage-induced histone H3 lysine 56 deacetylation regulates nucleotide excision repair and checkpoint signaling.

Abstract Nucleotide excision repair (NER) is the major pathway in cells for the removal of bulky photoproducts and ensuring integrity of the genome. NER constitutes lesion recognition, excision of damaged DNA, synthesis of a DNA fragment, and ligation of the repair patch. Nonetheless, DNA within the cells is compacted into chromatin and lesion recognition requires chromatin to be remodeled to permit access of repair and checkpoint machinery to the damage site. Consequent to repair of the lesions, chromatin structure is restored to its original state so that cells resume cell cycle progression. Although several reports have demonstrated the role of chromatin remodelers, histone modifications and histone chaperones in DNA damage response (DDR), knowledge of how these epigenetic mechanisms control the DDR is largely in its infancy. We and others have recently observed that histone H3 is deacetylated on lysine 56 immediately in response to UV-induced DNA damage. Acetylation is promptly restored upon completion of repair and is necessary for recovery from DNA damage induced cell cycle arrest. However, the function of H3K56 deacetylation in the DDR remains to be determined. We found that UV-induced deacetylation of H3K56 is abrogated in the presence of TSA and sodium butyrate, both of which inhibit class I histone deacetylases (HDACs). Knockdown of either HDAC1 or HDAC2 alone did not affect UV-induced deacetylation of H3K56. However, simultaneous knockdown of both HDAC1 and 2 resulted in retention of H3K56 acetylation after UV irradiation. Consistently, both HDAC1 and HDAC2 localized to sites of UV damage. These observations are consistent with that reported by Miller et. al., indicating that HDAC1 and HDAC2 are responsible for the deacetylation of H3K56 in response to DNA damage. Since the deacetylation of H3K56 is one of the most immediate responses to UV-irradiation, we assumed it to play a crucial role in NER. Accordingly, we observed that CPD removal was affected in H1299 cells with histone H3K56Q mutation that mimics constitutive acetylation. Furthermore, we found that localization of the checkpoint kinase ATM to UV damage sites was affected in these cells. Consequently, ATM-mediated H2AX phosphorylation was also found to be defective in the mutant cells. Based on our observations, we speculate that HDAC1 and 2 mediated H3K56 deacetylation regulates NER and checkpoint signaling cascade in response to UV damage. This work was supported by PHS grants ES2388, ES12991 and CA93413 and Pelotonia postdoctoral fellowship to AB. Citation Format: Aruna Battu, Altaf Ahmad Wani. UV damage-induced histone H3 lysine 56 deacetylation regulates nucleotide excision repair and checkpoint signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1786. doi:10.1158/1538-7445.AM2013-1786

Decreased translation of p21waf1 mRNA causes attenuated p53 signaling in some p53 wild-type tumors

DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. However, some p53 wild type tumor cell lines, such as HCT116 and U2OS, are also sensitive to inhibition of Chk1 due to attenuated p21waf1 induction upon DNA damage. The purpose of this study is to determine the cause of this attenuated p21waf1 protein induction. We find that neither the induction of p21waf1 mRNA nor protein half-life is sufficient to explain the low p21waf1 protein levels in HCT116 and U2OS cells. The induced mRNA associates with polysomes but little protein is made suggesting these two cell lines have a reduced rate of p21waf1 mRNA translation. This represents a novel mechanism for disruption of the p53-p21waf1 pathway as currently known mechanisms involve either mutation of p53 or reduction of p53 protein levels. As a consequence, this attenuated p21waf1 expression may render some p53 wild type tumors sensitive to a combination of DNA damage plus checkpoint inhibition.

Nitric oxide restrain root growth by DNA damage induced cell cycle arrest in Arabidopsis thaliana

Nitric oxide (NO) participates in the regulation of diverse functions in plant cells. However, different NO concentrations may trigger different pathways during the plant development. At basal levels of NO, plants utilize the NO signaling transduction pathway to facilitate plant growth and development, whereas higher concentrations trigger programmed cell death (PCD). Our results show that NO lower than the levels causing PCD, but higher than the basal levels induce DNA damage in root cells in Arabidopsis as witnessed by a reduction in root growth, rather than cell death, since cells retain the capacity to differentiate root hairs. The decrease in meristematic cells and increase in DNA damage signals in roots in responses to NO are in a dose dependent manner. The restraint of root growth is due to cell cycle arrest at G1 phase which is caused by NO induced DNA damage, besides a second arrest at G2/M existed in NO supersensitive mutant cue1. The results indicate that NO restrain root growth via DNA damage induced cell cycle arrest.

Context-Dependent Cell Cycle Checkpoint Abrogation by a Novel Kinase Inhibitor

BackgroundCheckpoint kinase 1 and 2 (Chk1/Chk2), and the Aurora kinases play a critical role in the activation of the DNA damage response and mitotic spindle checkpoints. We have identified a novel inhibitor of these kinases and utilized this molecule to probe the functional interplay between these two checkpoints.Principal FindingsFragment screening, structure guided design, and kinase cross screening resulted in the identification of a novel, potent small molecule kinase inhibitor (VER-150548) of Chk1 and Chk2 kinases with IC50s of 35 and 34 nM as well as the Aurora A and Aurora B kinases with IC50s of 101 and 38 nM. The structural rationale for this kinase specificity could be clearly elucidated through the X-ray crystal structure. In human carcinoma cells, VER-150548 induced reduplication and the accumulation of cells with >4N DNA content, inhibited histone H3 phosphorylation and ultimately gave way to cell death after 120 hour exposure; a phenotype consistent with cellular Aurora inhibition. In the presence of DNA damage induced by cytotoxic chemotherapeutic drugs, VER-150548 abrogated DNA damage induced cell cycle checkpoints. Abrogation of these checkpoints correlated with increased DNA damage and rapid cell death in p53 defective HT29 cells. In the presence of DNA damage, reduplication could not be observed. These observations are consistent with the Chk1 and Chk2 inhibitory activity of this molecule.ConclusionsIn the presence of DNA damage, we suggest that VER-150548 abrogates the DNA damage induced checkpoints forcing cells to undergo a lethal mitosis. The timing of this premature cell death induced by Chk1 inhibition negates Aurora inhibition thereby preventing re-entry into the cell cycle and subsequent DNA reduplication. This novel kinase inhibitor therefore serves as a useful chemical probe to further understand the temporal relationship between cell cycle checkpoint pathways, chemotherapeutic agent induced DNA damage and cell death.

Oncogenic Wip1 Phosphatase Is Inhibited by miR-16 in the DNA Damage Signaling Pathway

Wild-type p53-induced phosphatase 1 (Wip1) was identified as an oncogene amplified and overexpressed in several human cancers. Recent evidence suggested that Wip1 is a critical inhibitor in the ATM/ATR-p53 DNA damage signaling pathway. Wip1 dephosphorylates several key DNA damage-responsive proteins and reverses DNA damage-induced cell cycle checkpoints. Previous reports showed that Wip1 was transcriptionally induced by p53 at the early stage of the DNA damage response. To investigate the temporal and functional regulation of Wip1, we identified a microRNA, miR-16, that specifically targets the mRNA of Wip1 and thus negatively regulates the expression level of Wip1. miR-16 itself is induced immediately after DNA damage. Therefore, the increase in Wip1 protein level is significantly postponed compared with that of its mRNA level, preventing a premature inactivation of ATM/ATR signaling and allowing a functional completion of the early DNA damage response. To better understand miR-16 biological functions in the context of cancer cells, we examined its expression in mammary tumor stem cells and found it to be markedly downregulated in mammary tumor stem cells. Overexpression of miR-16 or inhibition of Wip1 suppresses the self-renewal and growth of mouse mammary tumor stem cells and sensitizes MCF-7 human breast cancer cells to the chemotherapeutic drug doxorubicin. Together, our results suggest an important role of miR-16 in the regulation of Wip1 phosphatase in the DNA damage response and mammary tumorigenesis.

Regulation of cell division and growth in roots of Lactuca sativa L. seedlings by the Ent-Kaurene diterpenoid rabdosin B.

Rabdosin B, an ent-kaurene diterpenoid purified from the air-dried aerial parts of Isodon japonica (Burm.f) Hara var. galaucocalyx (maxin) Hara, showed a biphasic, dose-dependent effect on root growth and a strong inhibitory effect on root hair development in lettuce seedlings (Lactuca sativa L.). Lower concentrations of rabdosin B (20-80 microM) significantly promoted root growth, but its higher levels at 120-200 microM, by contrast, had inhibitory effects. Additionally, all tested concentrations (10-40 microM) inhibited root hair development of seedlings in a dose-dependent manner. Further investigations on the underlying mechanism revealed that the promotion effect of rabdosin B at the lower concentrations resulted from increasing the cell length in the mature region and enhancing the mitotic activity of meristematic cells in seedlings' root tips. In contrast, rabdosin B at higher concentrations inhibited root growth by affecting both cell length in the mature region and division of meristematic cells. Comet assay and cell cycle analysis demonstrated that the decrease of mitotic activity of root meristematic cells was due to DNA damage induced cell cycle retardation of the G(2) phase and S phase at different times.

FOXO Transcription Factors Enforce Cell Cycle Checkpoints and Promote Survival of Hematopoietic Cells after DNA Damage

The PI3K/AKT signaling pathway contributes to cell cycle progression of cytokine-dependent hematopoietic cells under normal conditions, and it is absolutely required to override DNA damage-induced cell cycle arrest checkpoints in these cells. Phosphatidylinositol-3-kinase (PI3K)/AKT activity also correlates with Cdk2 activity in hematopoietic cells, suggesting that Cdk2 activation may be a relevant end point for this signaling pathway. However, mediators downstream of AKT in this pathway have not been defined. The forkhead transcription factor O (FOXO) family are negatively regulated by AKT-dependent phosphorylation and are known regulators of genes affecting cell cycle progression. We show that enhanced FOXO activity replicates the effect of PI3K inhibitors in enforcing G(1) and G(2) phase arrest after DNA damage. Conversely, knockdown of endogenous FOXO proteins increased Cdk2 activity and overrode DNA damage checkpoints in cells lacking PI3K activity. Moreover, loss of FOXO activity caused an increase in sensitivity to cisplatin-induced cell death, which was associated with failure to arrest cell cycle progression in the face of DNA damage caused by this chemotherapeutic agent. These cell cycle arrests were dependent on p27 expression when mediated by FOXO3a alone, but also involve p27-independent mechanisms when promoted by endogenous FOXO proteins. Together, these observations show that FOXO proteins enforce DNA damage-induced cell cycle arrest in hematopoietic cells. Inhibition of FOXO activity by cytokine-induced PI3K/AKT signaling is sufficient to override these DNA damage-induced cell cycle checkpoints, but may negatively impact hematopoietic cell viability.

Transcriptional profiling of the age-related response to genotoxic stress points to differential DNA damage response with age

The p53 DNA damage response attenuated with age and we have evaluated downstream factors in the DNA damage response. In old animals p21 protein accumulates in the whole cell fraction but significantly declines in the nucleus, which may alter cell cycle and apoptotic programs in response to DNA damage. We evaluated the transcriptional response to DNA damage in young and old and find 2692 genes are differentially regulated in old compared to young in response to oxidative stress ( p < 0.005). As anticipated, the transcriptional profile of young mice is consistent with DNA damage induced cell cycle arrest while the profile of old mice is consistent with cell cycle progression in the presence of DNA damage, suggesting the potential for catastrophic accumulation of DNA damage at the replication fork. Unique sets of DNA repair genes are induced in response to damage in old and young, suggesting the types of damage accumulating differs between young and old. The DNA repair genes upregulated in old animals point to accumulation of replication-dependent DNA double strand breaks (DSB). Expression data is consistent with loss of apoptosis following DNA damage in old animals. These data suggest DNA damage responses differ greatly in young and old animals.

P21Cip1/WAF1 mediates cyclin B1 degradation in response to DNA damage

p21Cip1/WAF1 is the principle mediator of cell cycle arrest in response to DNA damage. p21 primarily mediates G1 cell cycle arrest by inactivating G1-associated cyclin A- and cyclin E-containing cyclin/cdk complexes. In the present study we investigate the role of p21 in DNA damage-induced G2 cell cycle arrest, particularly with respect to the G2-associated cyclin, cyclin B1. We demonstrate that cells lacking p21 or deficient in their ability to upregulate p21 are unable to mediate the downregulation of cyclin B1 in response to DNA damage as compared to wild-type cells. Decreased levels of cyclin B1 in response to DNA damage seen in wild-type cells is due to p21-mediated degradation of cyclin B1 as this can be inhibited by a proteasomal inhibitor. Cell cycle analysis reveals that p21-null cells are unable sustain G2 cell cycle arrest and accumulate at greater than 4N DNA content. These results indicate that p21-mediated degradation of cyclin B1 in response to DNA damage is necessary for the maintenance of G2 cell cycle arrest.

UV Radiation Regulates Mi-2 through Protein Translation and Stability

Dermatomyositis (DM) is an autoimmune disease, which is often accompanied by the development of disease-specific autoantibodies directed against the SNF2-superfamily helicase, Mi-2. Recent evidence suggests that ultraviolet radiation exposure may be an important risk factor for the development of not only the disease but also specific autoimmunity against Mi-2. Consequently, we investigated the effects of ultraviolet radiation on Mi-2 protein expression. We observed an increase in protein levels upon ultraviolet radiation exposure in cell culture systems. These changes in expression occur quite rapidly, are maximized just 1 h following exposure, and are unique to Mi-2 when compared with other members of the NuRD complex. Changes in protein levels are not mediated through transcriptional mechanisms. Treatment results in a more efficiently translated message through regulatory elements in the 5'-UTR region of the transcript. Investigation into protein half-life further demonstrated increased stability of Mi-2 following UV exposure. Taken together, we describe a system by which Mi-2 protein expression can be quickly increased following UV exposure and then maintained up to 16 h later. These data provide a novel regulation of an important transcriptional regulator and provide insight into the possible mechanisms of the development of DM and associated autoantibodies.

The Ataxia Telangiectasia–Mutated Target Site Ser18 Is Required for p53-Mediated Tumor Suppression

The p53 tumor suppressor is phosphorylated at multiple sites within its NH(2)-terminal region. One of these phosphorylation sites (mouse Ser(18) and human Ser(15)) is a substrate for the ataxia telangiectasia-mutated (ATM) and ATM-related (ATR) protein kinases. Studies of p53(S18A) mice (with a germ-line mutation that replaces Ser(18) with Ala) have indicated that ATM/ATR phosphorylation of p53 Ser(18) is required for normal DNA damage-induced PUMA expression and apoptosis but not for DNA damage-induced cell cycle arrest. Unlike p53-null mice, p53(S18A) mice did not succumb to early-onset tumors. This finding suggested that phosphorylation of p53 Ser(18) was not required for p53-dependent tumor suppression. Here we report that the survival of p53(S18A) mice was compromised and that they spontaneously developed late-onset lymphomas (between ages 1 and 2 years). These mice also developed several malignancies, including fibrosarcoma, leukemia, leiomyosarcoma, and myxosarcoma, which are unusual in p53 mutant mice. Furthermore, we found that lymphoma development was linked with apoptotic defects. In addition, p53(S18A) animals exhibited several aging-associated phenotypes early, and murine embryonic fibroblasts from these animals underwent early senescence in culture. Together, these data indicate that the ATM/ATR phosphorylation site Ser(18) on p53 contributes to tumor suppression in vivo.

Attenuated Expression of Xeroderma Pigmentosum Group C Is Associated with Critical Events in Human Bladder Cancer Carcinogenesis and Progression

Xeroderma pigmentosum group C (XPC) is an important DNA damage recognition protein that binds to damaged DNA at a very early stage during DNA repair. The XPC protein is also involved in DNA damage-induced cell cycle checkpoint regulation and apoptosis. XPC defects are associated with many types of solid tumors. The mechanism of the XPC protein in cancer progression, however, remains unclear. In this report, we showed the strong correlation between bladder cancer progression and attenuated XPC protein expression using tissues derived from patients with bladder cancer. The results obtained from our immunohistochemical studies further revealed a strong correlation of XPC deficiency, p53 mutation, and the degree of malignancy of bladder tumors. In addition, the results obtained from our studies have also shown that HT1197 bladder cancer cells, which carry a low-level XPC protein, exhibited a decreased DNA repair capability and were resistant to cisplatin treatment. When an XPC gene cDNA-expression vector was stably transfected into the HT1197 cells, however, the cisplatin treatment-induced apoptotic cell death was increased. Increased p53 and p73 responses following cisplatin treatment were also observed in HT1197 cells stably transfected with XPC cDNA. Taken together, these results suggest that XPC deficiency is an important contributing factor in bladder tumor progression and bladder cancer cell drug resistance.

Selective Chk1 inhibitors differentially sensitize p53‐deficient cancer cells to cancer therapeutics

The majority of cancer therapeutics induces DNA damage to kill cells. Normal proliferating cells undergo cell cycle arrest in response to DNA damage, thus allowing DNA repair to protect the genome. DNA damage induced cell cycle arrest depends on an evolutionarily conserved signal transduction network in which the Chk1 kinase plays a critical role. In mammalian cells, the p53 and RB pathways further augment the cell cycle arrest response to prevent catastrophic cell death. Given the fact that most tumor cells suffer defects in the p53 and RB pathways, it is likely that tumor cells would depend more on the Chk1 kinase to maintain cell cycle arrest than would normal cells. Therefore Chk1 inhibition could be used to specifically sensitize tumor cells to DNA-damaging agents. We have previously shown that siRNA-mediated Chk1 knockdown abrogates DNA damage-induced checkpoints and potentiates the cytotoxicity of several DNA-damaging agents in p53-deficient cell lines. In this study, we have developed 2 potent and selective Chk1 inhibitors, A-690002 and A-641397, and shown that these compounds abrogate cell cycle checkpoints and potentiate the cytotoxicity of topoisomerase inhibitors and gamma-radiation in p53-deficient but not in p53-proficient cells of different tissue origins. These results indicate that it is feasible to achieve a therapeutic window with 1 or more Chk1 inhibitors in potentiation of cancer therapy based on the status of the p53 pathway in a wide spectrum of tumor types.