Checkpoint Adaptation Research Articles

Functional Characterization of a Novel Role for Rfa2 N‐terminal Hyper‐phosphorylation During Checkpoint Adaptation

Replication Protein A (RPA) is an essential heterotrimeric complex found in all eukaryotic organisms and has critical roles in DNA metabolism and cell cycle progression. In human cells, RPA is known to be hyper‐phosphorylated, primarily on the N‐terminus of the second subunit (Rpa2), in response to genotoxic stress. The significance and function of Rpa2 N‐terminal (NT) phosphorylation has been the subject of over 25 years of research; however, the physiological function of this modification has yet to be clearly determined. In budding yeast, Rpa2 N‐terminal phosphorylation is detectable upon prolonged exposure to commonly used clastogens or upon generation of an irreparable break at the MAT locus using HO endonuclease. Upon cleavage of the MAT locus, the master checkpoint kinase Rad53 becomes hyper‐phosphorylated, leading to signaling events that arrest yeast cells at the G2/M checkpoint to allow adequate time for DNA repair.During checkpoint adaptation, cell cycle progression restarts, despite the presence of unrepaired DNA. Checkpoint adaptation affords cells the ability to bypass an established checkpoint, with the potential negative consequence of cellular division with broken chromosomes, leading to mutations and/or chromosome loss. Through the use of functional and phenotypic analysis of yeast containing Rpa2 NT mutations, we have determined that the phospho‐state of the Rpa2 NT may play a key role in mediating checkpoint adaptation. We demonstrate here that the Rpa2 NT is necessary for, and readily hyper‐phosphorylated during, checkpoint adaptation. We have determined the subregions of the Rpa2 NT that promote checkpoint adaptation when phosphorylated, as well as some of the conserved kinases that appear to phosphorylate the Rpa2 NT during checkpoint adaptation. Finally, we propose a model by which Rfa2 NT hyper‐phosphorylation directly leads to dephosphorylation/deactivation of the master checkpoint signaling kinase Rad53 in order to coordinate checkpoint adaptation in yeast. Understanding the mechanistic role the Rpa2 NT plays in coordinated checkpoint exit in the face of persistent damage may shed light on events that lead to deregulation of checkpoint signaling, and subsequently, mechanisms that lead to aberrant cell growth.Support or Funding InformationThis research was supported by NSF‐CAREER 1253723 to SJH

Phosphorylation of Replication Factor A Promotes Checkpoint Adaptation in Adaptation‐Deficient Phosphatase Deletion Strains

The cell cycle has long been studied and characterized. Major events (G1, S, G2, and M) occurring within the cycle are well understood; however, the molecular mechanisms controlling how the cell cycle is regulated are still being deciphered. Since the events of the cell cycle must occur in a proper temporal order, the progression of each step in the cell cycle must be carefully monitored. In the event that a particular step does not occur properly or that the cell is not ready to move to the next step, arrest of the cell cycle occurs (through the activation of a “checkpoint”). In the case of the introduction of double‐stranded DNA breaks (DSBs), cells initiate a checkpoint, arresting cells prior to the completion of mitosis (G2/M checkpoint). Processing of a DSB requires initial DNA resection resulting in single‐stranded DNA (ssDNA), which is bound by the ssDNA‐binding complex Replication Factor A (RFA). This leads to the eventual phosphorylation on the N‐terminus (NT) of the second subunit of RFA (Rfa2).The signaling of the G2/M checkpoint involves several kinases, many of which also require phosphorylation to promote their activity. The phosphorylation/dephosphorylation of Rad53 (yeast Chk2) has long been used as a marker for checkpoint activation/exit. Previous results from our laboratory indicate that Rfa2 NT phosphorylation may play a key role in exit of the checkpoint through dephosphorylation of Rad53. Checkpoint exit can occur after complete repair of damaged DNA (checkpoint recovery), and it can also occur without the repair of difficult‐to‐repair DNA damage (checkpoint adaptation). Both checkpoint recovery and checkpoint adaptation require the dephosphorylation of Rad53 by a combination of phosphatases. Three phosphatases that have been shown to act on Rad53 previously include Pph3‐Psy2 (PP4), Ptc2, and Ptc3.To determine if Rfa2 phosphorylation acts through/promotes phosphatases to enable checkpoint adaptation, several phosphatase knockout (KO) strains were created. In combination with the phosphatase KO's, Rfa2 NT mutant forms that either mimic Rfa2 hyper‐phosphorylation or prevent Rfa2 phosphorylation were used to replace wild‐type Rfa2. Strain sensitivity to several DNA damaging agents was characterized, and some phosphatase KO's enhanced the sensitivities of particular Rfa2 mutants. Phosphatase KO's display an inability to exit from the G2/M checkpoint in our system (i.e., a checkpoint adaptation deficiency); however, the Rfa2 NT mutant mimicking phosphorylation or a hybrid Rfa2 form that can be hyper‐phosphorylated suppressed the adaptation deficiency in pph3Δ and psy2Δ cells, both microscopically and biochemically. Since suppression occurs in single pph3Δ and psy2Δ mutants, it is also important to determine if suppression is mediated through other phosphatases, Ptc2 and/or Ptc3 (currently ongoing). To date, the only mutations reported to suppress checkpoint adaptation deficiencies all lie within RFA, suggesting that RFA is ultimately what determines checkpoint exit. We further submit that the state of the Rfa2 N‐terminus plays a key role in driving checkpoint exit.Support or Funding InformationThis work was supported by award NSF‐CAREER‐1253723 from the National Science Foundation to SJH.

Regulatory functional territory of PLK-1 and their substrates beyond mitosis.

Polo-like kinase 1 (PLK-1) is a well-known (Ser/Thr) mitotic protein kinase and is considered as a proto-oncogene. As hyper-activation of PLK-1 is broadly associated with poor prognosis and cancer progression, it is one of the most extensively studied mitotic kinases. During mitosis, PLK-1 regulates various cell cycle events, such as spindle pole maturation, chromosome segregation and cytokinesis. However, studies have demonstrated that the role of PLK-1 is not only restricted to mitosis, but PLK-1 can also regulate other vital events beyond mitosis, including transcription, translation, ciliogenesis, checkpoint adaptation and recovery, apoptosis, chromosomes dynamics etc. Recent reviews have tried to define the regulatory role of PLK-1 during mitosis progression and tumorigenesis, but its’ functional role beyond mitosis is still largely unexplored. PLK-1 can regulate the activity of many proteins that work outside of its conventional territory. The dysregulation of these proteins can cause diseases such as Alzheimer’s disease, tumorigenesis etc. and may also lead to drug resistance. Thus, in this review, we discussed the versatile role of PLK-1 and tried to collect data to validate its’ functional role in cell cycle regulation apart from mitosis.

PLK1 inhibition enhances temozolomide efficacy in IDH1 mutant gliomas.

Despite multimodal therapy with radiation and the DNA alkylating agent temozolomide (TMZ), malignant gliomas remain incurable. Up to 90% of grades II-III gliomas contain a single mutant isocitrate dehydrogenase 1 (IDH1) allele. IDH1 mutant-mediated transformation is associated with TMZ resistance; however, there is no clinically available means of sensitizing IDH1 mutant tumors to TMZ. In this study we sought to identify a targetable mechanism of TMZ resistance in IDH1 mutant tumors to enhance TMZ efficacy. IDH1 mutant astrocytes rapidly bypassed the G2 checkpoint with unrepaired DNA damage following TMZ treatment. Checkpoint adaptation was accompanied by PLK1 activation and IDH1 mutant astrocytes were more sensitive to treatment with BI2536 and TMZ in combination (<20% clonogenic survival) than either TMZ (~60%) or BI2536 (~75%) as single agents. In vivo, TMZ or BI2536 alone had little effect on tumor size. Combination treatment caused marked tumor shrinkage in all mice and complete tumor regression in 5 of 8 mice. Mutant IDH1 promotes checkpoint adaptation which can be exploited therapeutically with the combination of TMZ and a PLK1 inhibitor, indicating PLK1 inhibitors may be clinically valuable in the treatment of IDH1 mutant gliomas.

Cover Picture

HT‐29 human colorectal adenocarcinoma cells treated with 30 ?M cisplatin for 72 h express high levels of cyclin B1 (red) prior to undergoing checkpoint adaptation (entry into mitosis with damaged DNA).

A Novel Role For RPA Phosphorylation In The Regulation Of G2 Exit In The Face Of Persistent DNA Damage

BackgroundReplication Protein A (RPA), a heterotrimeric complex with critical roles in DNA metabolism and cell cycle progression, is observed to be hyper‐phosphorylated in response to genotoxic stress. Hyper‐phosphorylation of human RPA (hRPA) occurs primarily on the N‐terminus (NT) of the 32 kDa Rpa2 (hRpa2) subunit. In contrast, phosphorylation of yeast RPA (yRPA) on the Rpa2 (yRpa2) NT is not detectable upon exposure to commonly used genotoxic agents (e.g., methyl methanesulfonate, hydroxyurea). In fact, the yRpa2 NT has never been identified as a major site of phosphorylation upon DNA damage treatment. However, the examination of yRpa2 NT phospho‐mutants suggests that the phospho‐state of yRpa2 NT may play a key role in cell cycle progression despite the continued presence of DNA damage (checkpoint adaptation). In some instances checkpoint adaptation might allow for the positive cellular consequence of continued cell growth. This comes with a potential negative organismal cost, as checkpoint adaptation is often detrimental to cell survival and is also associated with increased genomic instability, which can ultimately lead to mutations that cause cellular disease.Study ObjectiveTo elucidate the importance of the yeast Rpa2 NT in regulating exit from the G2/M checkpoint.MethodWe examined yRpa2 phosphorylation and the cell cycle progression of yRpa2 NT mutants in an inducible HO endonuclease system that generates a single double‐strand break (DSB) that cannot be repaired by homologous recombination (HR).Results and ConclusionsWe have demonstrated that the presence of the yRpa2 NT is necessary for checkpoint adaptation. Furthermore, we have recently discovered that in the presence of a persistent lesion, yRpa2 NT hyper‐phosphorylation is now readily detected. This is consistent with our published data demonstrating that checkpoint adaptation is affected in yRpa2 phospho‐mutant cells. Because N‐terminal phosphorylation is now detectable under this condition, we have employed alanine scanning mutagenesis to identify which sites on the yRpa2 NT are phosphorylated and important for promoting checkpoint adaptation. We have also examined the kinases responsible for phosphorylating the yRpa2 NT during checkpoint adaptation. In addition, we have evidence that phosphorylation of the yRpa2 NT occurs primarily upon prolonged exposure to other agents that specifically result in DSB formation. Finally, we propose a model that suggests that Rad53 deactivation and Rfa2 phosphorylation may be linked to coordinate checkpoint adaptation in yeast. Understanding how yRPA phospho‐states influence cellular division in the presence of persistent/prolonged genotoxic stress may help provide the framework for deciphering the molecular mechanism(s) cells employ to maintain their genome.Support or Funding InformationThis research was supported by NSF‐CAREER 1253723 to SJH.

Cytotoxic amounts of cisplatin induce either checkpoint adaptation or apoptosis in a concentration-dependent manner in cancer cells.

Checkpoint adaptation (entry into mitosis with damaged DNA) is a process that links arrest at the G2/M cell cycle checkpoint and cell death in cancer cells. It is not known, however, whether cells treated with the genotoxic agent, cisplatin, undergo checkpoint adaptation or if checkpoint adaptation is a major pathway leading to cell death or not. Therefore, we investigated the relationship between treatment with cisplatin and cytotoxicity in cancer cells. Treatment of HT-29 human colorectal adenocarcinoma cells with cisplatin can induce cell death by one of two different mechanisms. Cells treated with a cytotoxic 30 μM amount of cisplatin died after undergoing checkpoint adaptation. Before dying, however, almost all treated cells were positive for histone γH2AX staining and contained high levels of cyclin B1. Rounded cells appeared that were positive for phospho-Ser10 histone H3, with low levels of phospho-Tyr15 cyclin-dependent kinase 1, high levels of cyclin-dependent kinase 1 activity, and checkpoint kinase 1 that was not phosphorylated on Ser345. These cells were in mitosis with damaged DNA. Strikingly, with 30 μM cisplatin, 81% of cells had entered mitosis before dying. By contrast, after treatment with 100 μM cisplatin, nearly all cells died but only 7% of cells had entered mitosis. Instead, these cells died by apoptosis; they were positive for annexin-V staining, contained cleaved caspase 3, cleaved caspase 9 and cleaved PARP and did not contain Mcl-1. Our data demonstrate that cancer cells treated with cisplatin can undergo one of two modes of cell death depending upon concentration used. These findings suggest that checkpoint adaptation is likely a primary pathway in genotoxic cell death at pharmacological concentrations of cisplatin. Checkpoint adaptation might be a common biochemical pathway taken by human cancer cells in response to pharmacologically relevant, cytotoxic amounts of damaged DNA.

Sterigmatocystin-induced checkpoint adaptation depends on Chk1 in immortalized human gastric epithelial cells in vitro.

Sterigmatocystin (ST) is a common contaminant detected in food and animal feed that has been recognized as a possible human carcinogen. Our previous studies demonstrate that ST causes DNA damage and subsequently triggers cell cycle arrest in G2 and apoptosis in immortalized human gastric epithelial cells (GES-1). Recently, studies have shown that in certain contexts, cells with DNA damage may escape checkpoint arrest and enter mitosis without repairing the damage. The term for this process is "checkpoint adaptation," and it increases the risk of unstable genome propagation, which may contribute to carcinogenesis. Thus, we aimed to investigate whether checkpoint adaptation occurs in GES-1 cells treated with ST and explored the underlying molecular mechanisms that contribute to this phenotype. In this study, we found that ST treatment for 24h in GES-1 cells led to an initial G2 arrest; however, a fraction of GES-1 cells became large and rounded, and the number of p-H3-positive cells increased sharply after ST treatment for 48h. Moreover, collection of the large and rounded cells by mechanical shake-off revealed that the majority of these large cells were found in the mitotic phase of the cell cycle. Importantly, we found that these rounded cells entered mitosis despite damaged DNA and that a small subset of this cell population survived and continued to propagate. These results suggest that ST induces an initial G2 arrest that is subsequently followed by G2 phase checkpoint adaptation, which may potentially promote genomic instability and result in tumorigenesis. Furthermore, we showed that activation of Chk1 contributes to the G2 arrest in GES-1 cells that are treated with ST for 24h and that prolonged treatment of cells with ST for 48h led to a decrease in the total protein and phosphorylation levels of Chk1 in mitotic cells, indicating that checkpoint adaptation may be driven by inactivation of Chk1. Knockdown studies confirmed that cells entered mitosis following inactivation of Chk1. Taken together, we show that ST treatment for 24h activates Chk1 and induces a G2 arrest in GES-1 cells. However, prolonged ST treatment for 48h led to Chk1 inactivation in GES-1 cells, which promotes checkpoint adaptation and entry of cells into mitosis despite damaged DNA. Importantly, checkpoint adaptation in GES-1 cells treated with ST may potentially promote genomic instability and drive tumorigenesis.

DNA damage checkpoint adaptation genes are required for division of cells harbouring eroded telomeres.

In budding yeast, telomerase and the Cdc13p protein are two key players acting to ensure telomere stability. In the absence of telomerase, cells eventually enter a growth arrest which only few can overcome via a conserved process; such cells are called survivors. Survivors rely on homologous recombination-dependent mechanisms for telomeric repeat addition. Previously, we showed that such survivor cells also manage to bypass the loss of the essential Cdc13p protein to give rise to Cdc13-independent (or cap-independent) strains. Here we show that Cdc13-independent cells grow with persistently recognized DNA damage, which does not however result in a checkpoint activation; thus no defect in cell cycle progression is detectable. The absence of checkpoint signalling rather is due to the accumulation of mutations in checkpoint genes such as RAD24 or MEC1. Importantly, our results also show that cells that have lost the ability to adapt to persistent DNA damage, also are very much impaired in generating cap-independent cells. Altogether, these results show that while the capping process can be flexible, it takes a very specific genetic setup to allow a change from canonical capping to alternative capping. We hypothesize that in the alternative capping mode, genome integrity mechanisms are abrogated, which could cause increased mutation frequencies. These results from yeast have clear parallels in transformed human cancer cells and offer deeper insights into processes operating in pre-cancerous human cells that harbour eroded telomeres.

NSCLC cells demonstrate differential mode of cell death in response to the combined treatment of radiation and a DNA-PKcs inhibitor.

The current standard of care for lung cancer consists of concurrent chemotherapy and radiation. Several studies have shown that the DNA-PKcs inhibitor NU7441 is a highly potent radiosensitizer, however, the mechanism of NU7441's anti-proliferation effect has not been fully elucidated. In this study, the combined effect of NU7441 and ionizing radiation (IR) in a panel of non-small cell lung cancer cell lines (A549, H460 and H1299) has been investigated. We found that NU7441 significantly enhances the effect of IR in all cell lines. The notable findings in response to this combined treatment are (i) prolonged delay in IR-induced DNA DSB repair, (ii) induced robust G2/M checkpoint, (iii) increased aberrant mitosis followed by mitotic catastrophe specifically in H1299, (iv) dramatically induced autophagy in A549 and (v) IR-induced senescence specifically in H460. H1299 cells show greater G2 checkpoint adaptation after combined treatment, which can be attributed to higher expression level of Plk1 compared to A549 and H460. The enhanced autophagy after NU7441 treatment in A549 is possibly due to the higher endogenous expression of pS6K compared to H1299 and H460 cells. In conclusion, choice of cell death pathway is dependent on the mutation status and other genetic factors of the cells treated.

The DNA damage response and checkpoint adaptation in Saccharomyces cerevisiae: distinct roles for the replication protein A2 (Rfa2) N-terminus.

In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with “repairable” DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a “last-resort” mechanism for cell survival.

High Nutrient Levels and TORC1 Activity Reduce Cell Viability following Prolonged Telomere Dysfunction and Cell Cycle Arrest

Cells challenged with DNA damage activate checkpoints to arrest the cell cycle and allow time for repair. Successful repair coupled to subsequent checkpoint inactivation is referred to as recovery. When DNA damage cannot be repaired, a choice between permanent arrest and cycling in the presence of damage (checkpoint adaptation) must be made. While permanent arrest jeopardizes future lineages, continued proliferation is associated with the risk of genome instability. We demonstrate that nutritional signaling through target of rapamycin complex 1 (TORC1) influences the outcome of this decision. Rapamycin-mediated TORC1 inhibition prevents checkpoint adaptation via both Cdc5 inactivation and autophagy induction. Preventing adaptation results in increased cell viability and hence proliferative potential. In accordance, the ability of rapamycin to increase longevity is dependent upon the DNA damage checkpoint. The crosstalk between TORC1 and the DNA damage checkpoint may have important implications in terms of therapeutic alternatives for diseases associated with genome instability.

Polo-like kinase-1 in DNA damage response

Polo-like kinase-1 (Plk1) belongs to a family of serine-threonine kinases and plays a critical role in mitotic progression. Plk1 involves in the initiation of mitosis, centrosome maturation, bipolar spindle formation, and cytokinesis, well-reported as traditional functions of Plk1. In this review, we discuss the role of Plk1 during DNA damage response beyond the functions in mitotsis. When DNA is damaged in cells under various stress conditions, the checkpoint mechanism is activated to allow cells to have enough time for repair. When damage is repaired, cells progress continuously their division, which is called checkpoint recovery. If damage is too severe to repair, cells undergo apoptotic pathway. If damage is not completely repaired, cells undergo a process called checkpoint adaptation, and resume cell division cycle with damaged DNA. Plk1 targets and regulates many key factors in the process of damage response, and we deal with these subjects in this review. [BMB Reports 2014; 47(5): 249-255]

Genotoxic Anti-Cancer Agents and Their Relationship to DNA Damage, Mitosis, and Checkpoint Adaptation in Proliferating Cancer Cells

When a human cell detects damaged DNA, it initiates the DNA damage response (DDR) that permits it to repair the damage and avoid transmitting it to daughter cells. Despite this response, changes to the genome occur and some cells, such as proliferating cancer cells, are prone to genome instability. The cellular processes that lead to genomic changes after a genotoxic event are not well understood. Our research focuses on the relationship between genotoxic cancer drugs and checkpoint adaptation, which is the process of mitosis with damaged DNA. We examine the types of DNA damage induced by widely used cancer drugs and describe their effects upon proliferating cancer cells. There is evidence that cell death caused by genotoxic cancer drugs in some cases includes exiting a DNA damage cell cycle arrest and entry into mitosis. Furthermore, some cells are able to survive this process at a time when the genome is most susceptible to change or rearrangement. Checkpoint adaptation is poorly characterised in human cells; we predict that increasing our understanding of this pathway may help to understand genomic instability in cancer cells and provide insight into methods to improve the efficacy of current cancer therapies.

Flexibility could be important

Charles Keller and colleagues have found that a higher expression of PAX3–FOXO1A during G2/M phase is permissive for G2 checkpoint adaptation in rhabdomyosarcoma.

Cell-Cycle Dependent Expression of a Translocation-Mediated Fusion Oncogene Mediates Checkpoint Adaptation in Rhabdomyosarcoma

Rhabdomyosarcoma is the most commonly occurring soft-tissue sarcoma in childhood. Most rhabdomyosarcoma falls into one of two biologically distinct subgroups represented by alveolar or embryonal histology. The alveolar subtype harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. However, tumor cells have heterogeneous expression for the fusion gene. Using a conditional genetic mouse model as well as human tumor cell lines, we show that that Pax3:Foxo1a expression is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation under stress conditions such as irradiation in vitro and in vivo. Pax3:Foxo1a also tolerizes tumor cells to clinically-established chemotherapy agents and emerging molecularly-targeted agents. Thus, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer cells.

Deacetylase Rpd3 Facilitates Checkpoint Adaptation by Preventing Rad53 Overactivation

The DNA damage checkpoint is tightly controlled. After its activation, the checkpoint machinery is inactivated once lesions are repaired or undergoes adaptation if the DNA damage is unable to be repaired. Protein acetylation has been shown to play an important role in DNA damage checkpoint activation. However, the role of acetylation in checkpoint inactivation is unclear. Here we show that histone deacetylase Rpd3-mediated deacetylation of Rad53 plays an important role in checkpoint adaptation. Deletion of Rpd3 or inhibition of its activity impairs adaptation. RPD3 deletion also leads to a higher acetylation level and enhanced kinase activity of Rad53. Replacement of two major acetylation sites of Rad53 with arginine reduces its activity and further suppresses the adaptation defect of rpd3Δ cells, indicating that Rpd3 facilitates adaptation by preventing Rad53 overactivation. Similar to its role in adaptation, deletion of RPD3 or inhibition of its activity also suppressed checkpoint recovery. Altogether, our findings reveal an important role of Rpd3 in promoting checkpoint adaptation via deacetylation and inhibition of Rad53.

Repeated H2O2 exposure drives cell cycle progression in an in vitro model of ulcerative colitis

The production of hydrogen peroxide (H2O2) drives tumourigenesis in ulcerative colitis (UC). Recently, we showed that H2O2 activates DNA damage checkpoints in human colonic epithelial cells (HCEC) through c-Jun N-terminal Kinases (JNK) that induces p21WAF1. Moreover, caspases circumvented the G1/S and intra-S checkpoints, and cells accumulated in G2/M. The latter observation raised the question of whether repeated H2O2 exposures alter JNK activation, thereby promoting a direct passage of cells from G2/M arrest to driven cell cycle progression. Here, we report that increased proliferation of repeatedly H2O2-exposed HCEC cells (C-cell cultures) was associated with (i) increased phospho-p46 JNK, (ii) decreased total JNK and phospho-p54 JNK and (iii) p21WAF1 down-regulation. Altered JNK activation and p21WAF1 down-regulation were accompanied by defects in maintaining G2/M and mitotic spindle checkpoints through adaptation, as well as by apoptosis resistance following H2O2 exposure. This may cause increased proliferation of C-cell cultures, a defining initiating feature in the inflammation-carcinoma pathway in UC. We further suggest that dysregulated JNK activation is attributed to a non-apoptotic function of caspases, causing checkpoint adaptation in C-cell cultures. Additionally, loss of cell-contact inhibition and the overcoming of senescence, hallmarks of cancer, contributed to increased proliferation. Furthermore, there was evidence that p54 JNK inactivation is responsible for loss of cell-contact inhibition. We present a cellular model of UC and suggest a sinusoidal pattern of proliferation, which is triggered by H2O2-induced reactive oxygen species generation, involving an interplay between JNK activation/inactivation, p21WAF1, c-Fos, c-Jun/phospho-c-Jun, ATF2/phospho-ATF2, β-catenin/TCF4-signalling, c-Myc, CDK6 and Cyclin D2, leading to driven cell cycle progression.

Abstract 1747: The Pax3:Foxo1a translocation product is a cell cycle-specific modifier of the alveolar rhabdomyosarcoma tumor phenotype.

Abstract BACKGROUND: Rhabdomyosarcoma is the most common soft-tissue sarcoma in childhood, which falls into one of two biologically distinct subtypes, alveolar (aRMS) or embryonal rhabdomyosarcoma. aRMS harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. MATERIALS AND METHODS: Primary murine aRMS cultures were obtained from the Myf6Cre, Pax3:Foxo1a, p53 conditional mouse aRMS model, in which eYFP is expressed as a second cistron in the targeted Pax3:Foxo1a-ires-eYFP allele. Limited dilution transplantation experiments were performed using Pax3:Foxo1a knockdown by siRNA of eYFP or control aRMS primary cultures. To elucidate the dynamical function of Pax3:Foxo1a, time-lapse confocal microscopy experiments, cell cycle analysis, quantitative RT-PCR, western blotting, immunohistochemistry and mRNA array were performed using murine aRMS primary cell cultures with or without Pax3:Foxo1a knockdown treated by 6 Gy irradiation, or selected for ploidy using hoechst33342 sorted cell cycle-specific cells. RESULTS: Pax3:Foxo1a is not required for tumor repopulating ability in mouse limiting-dilution transplantation experiments. The expression level of Pax3:Foxo1a was discovered to be dynamic and varied during the cell cycle in murine aRMS primary cell cultures and human aRMS cell lines. Pax3:Foxo1a is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation in genome-wide expression analysis and quantitative RT-PCR. Radiation resulted in a higher fraction of DNA breaks amongst mitotic cells (as represented by dual pHH3 positive, H2AX positive cells) under conditions of Pax3:Foxo1a expression compared with knockdown, suggesting that Pax3:Foxo1a facilitates G2/M transit, consistent with checkpoint adaptation. CONCLUSION: We demonstrated that Pax3:Foxo1a does not function in tumor cell maintenance, the expression level of Pax3:Foxo1a is dynamic and varies during the cell cycle, and Pax3:Foxo1a facilitates checkpoint adaptation under stress conditions such as irradiation. Furthermore, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer biology. Citation Format: Ken Kikuchi, Simone Hettmer, M. Imran Aslam, Joel E. Michalek, Wolfram Laub, Brian P. Rubin, Amy J. Wagers, Charles Keller. The Pax3:Foxo1a translocation product is a cell cycle-specific modifier of the alveolar rhabdomyosarcoma tumor phenotype. [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 1747. doi:10.1158/1538-7445.AM2013-1747

Tid1/Rdh54 translocase is phosphorylated through a Mec1- and Rad53-dependent manner in the presence of DSB lesions in budding yeast

Saccharomyces cerevisiae cells with a single double-strand break (DSB) activate the ATR/Mec1-dependent checkpoint response as a consequence of extensive ssDNA accumulation. The recombination factor Tid1/Rdh54, a member of the Swi2-like family proteins, has an ATPase activity and may contribute to the remodelling of nucleosomes on DNA. Tid1 dislocates Rad51 recombinase from dsDNA, can unwind and supercoil DNA filaments, and has been implicated in checkpoint adaptation from a G2/M arrest induced by an unrepaired DSB.Here we show that both ATR/Mec1 and Chk2/Rad53 kinases are implicated in the phosphorylation of Tid1 in the presence of DNA damage, indicating that the protein is regulated during the DNA damage response. We show that Tid1 ATPase activity is dispensable for its phosphorylation and for its recruitment near a DSB, but it is required to switch off Rad53 activation and for checkpoint adaptation. Mec1 and Rad53 kinases, together with Rad51 recombinase, are also implicated in the hyper-phosphorylation of the ATPase defective Tid1-K318R variant and in the efficient binding of the protein to the DSB site.In summary, Tid1 is a novel target of the DNA damage checkpoint pathway that is also involved in checkpoint adaptation.