Resume Cell Cycle Progression Research Articles

Autorepression-Based Conditional Gene Expression System in Yeast for Variation-Suppressed Control of Protein Dosage.

Conditional control of gene expression allows an experimenter to investigate many aspects of a gene's function. In the model organism Saccharomyces cerevisiae, a number of methods to control gene expression are widely practiced, including induction by metabolites, small molecules, and even light. However, all current methods suffer from at least one of a set of drawbacks, including need for specialized growth conditions, leaky expression, or requirement of specialized equipment. Here we describe protocols using two transformations to construct strains that carry a new controller in which all these drawbacks are overcome. In these strains, the expression of a controlled gene of interest is repressed by the bacterial repressor TetR and induced by anhydrotetracycline. TetR also regulates its own expression, creating an autorepression loop. This autorepression allows tight control of gene expression and protein dosage with low cell-to-cell variation in expression. A second repressor, TetR-Tup1, prevents any leaky expression. We also present a protocol showing a particular workhorse application of such strains to generate synchronized cell populations. We turn off expression of the cell cycle regulator CDC20 completely, arresting the cell population, and then we turn it back on so that the synchronized cells resume cell cycle progression. This control system can be applied to any endogenous or exogenous gene for precise expression. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Generating a parent WTC846 strain Basic Protocol 2: Generating a WTC846 strain with controlled expression of the targeted gene Alternate Protocol: CRISPR-mediated promoter replacement Basic Protocol 3: Cell cycle synchronization/arrest and release using the WTC846- K3 ::CDC20 strain.

Saikosaponin A enhances Docetaxel efficacy by selectively inducing death of dormant prostate cancer cells through excessive autophagy

Quiescent cancer cells (QCCs), also known as dormant cancer cells, resist and survive chemo- and radiotherapy, resulting in treatment failure and later cancer recurrence when QCCs resume cell cycle progression. However, drugs selectively targeting QCCs are lacking. Saikosaponin A (SSA) derived from Bupleurum DC., is highly potent in eradicating multidrug-resistant prostate QCCs compared with proliferative prostate cancer cells. By further exacerbating the already increased autophagy through inactivation of Akt-mTOR signaling, SSA triggered cell death in QCCs. Contrarily, inhibition of autophagy or activation of Akt signaling pathway prevented SSA-induced cell death. The multicycle of Docetaxel treatments increased the proportion of QCCs, whereas administering SSA at intervals of Docetaxel treatments aggravated cell death in vitro and led to tumor growth arrest and cell death in vivo. In conclusion, SSA is posed as a novel QCCs-eradicating agent by aggravating autophagy in QCCs. In combination with the current therapy, SSA has potential to improve treatment effectiveness and to prevent cancer recurrence.

DNA damage checkpoint execution and the rules of its disengagement.

Chromosomes are susceptible to damage during their duplication and segregation or when exposed to genotoxic stresses. Left uncorrected, these lesions can result in genomic instability, leading to cells’ diminished fitness, unbridled proliferation or death. To prevent such fates, checkpoint controls transiently halt cell cycle progression to allow time for the implementation of corrective measures. Prominent among these is the DNA damage checkpoint which operates at G2/M transition to ensure that cells with damaged chromosomes do not enter the mitotic phase. The execution and maintenance of cell cycle arrest are essential aspects of G2/M checkpoint and have been studied in detail. Equally critical is cells’ ability to switch-off the checkpoint controls after a successful completion of corrective actions and to recommence cell cycle progression. Interestingly, when corrective measures fail, cells can mount an unusual cellular response, termed adaptation, where they escape checkpoint arrest and resume cell cycle progression with damaged chromosomes at the cost of genome instability or even death. Here, we discuss the DNA damage checkpoint, the mitotic networks it inhibits to prevent segregation of damaged chromosomes and the strategies cells employ to quench the checkpoint controls to override the G2/M arrest.

Acute Myeloid Leukaemia in Elderly: Expression Analysis of DNA Damage Response (DDR) and Anti-Apoptosis Genes Identify Novel Targets for Potential “Synergistic Lethality”

Background: Genetic instability and heterogeneity are fundamental to AML biology and pivotal to its prognosis. Acute myeloid leukaemia (AML) is most frequent in older adults (>60 years) and a significant majority are considered "treatment-naïve" given medical comorbidities, high-risk disease biology, and poor tolerance to chemotherapy. These patients receive low-intensity regimens, mostly hypomethylating agents due to impaired tolerance to induction chemotherapy, resulting in low overall survival. Thus, there is a critical need to develop targeted therapies capable of rapidly inducing a high rate of clinical response, with better tolerability and durable responses for these elderly AML patients. DNA damage response (DDR) is a specialized and highly orchestrated signalling cascade to maintain genetic stability and is closely linked to the cell cycle. The cell cycle checkpoints result in arrest and the resumption of cell cycle progression when DNA damage has been repaired. However, the DNA repair failure will direct cells towards cellular senescence or apoptosis. Hence, DDR and apoptosis are intricately related physiological processes. In AML, mutations in key regulators of gene expression and/or chromatin structure, such as p53, K-RAS, and isocitrate dehydrogenase 1 and 2 (IDH1/2) results in defective DDR. Defects in DDR are also age-related; therefore, resulting in an exponential increase in the incidence of cancer with age. The emerging treatment regimens for elderly AML are focusing on synergistic lethality using a combination of DDR inhibitors (PARP1) and/ or anti-apoptotic inhibitors (BCL2) in combination with low dose anthracycline and additional targeted therapies like (IDH2/DNTM etc.). However, the wider implication and influence of DDR genes either alone or in combination with anti-apoptotic genes remains uncharacterized in elderly AML. In this pilot study, we screened a large cohort of AML samples for mRNA expression for a series of DDR and apoptotic genes to investigate the age-related variation in expression of these molecules. We observed distinct differences in DDR and anti-apoptotic gene expression between elderly AML compared to paediatric patients. We anticipate, that these preliminary observations will pave the pathway for future comprehensive studies to expand the employment of synergistic lethality strategy for elderly AML patients. Methods: We employed RNA extracted from formalin-fixed paraffin-embedded (FFPE) diagnostic tissuesamples in 100 AML patients. Age defined the categorization of patients into three groups; a, <18 years (n=34); b,18-60 years (n=32) and c, >60 years (n=34). nCounter (NanoString Technologies) platform was used for the quantification of mRNA. Qlucore Omics Explorer software was employed with defined criteria (fold change >2.0; p<0.01 and q <0.05) for statistical analysis. Results:We noted that several DDR mediators (BRIP1, POLD1, XRCC4) are differentially up-regulated in the AML samples linked with elderly patient group, alongside anti-apoptotic genes (IDH2, IKBKB) when compared with samples from patient <18 years (Figure 1). Moreover, DDR effectors (BRCA1& ATM) were up-regulated in the elderly cohort relative to paediatric AML samples, suggesting potential targets for treating elderly AML (Figure2). Conclusions:This pilot study identifies several additional and novel DDR targets; which can be exploited to enhance chemotherapeutic efficacy in "treatment fit" as well as "treatment naïve" elderly AML patients. Besides, we also report numerous novel anti-apoptotic molecules up-regulated in elderly AML. Taken together the preliminary data, presented here, expand the repertoire for targeted therapy in elderly AML with a specific focus on synergistic lethality. Disclosures No relevant conflicts of interest to declare.

Systems biology approach reveals a link between mTORC1 and G2/M DNA damage checkpoint recovery

Checkpoint recovery, the process that checkpoint-arrested cells with normal DNA repair capacity resume cell cycle progression, is essential for genome stability. However, the signaling network of the process has not been clearly defined. Here, we combine functional proteomics, mathematical modeling, and molecular biology to identify mTORC1, the nutrient signaling integrator, as the determinant for G2/M checkpoint recovery. Inhibition of the mTORC1 pathway delays mitotic entry after DNA damage through KDM4B-mediated regulation of CCNB1 and PLK1 transcription. Cells with hyper-mTORC1 activity caused by TSC2 depletion exhibit accelerated G2/M checkpoint recovery. Those Tsc2-null cells are sensitive to WEE1 inhibition in vitro and in vivo by driving unscheduled mitotic entry and inducing mitotic catastrophe. These results reveal that mTORC1 functions as a mediator between nutrition availability sensing and cell fate determination after DNA damage, suggesting that checkpoint inhibitors may be used to treat mTORC1-hyperactivated tumors such as those associated with tuberous sclerosis complex.

Dynamic regulation of Cdr1 kinase localization and phosphorylation during osmotic stress

Environmental conditions modulate cell cycle progression in many cell types. A key component of the eukaryotic cell cycle is the protein kinase Wee1, which inhibits the cyclin-dependent kinase Cdk1 in yeast through human cells. In the fission yeast Schizosaccharomyces pombe, the protein kinase Cdr1 is a mitotic inducer that promotes mitotic entry by phosphorylating and inhibiting Wee1. Cdr1 and Wee1 both localize to punctate structures, termed nodes, on the medial cortex, but it has been unknown whether node localization can be altered by physiological signals. Here we investigated how environmental conditions regulate Cdr1 signaling for cell division. Osmotic stress induced hyperphosphorylation of the mitotic inducer Cdr1 for several hours, and cells delayed division for the same time period. This stress-induced hyperphosphorylation required both Cdr1 autophosphorylation and the stress-activated protein kinase Sty1. During osmotic stress, Cdr1 exited cortical nodes and localized in the cytoplasm. Using a series of truncation mutants, we mapped a C-terminal domain that is necessary and sufficient for Cdr1 node localization and found that Sty1 directly phosphorylates this domain in vitro Sty1 was not required for Cdr1 exit from nodes, indicating the existence of additional regulatory signals. Both Cdr1 phosphorylation and node localization returned to basal levels when cells adapted to osmotic conditions and resumed cell cycle progression. In summary, we identified a mechanism that prevents Cdr1 colocalization with its inhibitory target Wee1 during osmotic stress. Dynamic regulation of protein localization to cortical nodes might represent a strategy to modulate entry into mitosis under differing environmental conditions.

SKL2001 suppresses colon cancer spheroid growth through regulation of the E-cadherin/β-Catenin complex

Aberrant activation of Wnt signaling plays a pivotal role in the development of human cancers including colon cancer. Small compounds that regulate Wnt signaling are attractive candidate for the colon cancer therapy. Here, we showed that SKL2001, which has been identified as an activator for Wnt signaling by disrupting the Axin/β-Catenin complex, negatively regulates growth of colon cancer spheroids cultured in the 3D condition that simulates tumor microenvironment in vivo. SKL2001 inhibited proliferation of colon cancer cells cultured in 3D spheroid and induced them accumulation in the G0/G1 phase of the cell cycle with a reduced c-myc level. To examine the potential of arrested cells to recover, colon cancer spheroids that were treated with SKL2001 were then cultured in the SKL2001-free medium. We found that SKL2001-treated cells were resumed cell cycle progression and proliferated in the SKL2001-free medium. Notably, SKL2001 facilitated round-shape spheroid formation. This was associated with upregulated expressions of E-cadherin and β-Catenin. These findings suggest that SKL2001 can suppress colon cancer spheroid growth through regulating cell cycle progression and cadherin/catenin mediated cell-cell contact.

Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair.

To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair.

Abstract 578: Plk1 decreases DNA double-strand break (DSB) formation during replication-stalling genotoxic stress.

Abstract Polo-like kinase 1 (Plk1) is a key regulator of mitosis and its over-expression/ deregulation is linked to cellular transformation. Recent studies highlight its role in resumption of cell cycle progression after DNA damage. Plk1 has also been shown to enhance DNA replication under stress of stalled replication forks. We reported Plk1 activation-induced G2/M checkpoint bypass in normal human lung cells (HLFs), in the presence of acute genotoxic stress. The present aim was to determine the effect of Plk1 on DNA double strand breaks (DSBs) during genotoxic stress. In the present study, we measured DNA DSB induction in HLFs by Cr(VI), etoposide, and H2O2 after Plk1 activation using the comet assay and γH2AX immunofluorescence staining. We used equitoxic concentrations of the different genotoxins and found that DNA DSBs increased > 2 fold after exposure to 3 μM Cr(VI), 12.5 μM etoposide, and 150 μM H2O2, respectively, and persisted for at least 4h. Transfection of a Plk1 constitutively active (c/a) mutant (T210D) abrogated both Cr(VI)- and etoposide-induced DNA DSBs as early as 30 min for the duration of treatment, in comparison to the vector-transfected cells. In sharp contrast, Plk1 had no effect on H2O2-induced DNA DSBs. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Moreover, Plk1 enhanced S phase progression during Cr(VI) treatment, which was accompanied by increased replication fork velocity and longer inter-origin distances as determined by using a single-molecule approach based on molecular combing. Expression of a Plk1 “kinase-dead” mutant (K82R) also abrogated DNA DSBs during Cr(VI) treatment, suggesting that this effect may be independent of Plk1 kinase activity. In summary, our data underline the ability of Plk1 to override DNA DSB-induced checkpoints, and to decrease DSBs after replication-stalling genotoxin exposure. We postulate these effects to be due to the replication-enhancing ability of Plk1, which may occur at the expense of genomic stability. This work was supported in part by NIH grants CA107972 and ES017334 (S.C.); ES05304 (S.R.P.); and a PhRMA Foundation predoctoral fellowship (G.C.K.). Citation Format: Daniel Gebreselassie, Gina Chun-Kost, Elisabetta Leo, Yves Pommier, Erika Saito, Steven Patierno, Susan M. Ceryak. Plk1 decreases DNA double-strand break (DSB) formation during replication-stalling genotoxic stress. [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 578. doi:10.1158/1538-7445.AM2013-578

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

The DNA damage checkpoint triggers autophagy to regulate the initiation of anaphase

Budding yeast cells suffering a single, unrepaired DNA double strand break (DSB) arrest prior to anaphase for 12–15 h, in a Mec1 (ATR)‐dependent manner, following which checkpoint signaling is down‐regulated to allow resumption of cell cycle progression in a phenomenon known as adaptation. A similar, but not genetically equivalent process of recovery occurs after the DSB has been repaired. In this study, we found that inducing autophagy in wild‐type cells suffering DNA damage, either by the addition of rapamycin, by overexpression of a dominant‐active version of Atg13 (ATG13–8SA) or by deletion of genes required for Golgi‐Associated Retrograde Transport (GARP) prevented both adaptation and recovery. These effects were due to the DNA damage‐dependent mislocalization of the mitotic activator Esp1 (Separase) via the degradation of its chaperone/inhibitor Pds1 (Securin). Failure to adapt is dependent on vacuolar proteases and autophagy, as these defects were suppressed by 1) deleting the vacuolar protease genes or by 2) deleting Autophagy related genes (specifically those required for the Cytoplasm‐to‐Vacuole transport (CVT) pathway). Overexpression of ATG13–8SA also promoted the mislocalization of Pds1 and Esp1 after induction of a DSB. Arrest persisted even as Rad53 (Chk2) hyperphosphorylation diminished after 12–15 h. Finally we note that ATG13–8SA overexpressing cells were sensitive to a wide‐array of DNA damaging agents and autophagy mutants displayed a reduced duration of checkpoint arrest. Overall, our results suggest a role for DNA damage‐induced autophagy in maintaining robust checkpoint arrest by targeting mitotic activators for degradation and specifically implicate the CVT pathway as a target of the DNA damage checkpoint.

The Saccharomyces cerevisiae Chromatin Remodeler Fun30 Regulates DNA End Resection and Checkpoint Deactivation

Fun30 is a Swi2/Snf2 homolog in budding yeast that has been shown to remodel chromatin both in vitro and in vivo. We report that Fun30 plays a key role in homologous recombination, by facilitating 5'-to-3' resection of double-strand break (DSB) ends, apparently by facilitating exonuclease digestion of nucleosome-bound DNA adjacent to the DSB. Fun30 is recruited to an HO endonuclease-induced DSB and acts in both the Exo1-dependent and Sgs1-dependent resection pathways. Deletion of FUN30 slows the rate of 5'-to-3' resection from 4 kb/h to about 1.2 kb/h. We also found that the resection rate is reduced by DNA damage-induced phosphorylation of histone H2A-S129 (γ-H2AX) and that Fun30 interacts preferentially with nucleosomes in which H2A-S129 is not phosphorylated. Fun30 is not required for later steps in homologous recombination. Like its homolog Rdh54/Tid1, Fun30 is required to allow the adaptation of DNA damage checkpoint-arrested cells with an unrepaired DSB to resume cell cycle progression.

Whole transcriptome analysis of the silicon response of the diatom Thalassiosira pseudonana.

BackgroundSilicon plays important biological roles, but the mechanisms of cellular responses to silicon are poorly understood. We report the first analysis of cell cycle arrest and recovery from silicon starvation in the diatom Thalassiosira pseudonana using whole genome microarrays.ResultsThree known responses to silicon were examined, 1) silicified cell wall synthesis, 2) recovery from silicon starvation, and 3) co-regulation with silicon transporter (SIT) genes. In terms of diatom cell wall formation, thus far only cell surface proteins and proteins tightly associated with silica have been characterized. Our analysis has identified new genes potentially involved in silica formation, and other genes potentially involved in signaling, trafficking, protein degradation, glycosylation and transport, which provides a larger-scale picture of the processes involved. During silicon starvation, an overrepresentation of transcription and translation related genes were up-regulated, indicating that T. pseudonana is poised to rapidly recover from silicon starvation and resume cell cycle progression upon silicon replenishment. This is in contrast to other types of limitation, and provides the first molecular data explaining the well-established environmental response of diatoms to grow as blooms and to out-compete other classes of microalgae for growth. Comparison of our data with a previous diatom cell cycle analysis indicates that assignment of the cell cycle specific stage of particular cyclins and cyclin dependent kinases should be re-evaluated. Finally, genes co-varying in expression with the SITs enabled identification of a new class of diatom-specific proteins containing a unique domain, and a putative silicon efflux protein.ConclusionsAnalysis of the T. pseudonana microarray data has provided a wealth of new genes to investigate previously uncharacterized cellular phenomenon related to silicon metabolism, silicon’s interaction with cellular components, and environmental responses to silicon.

Antagonism between curcumin and the topoisomerase II inhibitor etoposide

The use of combinations of chemotherapy and natural products has recently emerged as a new method of cancer therapy, relying on the capacity of certain natural compounds to trigger cell death with low doses of chemotherapeutic agents and few side effects. The current study aims to evaluate the modulatory effects of curcumin (CUR), Nigella sativa (NS) and taurine on etoposide (ETP) cytotoxicity in a panel of cancer cell lines and to identify their underlying mechanisms. CUR alone showed potent antitumor activity, but surprisingly, its interaction with ETP was antagonistic in four out of five cancer cell lines. Neither taurine nor Nigella sativa affect the sensitivity of cancer cells to ETP. Examination of the DNA damage response machinery (DDR) showed that both ETP and CUR elicited DNA double-strand breaks (DSB) and evoked γ-H2AX foci formation at doses as low as 1 µg/ml. Cell cycle analysis revealed S phase arrest after ETP or CUR application, whereas co-treatment with ETP and CUR led to increased arrest of the cell cycle in S phase (MCF-7 cells) or the accumulation of cells in G2/M phases (HCT116, and HeLa cells). Furthermore, cotreatment with ETP and CUR resulted in modulation of the level of DNA damage induction and repair compared with either agent alone. Electron microscopic examination demonstrated that different modalities of cell death occurred with each treatment. CUR alone induced autophagy, apoptosis and necrosis, whereas ETP alone or in combination with CUR led to apoptosis and necrosis. Conclusions: Cotreatment with ETP and CUR resulted in an antagonistic interaction. This antagonism is related, in part, to the enhanced arrest of tumor cells in both S and G2/M phases, which prevents the cells from entering M-phase with damaged DNA and, consequently, prevents cell death from occurring. This arrest allows time for the cells to repair DNA damage so that cell cycle -arrested cells can eventually resume cell cycle progression and continue their physiological program.

Abstract 2131: Plk1 decreases DNA double strand break (DSB) formation during replication- stalling genotoxic stress

Abstract Polo-like kinase 1 (Plk1) is a regulator of the mitotic checkpoint, and is involved in adaptation, which is the progression of cells into mitosis with damaged DNA. Deregulation of Plk1 activity is linked to cellular transformation. Recent studies suggest Plk1 activity to be critical for resumption of cell cycle progression during recovery from DNA damage. However, the ability of Plk1 to affect the DNA damage response remains relatively unclear. The aim of the present study was to determine the ability of Plk1 to affect the DNA damage response in the face of different forms of DSB-inducing genotoxic stress. We employed the respiratory carcinogen, hexavalent chromium [Cr(VI)], the anticancer drug and topoisomerase II inhibitor, etoposide, as well as the oxidizer, H2O2. We have previously shown that Plk1 activation was sufficient to bypass the G2/M checkpoint in normal human lung cells (HLFs), in the presence of Cr(VI)-induced acute genotoxic stress. In the present study, we measured DNA DSB induction in HLFs by the respective genotoxins under the condition of Plk1 activation by using the comet assay and γH2AX immunofluorescence staining. Transfection of HLFs with the constitutively active (c/a) Plk1 T210D mutant resulted in increased Plk1 protein expression 24h-48h post transfection. We used equitoxic concentrations of the different genotoxins and found that DNA DSBs increased > 2 fold after exposure to 3 μM Cr(VI), 12.5 μM etoposide, and 150 μM H2O2, respectively, and persisted for at least 4h. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Expression of the c/a T210D mutant increased Plk1 activity in vitro, and abrogated both Cr(VI) and etoposide-induced DNA DSBs as early as 30 min, and up to 4h post-treatment, in comparison to the vector control-transfected cells. In sharp contrast, treatment with 150 μM H2O2 induced a similar level of DNA DSBs in both the c/a T210D mutant- and vector-transfected cells. These data highlight the ability of Plk1 to decrease DNA DSBs uniquely under conditions of replication-stalling genotoxic stress. Given the documented roles of aberrant DNA damage response and Plk1 activation in cellular transformation, there is a critical need to define the molecular mechanism(s) by which Plk1 mediates decreased DNA DSBs after genotoxin exposure and delineate the pathways responsible for Plk1 activation. We postulate that the ability of Plk1 activation to abrogate DNA DSB formation after either Cr(VI) or etoposide exposure may occur at the expense of genomic stability. Supported by NIH grants CA107972 and ES017334 to SC and ES09961 and ES05304 to SRP and PhRMA foundation to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2131. doi:1538-7445.AM2012-2131

Staging a recovery from mitotic arrest

Checkpoint controls, the surveillance pathways that impose "an order of execution" on the major cell cycle events, are critical to the maintenance of genome stability. When cells fail to execute a cellular event or do so erroneously due to misregulation or exposure to genotoxic stresses, these evolutionarily conserved regulatory circuits prevent passage to the subsequent event, thus bringing the cell cycle to a halt. Once the checkpoint stimulus is removed, cells recover from the arrest and eventually resume cell cycle progression. While the activation, execution and maintenance, the three major aspects of the checkpoint controls, have been investigated in detail, the recovery process remains underexplored. It is not clear if cells recover passively upon dissipation of the checkpoint signals or require an active participation by specific effectors. A recent study in the yeast Saccharomyces cerevisiae uncovered two previously unsuspected functions of Cdk1 in efficient recovery from the spindle assembly checkpoint (SAC) imposed arrest. An inability to fulfil these requirements in the absence of Cdk1 makes it virtually impossible for cells to recover from the mitotic arrest. Given the conserved nature of the SAC, these findings may have implications for vertebrate cells.

Abstract 3914: Plk1 enhances DNA double strand break (DSB) repair during acute genotoxin exposure

Abstract Polo-like kinase 1 (Plk1) is a regulator of the mitotic checkpoint, and is involved in adaptation, which is the progression of cells into mitosis with damaged DNA. Deregulation of Plk1 activity is linked to cellular transformation. Recent studies suggest Plk1 activity to be critical for resumption of cell cycle progression in cancer cells during recovery from DNA damage. However, the ability of Plk1 to affect the DNA damage response remains relatively unclear. In this study we employed the respiratory carcinogen, hexavalent chromium [Cr(VI)], which is a well-documented genotoxin of occupational and environmental concern. We have previously shown that Plk1 activation was sufficient to bypass the G2/M checkpoint in normal human lung cells (HLFs), in the presence of Cr(VI)-induced acute genotoxic stress. In the present study we measured DNA DSB induction by Cr(VI) in HLFs under the condition of Plk1 activation by using the comet assay and gamma H2AX immunofluorescence staining. Transfection of HLFs with the constitutively active (c/a) Plk1 T210D mutant resulted in increased Plk1 protein expression 24h-48h post transfection. We found that DNA DSBs increased > 2 fold 15 min after 3 μM Cr(VI) exposure, and persisted for at least 4h. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Expression of the c/a T210D mutant increased Plk1 activity in vitro, and abrogated Cr(VI)-induced DNA DSBs as early as 30 min, and up to 4h post-treatment, in comparison to the vector control transfected cells. Since Cr(VI)-induced DNA DSBs were equally formed by 15 min in both vector- and Plk1 T210D-transfected cells, these data highlight the heretofore unreported ability of Plk1 to enhance DNA repair. Our previous report found that Plk1 expression increased survival and mutagenesis of wt S. cerevisiae treated with Cr(VI). Therefore, we also determined the ability of Plk1 to “rescue” repair deficient rad52 yeast from Cr(VI)-induced lethality. S. cerevisae transformed with Plk1-Gal-HA-EGFP plasmid grown under inducible conditions overexpress Plk1. We found that induction of Plk1 in rad52 yeast significantly protected them from Cr(VI) clonogenic lethality. Given the documented roles of aberrant DNA damage response and Plk1 activation in cellular transformation, there is a critical need to define the molecular mechanism(s) by which Plk1 mediates DNA damage repair after genotoxin exposure and delineate the pathways responsible for Plk1 activation. We postulate that the ability of Plk1 activation to provoke DNA DSB repair after Cr(VI) exposure occurs at the expense of genomic stability. Supported by NIH grants CA107972 to SC and ES09961 and ES05304 to SRP and PhRMA foundation to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3914. doi:10.1158/1538-7445.AM2011-3914

Protein Phosphatases Pph3, Ptc2, and Ptc3 Play Redundant Roles in DNA Double-Strand Break Repair by Homologous Recombination

In response to a DNA double-strand break (DSB), cells undergo a transient cell cycle arrest prior to mitosis until the break is repaired. In budding yeast (Saccharomyces cerevisiae), the DNA damage checkpoint is regulated by a signaling cascade of protein kinases, including Mec1 and Rad53. When DSB repair is complete, cells resume cell cycle progression (a process called "recovery") by turning off the checkpoint. Recovery involves two members of the protein phosphatase 2C (PP2C) family, Ptc2 and Ptc3, as well as the protein phosphatase 4 (PP4) enzyme, Pph3. Here, we demonstrate a new function of these three phosphatases in DSB repair. Cells lacking all three phosphatases Pph3, Ptc2, and Ptc3 exhibit synergistic sensitivities to the DNA-damaging agents camptothecin and methyl methanesulfonate, as well as hydroxyurea but not to UV light. Moreover, the simultaneous absence of Pph3, Ptc2, and Ptc3 results in defects in completing DSB repair, whereas neither single nor double deletion of the phosphatases causes a repair defect. Specifically, cells lacking all three phosphatases are defective in the repair-mediated DNA synthesis. Interestingly, the repair defect caused by the triple deletion of Pph3, Ptc2, and Ptc3 is most prominent when a DSB is slowly repaired and the DNA damage checkpoint is fully activated.

Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.

Mechanisms of ATR-mediated checkpoint signalling

Cell cycle checkpoints maintain genomic integrity by delaying cell division in the presence of DNA damage or replication problems. A crucial player in this process is the ATR kinase. The rapid localisation of ATR to sites of genotoxic stress and the central role of this kinase in the checkpoint response lead to the suggestion that ATR functions as a sensor of DNA lesions. After activation, ATR phosphorylates and activates the effector kinase Chk1, thereby causing an inhibition in cell cycle progression. However, this would not be possible without the existence of many other proteins operating in this pathway. Here we review current progress in our understanding of the regulatory factors involved in the ATR-mediated checkpoint response, as well as resumption of cell cycle progression upon repair of the damage, thereby focussing on the mechanisms in mammalian cells.