Cell Cycle Checkpoint Responses Research Articles

Senescence-associated secretory phenotype and its possible role in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a major disease of the lungs. It primarily occurs after a prolonged period of cigarette smoking. Chronic inflammation of airways and the alveolar space as well as lung tissue destruction are the hallmarks of COPD. Recently it has been shown that cellular senescence might play a role in the pathogenesis of COPD. Cellular senescence comprises signal transduction program, leading to irreversible cell cycle arrest. The growth arrest in senescence can be triggered by many different mechanisms, including DNA damage and its recognition by cellular sensors, leading to the activation of cell cycle checkpoint responses and activation of DNA repair machinery. Senescence can be induced by several genotoxic factors apart from telomere attrition. When senescence induction is based on DNA damage, senescent cells display a unique phenotype, which has been termed "senescence-associated secretory phenotype" (SASP). SASP may be an important driver of chronic inflammation and therefore may be part of a vicious cycle of inflammation, DNA damage, and senescence. This research perspective aims to showcase cellular senescence with relevance to COPD and the striking similarities between the mediators and secretory phenotype in COPD and SASP.

TheArabidopsisSIAMESE-RELATED Cyclin-Dependent Kinase Inhibitors SMR5 and SMR7 Regulate the DNA Damage Checkpoint in Response to Reactive Oxygen Species

Whereas our knowledge about the diverse pathways aiding DNA repair upon genome damage is steadily increasing, little is known about the molecular players that adjust the plant cell cycle in response to DNA stress. By a meta-analysis of DNA stress microarray data sets, three family members of the SIAMESE/SIAMESE-RELATED (SIM/SMR) class of cyclin-dependent kinase inhibitors were discovered that react strongly to genotoxicity. Transcriptional reporter constructs corroborated specific and strong activation of the three SIM/SMR genes in the meristems upon DNA stress, whereas overexpression analysis confirmed their cell cycle inhibitory potential. In agreement with being checkpoint regulators, SMR5 and SMR7 knockout plants displayed an impaired checkpoint in leaf cells upon treatment with the replication inhibitory drug hydroxyurea (HU). Surprisingly, HU-induced SMR5/SMR7 expression depends on ATAXIA TELANGIECTASIA MUTATED (ATM) and SUPPRESSOR OF GAMMA RESPONSE1, rather than on the anticipated replication stress-activated ATM AND RAD3-RELATED kinase. This apparent discrepancy was explained by demonstrating that, in addition to its effect on replication, HU triggers the formation of reactive oxygen species (ROS). ROS-dependent transcriptional activation of the SMR genes was confirmed by different ROS-inducing conditions, including high-light treatment. We conclude that the identified SMR genes are part of a signaling cascade that induces a cell cycle checkpoint in response to ROS-induced DNA damage.

Analyzing cell cycle checkpoints in response to ionizing radiation in mammalian cells.

Exposure of cells to DNA-damaging agents, such as ionizing radiation (IR), results in perturbation of cell cycle progression. IR activates cell cycle checkpoints that arrest the cell cycle at the G1/S, S, and G2/M phases. The DNA damage-signaling network involves a number of important DNA damage response factors that are required for maintaining genome stability and prevention of cancer. These factors are involved in the regulation of cell cycle checkpoints and include ATM, NBS1, BRCA1, Chk2, and p53. Here we describe a series of assays that are often used to analyze cell cycle checkpoints after IR. These assays include a G1/S checkpoint assay that measures 5-bromodeoxyuridine incorporation into DNA, an S-phase checkpoint assay that measures DNA synthesis at a very early time point after IR, and a G2/M checkpoint assay that quantitates histone H3 phosphorylation. This collection of assays allows us to investigate the specific functions of proteins involved in regulating different cell cycle checkpoints in mammalian cells as a response to IR.

RAD51B Activity and Cell Cycle Regulation in Response to DNA Damage in Breast Cancer Cell Lines.

Common genetic variants mapping to two distinct regions of RAD51B, a paralog of RAD51, have been associated with breast cancer risk in genome-wide association studies (GWAS). RAD51B is a plausible candidate gene because of its established role in the homologous recombination (HR) process. How germline genetic variation in RAD51B confers susceptibility to breast cancer is not well understood. Here, we investigate the molecular function of RAD51B in breast cancer cell lines by knocking down RAD51B expression by small interfering RNA and treating cells with DNA-damaging agents, namely cisplatin, hydroxyurea, or methyl-methanesulfonate. Our results show that RAD51B-depleted breast cancer cells have increased sensitivity to DNA damage, reduced efficiency of HR, and altered cell cycle checkpoint responses. The influence of RAD51B on the cell cycle checkpoint is independent of its role in HR and further studies are required to determine whether these functions can explain the RAD51B breast cancer susceptibility alleles.

Abstract P3-04-04: Amplification and overexpression of TLK2 promote genomic instability in invasive breast cancer

Abstract The accumulation of numerous chromosomal aberrations is a hallmark characteristic of invasive breast cancer (IBC). It is thus critical to find specific chromosomal aberrations that may drive breast cancer initiation and progression, particularly those that are drivers of the genomic instability. By integrative analysis of genomic data from The Cancer Genome Atlas (TCGA), we have nominated Tousled-Like Kinase 2 (TLK2) as a candidate oncogene target for IBC. Ultra-high resolution copy-number data from TCGA suggest that TLK2 was amplified in approximately 10% of IBC, which are more frequent in luminal B than luminal A tumors. These data also revealed that TLK2-high breast tumors harbor significantly more global copy-number aberrations than the TLK2 low-tumors. TLK2 is a nuclear serine/threonine kinase that serves as a cell cycle checkpoint regulating chromatin assembly, and in response to DNA damage, TLK2 can be inactivated by phosphorylation through the ATM-dependent pathway. We thus postulated that up-regulation of TLK2 may help cancer cells to surpass the cell cycle checkpoint upon DNA damage, which may promote genomic instability in breast cancer cells. To analyze the effect of TLK2 overexpression, we introduced an inducible expression vector containing the TLK2 open-reading frame into the MCF10A benign epithelial cells and the T47D breast cancer cells. As shown by fluorescence activated cell sorting (FACS), ectopic expression of TLK2 in these cells overcame the cell cycle checkpoint in response to the DNA damage induced by gamma irradiation. gamma-H2AX foci formation revealed increased DNA double strand breaks and prolonged DNA repair window after irradiation in the TLK2 overexpressing cells. Interestingly, immunofluorescence localized TLK2 to the nucleus of breast cancer cells, which was markedly enriched in DNA double-strand break foci (or gamma-H2AX foci). To examine the effect of TLK2 knockdown, the MCF7 and MDAMB361 breast cancer cells overexpressing TLK2 were transfected with different TLK2 siRNAs. TLK2 inhibition led to G1/S cell cycle arrest (FACS analysis), profound decrease in cell growth (MTT assay), and enhanced apoptosis (Annexin V assay) in the MCF7 and MDAMB361 breast cancer cells over-expressing TLK2. Together, this study supports the role of TLK2 deregulation in the increased genomic instability of TLK2-high breast cancers, and suggests its potential as an attractive therapeutic target. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-04.

The Wee1 Inhibitor MK1775 In Combination With Cytarabine (AraC) Has Potent Activity In AML By Completely Abrogating DNA Damage and Cell Cycle Checkpoint Repair Capacity Via The Mrn/NBS1 Complex

Cytarabine (AraC) resistance is a fundamental feature of refractory/relapsed AML. RNA interference (RNAi) screens conducted in our laboratory recently identified WEE1 kinase (WEE1) as one of the top candidate genes and target in leukemias in combination with AraC. WEE1 is a tyrosine kinase belonging to the Ser/Thr family of protein kinases and acts as a negative regulator of mitotic entry by controlling DNA damage (DDR) and cell cycle checkpoint responses. The WEE1 inhibitor MK1775 potently synergizes with AraC ex vivo and in vitro and clinical trials are in preparation. However, the mechanism of action for the anti-leukemic activity of MK1775 with AraC remains unknown. To elucidate genes mediating activity of the combination, we first performed siRNA rescue screens silencing a custom set of 44 genes involved in WEE1 regulation under combined AraC + MK1775 to identify sensitizers and markers of resistance. The MRN (MRE11, Rad51, NBS1) complex and particularly NBS1 were potent modifiers of AraC and MK1775.Focusing on NBS1 since it is proposed to centrally regulate the defense capacity of leukemic cells, we identified that NBS1 phosphorylation at Ser343 (the ATM regulation site) is significantly altered both in cell lines and primary AML samples under combined AraC+MK1775 treatment as compared to single agent MK1775. In parallel, lower phosphorylation of ATMS1981(an autophosphorylation site in response to DNA strand breaks), was observed indicating that the ATM-CHEK1 pathway is not activated under co-treatment. Further Homologous recombination (HR)-mediated repair was compromised by AraC+MK1775 shown by DR-GFP expression vector to measure intracellular HR capacity: post-transfection of the I-SceI nuclease which cleaves non-functioning GFP tandem repeats to form a functional GFP unit, the HR was reduced with the combination. Consistently other HR markers decreased as well.Delayed accumulation of Cyclin A (indicative of S-phase progression) and greater inhibition of phospho-Cdk2Y15in synchronized cells treated with AraC + MK1775 in comparison to controls was observed. In addition the cell cycle was globally dysregulated by slower S-phase kinetics (progression), a completely abrogated G2/M checkpoint/phase as well as de-regulated DNA replication origin formation and firing as evidenced by Cdt1 and Mus81. As a consequence high single and double strand breaks (ɣH2AX) were observed with an increase in phospho-histone H3 in AraC + MK1775 treated cells compared to untreated cells or MK1775 single agent, confirming faster mitotic entry. Changes were followed by massive induction of apoptosis.Since WEE1 is implicated in leukemic stem cell maintenance we examined the long term effects of the combination in colony forming assays. AraC + MK1775 treated leukemic cells obtained from patients with AML were re-plated on Methocult after drug washout and colonies counted after 14 days. While MK1775 as a single agent could reduce colony formation by 4 fold compared to controls and lower dose AraC, co-treatment with low to moderate doses of AraC and MK1775 reduced colony formation by more than 7 fold and to almost zero in some primary specimens.Taken together, these results suggest that leukemia cells co-treated with AraC + MK1775 lost their ability to activate DNA damage and repair pathways mainly by compromising the MRN complex via NBS1 with subsequently reduced HR. The combination (as opposed to single agents) almost complete dysregulated the cell cycle and its checkpoints lead to DNA damage, genomic instability and rapid exit from the cell cycle with cell death via apoptosis. Thus we have molecularly characterized the detailed mechanisms underlying the potent AraC+WEE1 inhibition in AML and describe for the first time a therapeutic combination that has the potential to abrogate the MRN and NBS1 repair capacity which is central for drug resistance in AML. A key implication of our work is to provide a clinical rationale, mechanistic understanding and suggestions for biomarkers to clinically evaluate AraC + MK1775 in patients with AML. Disclosures:No relevant conflicts of interest to declare.

DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5‐ethynyl‐2′‐deoxyuridine incorporated into DNA

The "click chemistry" approach utilizing 5-ethynyl-2'-deoxyuridine (EdU) as a DNA precursor was recently introduced to assess DNA replication and adapted to flow- and imaging-cytometry. In the present study, we observed that EdU, once incorporated into DNA, induces DNA damage signaling (DDS) such as phosphorylation of ATM on Ser1981, of histone H2AX on Ser139, of p53 on Ser15, and of Chk2 on Thr68. It also perturbs progression of cells through the cell cycle and subsequently induces apoptosis. These effects were observed in non-small cell lung adenocarcinoma A549 as well as in B-cell human lymphoblastoid TK6 and WTK1 cells, differing in the status of p53 (wt versus mutated). After 1 h EdU pulse-labeling, the most affected was cells progression through the S phase subsequent to that at which they had incorporated EdU. This indicates that DNA replication using the template containing incorporated EdU is protracted and triggers DDS. Furthermore, progression of cells having DNA pulse-labeled with EdU led to accumulation of cells in G2 , likely by activating G2 checkpoint. Consistent with the latter was activation of p53 and Chk2. Although a correlation was observed in A549 cells between the degree of EdU incorporation and the extent of γH2AX induction, such correlation was weak in TK6 and WTK1 cells. The degree of perturbation of the cell cycle kinetics by the incorporated EdU was different in the wt p53 TK6 cells as compared to their sister WTK1 cell line having mutated p53. The data are thus consistent with the role of p53 in modulating activation of cell cycle checkpoints in response to impaired DNA replication. The confocal microscopy analysis of the 3D images of cells exposed to EdU for 1 h pulse and then grown for 24 or 48 h revealed an increased number of colocalized γH2AX and p53BP1 foci considered to be markers of DNA double-strand breaks and enlarged nuclei.

Molecular cloning, characterization and expression analysis of tumor suppressor protein p53 from orange-spotted grouper, Epinephelus coioides in response to temperature stress

The tumor suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. In the present study, the cDNA of p53 from the orange-spotted grouper (Epinephelus coioides) (Ec-p53) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Ec-p53 was of 1921 bp, including an open reading frame (ORF) of 1143 bp encoding a polypeptide of 380 amino acids with predicted molecular weight of 42.3 kDa and theoretical isoelectric point of 7.0. Quantitative real-time PCR (qRT-PCR) assays revealed that Ec-p53 was ubiquitously expressed in all the examined tissues but with high levels in intestine and liver of the orange-spotted grouper. In addition, we measured the DNA damage and apoptosis in the blood cells and the percentage of dead and damaged blood cells. Our results suggest that oxidative stress and DNA damage occurred in grouper in conditions where the temperature was 15 ± 0.5 °C. Furthermore, qRT-PCR and western blot confirmed that low temperature stress induced upregulation of Ec-p53 in the mRNA and protein levels. These results suggest that low temperature-induced oxidative stress may cause DNA damage or apoptosis, and cooperatively stimulate the expression of Ec-p53, which plays a critical role in immune defense and antioxidant responses.

The Smc5/Smc6/MAGE Complex Confers Resistance to Caffeine and Genotoxic Stress in Drosophila melanogaster

The SMC5/6 protein complex consists of the Smc5, Smc6 and Non-Smc-Element (Nse) proteins and is important for genome stability in many species. To identify novel components in the DNA repair pathway, we carried out a genetic screen to identify mutations that confer reduced resistance to the genotoxic effects of caffeine, which inhibits the ATM and ATR DNA damage response proteins. This approach identified inactivating mutations in CG5524 and MAGE, homologs of genes encoding Smc6 and Nse3 in yeasts. The fact that Smc5 mutants are also caffeine-sensitive and that Mage physically interacts with Drosophila homologs of Nse proteins suggests that the structure of the Smc5/6 complex is conserved in Drosophila. Although Smc5/6 proteins are required for viability in S. cerevisiae, they are not essential under normal circumstances in Drosophila. However, flies carrying mutations in Smc5, Smc6 and MAGE are hypersensitive to genotoxic agents such as ionizing radiation, camptothecin, hydroxyurea and MMS, consistent with the Smc5/6 complex serving a conserved role in genome stability. We also show that mutant flies are not compromised for pre-mitotic cell cycle checkpoint responses. Rather, caffeine-induced apoptosis in these mutants is exacerbated by inhibition of ATM or ATR checkpoint kinases but suppressed by Rad51 depletion, suggesting a functional interaction involving homologous DNA repair pathways that deserves further scrutiny. Our insights into the SMC5/6 complex provide new challenges for understanding the role of this enigmatic chromatin factor in multi-cellular organisms.

Candidate tumor suppressor BTG3 maintains genomic stability by promoting Lys63-linked ubiquitination and activation of the checkpoint kinase CHK1

B-cell translocation gene 3 (BTG3) is a member of the antiproliferative BTG/ Transducer of ErbB2 gene family and is induced by genotoxic stress in a p53- and Checkpoint kinase 1 (CHK1)-dependent manner. Down-regulation of BTG3 has been observed in human cancers, suggesting that it plays an important role in tumor suppression, although the underlying mechanisms are unclear. Here, we report that BTG3 interacts with CHK1, a key effector kinase in the cell cycle checkpoint response, and regulates its phosphorylation and activation. Upon interaction, BTG3 mediates K63-linked ubiquitination of CHK1 at Lys132 through the cullin-RING ligase 4(Cdt2) E3 complex, thus facilitating CHK1 chromatin association. We show that BTG3-depleted cells phenocopy those CHK1-deficient cells, exhibiting increased cell death after replication block and impaired chromosome alignment and segregation. These defects could be corrected by wild-type BTG3 but not by a mutant impaired in CHK1 interaction. We propose that BTG3-dependent CHK1 ubiquitination contributes to its chromatin localization and activation and that a defect in this regulation may increase genome instability and promote tumorigenesis.

DNA mismatch repair protein MSH2 dictates cellular survival in response to low dose radiation in endometrial carcinoma cells

DNA repair and G2-phase cell cycle checkpoint responses are involved in the manifestation of hyper-radiosensitivity (HRS). The low-dose radioresponse of MSH2 isogenic endometrial carcinoma cell lines was examined. Defects in cell cycle checkpoint activation and the DNA damage response in irradiated cells (0.2Gy) were evaluated. HRS was expressed solely in MSH2+ cells and was associated with efficient activation of the early G2-phase cell cycle checkpoint. Maintenance of the arrest was associated with persistent MRE11, γH2AX, RAD51 foci at 2h after irradiation. Persistent MRE11 and RAD51 foci were also evident 24h after 0.2Gy. MSH2 significantly enhances cell radiosensitivity to low dose IR.

Elevated Cyclin G2 Expression Intersects with DNA Damage Checkpoint Signaling and Is Required for a Potent G2/M Checkpoint Arrest Response to Doxorubicin

To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G(1)/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G(2)/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.

Abstract 4350: Normal stem cells exhibit hypersensitivity and cell death in response to ionizing radiation

Abstract Radiotherapy is an efficient method of treating cancer. Several decades of clinical observation in patients treated with radiation/ radiomimetic drugs has, however, revealed undesirable consequences to the normal tissue. Ionizing radiation induced normal tissue injury manifests through symptoms such as hair loss, skin regeneration defects, erosion of intestinal epithelium, hematopoietic anomalies, diminished sperm counts, infertility, neuro-cognitive impairments etc. The phenomenon of “stem cell drop-out” can be attributed as the underlying cause for these side effects. However, insufficient research has been conducted for investigating the relative radiosensitivity of undifferentiated stem/ progenitor cells compared to their differentiated progenies and the underlying molecular mechanisms of this differential radiation response (DNA damage response and IR induced apoptotic response). We demonstrate stem cell specific apoptosis and hypersensitivity to low dose IR in various stem cell niches in vivo. Further, this stem cell hypersensitivity observed in the mouse tissue niches, is recapitulated in multiple primary cell culture models of undifferentiated stem cells and isogenic differentiated cells grown in appropriate stem cell culture/ differentiation conditions. Cell cycle parameters, checkpoint response and radiosensitive phases of these cells have been studied. Towards characterizing the novel associations between cellular differentiation, DNA damage response and apoptotic response, we have studied alterations in transcription profiles of stem and isogenic differentiated cells with and without radiation. Our investigation has uncovered transcriptional induction and suppression of genes that are unique to differentiation states. Results of our studies will be presented. 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 4350. doi:1538-7445.AM2012-4350

Abstract SY11-03: Mechanisms of ATM activation

Abstract The Ataxia-Telangiectasia Mutated (ATM) kinase initiates a rapid cascade of protein phosphorylation in response to double-strand breaks in chromosomal DNA. The critical targets of ATM during this response include many tumor suppressors involved in cell cycle progression and apoptosis. The DNA damage response is thus defective in cells lacking ATM; however, there are also many indications that cells lacking ATM are defective in responses to other forms of stress, particularly oxidative stress. Mammalian cells lacking ATM exhibit high levels of reactive oxygen species and indications of chronic oxidative stress. In addition, the shortened lifespan, haematopoietic stem cell loss, and increased lymphomagenesis seen in A-T mouse models can be suppressed by antioxidants, suggesting that oxidative stress constitutes an important part of the A-T phenotype and is important for cancer progression. We have observed robust ATM activation in human cell lines in response to oxidative stress, in the absence of apparent DNA damage. In addition, oxidation of purified recombinant ATM in vitro induces ATM activation in the absence of DNA DSBs and the MRN complex. The oxidized form of ATM is a disulfide-crosslinked dimer containing several disulfide bonds. Mutation of a critical cysteine residue involved in disulfide bond formation specifically blocks activation via the oxidation pathway, both in vitro with purified components and in human cells. Identification of this pathway explains many observations of ATM activation under conditions of oxidative stress, and shows that ATM is an important sensor of reactive oxygen species (ROS) in vivo. Here we investigate how the oxidation pathway of ATM activation is affected by small molecules, specifically by resveratrol and related compounds, and how this affects cell cycle checkpoint responses in human cells. We also show in vitro that ATM is a direct target of these compounds and investigate the mechanism underlying the effects on ATM function. 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 SY11-03. doi:1538-7445.AM2012-SY11-03

Loss of Cyclin-Dependent Kinase 2 (CDK2) Inhibitory Phosphorylation in a CDK2AF Knock-In Mouse Causes Misregulation of DNA Replication and Centrosome Duplication

Cyclin-dependent kinase 1 (CDK1) inhibitory phosphorylation controls the onset of mitosis and is essential for the checkpoint pathways that prevent the G(2)- to M-phase transition in cells with unreplicated or damaged DNA. To address whether CDK2 inhibitory phosphorylation plays a similar role in cell cycle regulation and checkpoint responses at the start of the S phase, we constructed a mouse strain in which the two CDK2 inhibitory phosphorylation sites, threonine 14 and tyrosine 15, were changed to alanine and phenylalanine, respectively (CDK2AF). This approach showed that inhibitory phosphorylation of CDK2 had a major role in controlling cyclin E-associated kinase activity and thus both determined the timing of DNA replication in a normal cell cycle and regulated centrosome duplication. Further, DNA damage in G(1) CDK2AF cells did not downregulate cyclin E-CDK2 activity when the CDK inhibitor p21 was also knocked down. We were surprised to find that this was insufficient to cause cells to bypass the checkpoint and enter the S phase. This led to the discovery of two previously unrecognized pathways that control the activity of cyclin A at the G(1) DNA damage checkpoint and may thereby prevent S-phase entry even when cyclin E-CDK2 activity is deregulated.

Multiple Roles of Cyclin-Dependent Kinase 4/6 Inhibitors in Cancer Therapy

Cyclin-dependent kinases (CDKs) regulate cell proliferation and coordinate the cell cycle checkpoint response to DNA damage. Although inhibitors with varying selectivity to specific CDK family members have been developed, selective CDK4/6 inhibitors have emerged as the most attractive antineoplastic agents because of the importance of CDK4/6 activity in regulating cell proliferation and the toxic effects associated with inhibition of other CDKs (eg, CDK1 and CDK2). FVB/N wild-type mice (n = 13) were used to evaluate carboplatin-induced myelosuppression in bone marrow by complete blood cell counts after treatment with the CDK4/6 inhibitor PD0332991. Genetically engineered murine models of retinoblastoma (Rb)-competent (MMTV-c-neu) and Rb-incompetent (C3-TAg) breast cancer (n = 16 MMTV-c-neu mice in the carboplatin plus vehicle control group, n = 17 MMTV-c-neu mice in the carboplatin plus PD0332991 group, n = 17 C3-TAg mice in the carboplatin plus vehicle control group, and n = 14 C3-TAg mice in the carboplatin plus PD0332991 group) were used to investigate the antitumor activity of PD0332991 alone or in combination with chemotherapy. All statistical tests were two-sided. Coadministration of PD0332991 with carboplatin compared with carboplatin alone in FVB/N wild-type mice increased hematocrit (51.2% vs 33.5%, difference = 17.7%, 95% confidence interval [CI] = -26.7% to -8.6%, P < .001), platelet counts (1321 vs 758.5 thousand cells per μL, difference = 562.5 thousand cells per μL, 95% CI = -902.8 to -222.6, P = .002), myeloid cells (granulocytes and monocytes; 3.1 vs 1.6 thousand cells per μL, difference = 1.5 thousand cells per μL, 95% CI = -2.23 to -0.67, P < .001), and lymphocytes (7.9 vs 5.4 thousand cells per μL, difference = 2.5 thousand cells per μL, 95% CI = -4.75 to -0.18, P = .02). Daily administration of PD0332991 exhibited antitumor activity in MMTV-c-neu mice as a single agent. However, the combination of carboplatin plus PD0332991 decreased antitumor activity compared with carboplatin alone in Rb-competent mice (mean percent change in tumor volume at day 21 = -52.6% vs 3.7% for carboplatin and carboplatin plus PD0332991, respectively, difference = 56.3%, 95% CI = -109.0% to -3.6%, P = .04). In contrast, Rb-deficient tumors in C3-Tag mice were resistant to PD0332991, and coadministration of PD0332991 plus carboplatin had no effect on in vivo tumor growth (mean percent change in tumor volume at day 21 = 118.8% and 109.1% for carboplatin and carboplatin plus PD0332991, respectively, difference = 9.7%, 95% CI = -183.5% to 202.9%, P = .92). Finally, in tumor-bearing mice, coadministration of PD0332991 with carboplatin provided statistically significant protection of platelets (P = .04). We believe that the present data support a possible role for CDK4/6 inhibitors in a majority of patients with advanced cancer: to either inhibit tumor growth in CDK4/6-dependent tumors or ameliorate the dose-limiting toxicities of chemotherapy in CDK4/6-indepdendent tumors. Our data also suggest CDK4/6 inhibitors should not be combined with DNA-damaging therapies, such as carboplatin, to treat tumors that require CDK4/6 activity for proliferation.

Lig4 R278H Mutation Impacts on LT-HSC Functions, Cancer Development and Aging

Abstract 1350DNA Ligase IV (Lig4) is an essential component of the non-homologous end-joining (NHEJ) repair pathway, essential for the end-ligation of broken DNA ends generated during V(D)J recombination and IgH Class Switch Recombination (CSR) in B lymphocytes. Mutations in Lig4 underlie the DNA Lig4 Syndrome, a rare herited immunodeficiency disease. We have modeled one such mutation in mice, a homozygous arginine to histidine 278 (R278H) change with the enzyme active site, which others and we have shown significantly impairs the catalytic end-ligation activity of the LigIV protein. These mice show significant immune-specific defects, including decreased T and B lymphocytes, in part associated with defects in development, increased cell death, and/or autophagy. Bypass of B lymphocyte development leads to increased numbers of total peripheral B lymphocytes that can undergo robust CSR at near normal levels, which however contain high levels of IgH specific DNA breaks and translocations. Both young and older mice display significant reduction in LT-HSCs, which may underlie their B and T lymphocyte associated defects, which in LT-HSC reconstitution experiments in SCID mice, fail to engraft. The decreased R278H mice LT-HSCs observed are in contrast to previous reported findings for Ku80 and DNA-PKcs deficient mice, and also ATM deficiency, which we further are investigating to elucidate the normal and pathological functions of aberrant Lig4. We hypothesize the cause of the LT-HSC defects to be caused by changes in DNA damage, cell cycle checkpoint responses and genomic stability maintenance associated with the R278H mutation, that promote the restriction or selection of LT-HSC cell populations that are prone to genomic instability during differentiation. As such, the majority of relatively, young adult R278H mice routinely spontaneously develop T cell lymphomas, and mice that do not succumb to lymphomas subsequently go on to develop other solid tumors and other pathological consequences which are characteristic of premature aging. Our findings implicate Lig4 is required for genomic stability maintenance and proliferative expansion of earliest HSCs and other stem cell populations. The implications of our findings in the context of impacts of the R278H mutation on genomic instability and DNA damage response in the context of HSCs, cancer development and aging in the DNA Lig4 Syndrome, and its relation to other human immunodeficiency and progeroid syndromes will be discussed. Disclosures:No relevant conflicts of interest to declare.

P53 Facilitates BH3-only BID nuclear export to induce apoptosis in the irrepairable DNA damage response

Over the past decade, the BH3-only BID likes p53, has emerged as a central player in linking death signals through surface death receptors to the core apoptotic mitochondrial pathway and life signals through cell cycle arrest. Recent studies indicate that pro-apoptotic activation of BID may be negatively regulated by its phosphorylation in response to DNA damage. BID itself plays a role in cell cycle checkpoint response, in DNA repair, or in integrating apoptosis and the DNA damage response, which indicate BID is a nuclear-cytoplasmic protein. However, BID does not have any obvious nuclear localization signals (NLS), and only carries nuclear export signals (NES). Mutating BID NES does not affect the nuclear exit of BID, suggesting that BID NES does not seem to function as a NES. Therefore, BID is transported into the nucleus and its export is probably mediated by other NES-carrying proteins. As a well-characterized transcription factor, p53 carries typical NLS and NES. Bid is transcriptionally regulated by p53, and both can be exported from nucleus to the mitochondria in response to DNA damage. Moreover, p53 can, through the interaction with BID in the mitochondria to induce apoptosis. Given the above background, we hypothesize that p53 facilitates BID nuclear export to induce apoptosis in response to irreparable DNA damage.

Characterization of p53 expression in rainbow trout

The tumour suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. Given the high incidence of p53 mutations in human cancers, it has been extensively studied, though only a small fraction of these investigations have been in non-mammalian systems. For the present study, an anti-rainbow trout p53 polyclonal antibody was generated. A variety of rainbow trout (Oncorhynchus mykiss) tissues and cell lines were examined through western blot analysis of cellular protein extracts, which revealed relatively high p53 levels in brain and gills. To evaluate the checkpoint response of rainbow trout p53, RTbrain-W1 and RTgill-W1 cell lines were exposed to varying concentrations of the DNA damaging agent bleomycin and ribonucleotide reductase inhibitor hydroxyurea. In contrast to mammals, these checkpoint-inducing agents provoked no apparent increase in rainbow trout p53 levels. These results infer the presence of alternate DNA damage checkpoint mechanisms in rainbow trout cells.

RB and p53 Cooperate to Prevent Liver Tumorigenesis in Response to Tissue Damage

The tumor suppressors retinoblastoma (RB) and p53 are important regulators of the cell cycle. Although human cancer cells inactivate RB and p53 by many mechanisms, the cooperative roles of these proteins in tumorigenesis are complex and tissue specific. We analyzed the cooperation of RB and p53 in liver development and pathogenesis of hepatocellular carcinoma. Spontaneous and carcinogen-induced (diethylnitrosamine) tumorigenesis were studied in mice with liver-specific deletions of Rb and/or p53 (Rbf/f;albcre+, p53f/f;albcre+ and Rbf/f; p53f/f;albcre+ mice). Genotype, histologic, immunohistochemical, microarray, quantitative polymerase chain reaction, immunoblot, and comparative genomic hybridization analyses were performed using normal and tumor samples. Comparative microarray analyses were performed against publicly available human microarray data sets. Deletion of RB and p53 from livers of mice deregulated the transcriptional programs associated with human disease. These changes were not sufficient for spontaneous tumorigenesis; potent quiescence mechanisms compensated for loss of these tumor suppressors. In response to hepatocarcinogen-induced damage, distinct and cooperative roles of RB and p53 were revealed; their loss affected cell cycle control, checkpoint response, and genome stability. In damaged tissue, combined loss of RB and p53 resulted in early lesion formation, aggressive tumor progression, and gene expression signatures and histologic characteristics of advanced human hepatocellular carcinoma. The effects RB and p53 loss are determined by the tissue environment; cell stresses that promote aggressive disease reveal the functions of these tumor suppressors.