Non-cycling Cells Research Articles

Clofarabine as a Novel Nucleoside Analogue Approved to Treat Patients with Haematological Malignancies: Mechanism of Action and Clinical Activity

Clofarabine is a second generation of purine nucleoside analogues designed to combine the most favorable pharmacokinetic properties of fludarabine and cladribine. Clofarabine acts by inhibiting DNA polymerases and ribonucleotide reductase as well as by inducing apoptosis in cycling and non-cycling cells. Phase I/II clinical studies revealed its efficacy in hematological malignancies, and in 2004 clofarabine was approved by the United States Food and Drug Administration for the treatment of pediatric relapsed or refractory acute lymphoblastic leukemia after at least two prior chemotherapy regimens. The mechanism of action, pharmacology and clinical activity of clofarabine is the subject of this review.

CD133 (Prominin) Negative Human Neural Stem Cells Are Clonogenic and Tripotent

BackgroundCD133 (Prominin) is widely used as a marker for the identification and isolation of neural precursor cells from normal brain or tumor tissue. However, the assumption that CD133 is expressed constitutively in neural precursor cells has not been examined.Methodology/Principal FindingsIn this study, we demonstrate that CD133 and a second marker CD15 are expressed heterogeneously in uniformly undifferentiated human neural stem (NS) cell cultures. After fractionation by flow cytometry, clonogenic tripotent cells are found in populations negative or positive for either marker. We further show that CD133 is down-regulated at the mRNA level in cells lacking CD133 immunoreactivity. Cell cycle profiling reveals that CD133 negative cells largely reside in G1/G0, while CD133 positive cells are predominantly in S, G2, or M phase. A similar pattern is apparent in mouse NS cell lines. Compared to mouse NS cells, however, human NS cell cultures harbour an increased proportion of CD133 negative cells and display a longer doubling time. This may in part reflect a sub-population of slow- or non-cycling cells amongst human NS cells because we find that around 5% of cells do not take up BrdU over a 14-day labelling period. Non-proliferating NS cells remain undifferentiated and at least some of them are capable of re-entry into the cell cycle and subsequent continuous expansion.ConclusionsThe finding that a significant fraction of clonogenic neural stem cells lack the established markers CD133 and CD15, and that some of these cells may be dormant or slow-cycling, has implications for approaches to identify and isolate neural stem cells and brain cancer stem cells. Our data also suggest the possibility that CD133 may be specifically down-regulated during G0/G1, and this should be considered when this marker is used to identify and isolate other tissue and cancer stem cells.

Flow cytometry methods for the study of cell‐cycle parameters of planarian stem cells

Due to their characteristic inaccessibility and low numbers, little is known about the cell-cycle dynamics of most stem cells in vivo. A powerful, established methodology to study cell-cycle dynamics is flow cytometry, which is used routinely to study the cell-cycle dynamics of proliferating cells in vitro. Its use in heterogeneous mixtures of cells obtained from whole animals, however, is complicated by the relatively low abundance of cycling to non-cycling cells. We report on flow cytometric methods that take advantage of the abundance of proliferating stem cells in the planarian Schmidtea mediterranea. The optimized protocols allow us to measure cell-cycle dynamics and follow BrdU-labeled cells specifically in complex mixtures of cells. These methods expand on the growing toolkit being developed to study stem cell biology in planarians, and open the door to detailed cytometric studies of a collectively totipotent population of adult stem cells in vivo.

The small-molecule CDK inhibitor, SNS-032, enhances cellular radiosensitivity in quiescent and hypoxic non-small cell lung cancer cells

In solid tumors, including non-small cell lung carcinomas (NSCLC) the existence of radioresistant subpopulations, such as quiescent or hypoxic tumor cells, is well established, thus posing a critical therapeutic problem. Although small-molecule inhibitors targeting cyclin-dependent kinases (CDK) were demonstrated to enhance cellular radiosensitivity preferentially in proliferating tumor cells, cell cycle-independent activities of these substances were recently suggested. In this study, the potential of a newer generation small-molecule CDK inhibitor, SNS-032, to sensitize radioresistant tumor cells to ionizing radiation was tested in vitro using two NSCLC cell lines (NCI-H460 and A549). Exposure of quiescent and hypoxic lung tumor cells to SNS-032 at a clinically achievable concentration (500 nM) prior to irradiation resulted in a significant increase in cellular radiosensitivity indicating cell cycle-unrelated mechanisms. The effect of SNS-032 on non-cycling cells was not attributed to an enhanced toxicity of the drug. A SNS-032 mediated delay in the resolution of radiation-induced γH2AX foci a surrogate for DNA double-strand breaks was determined in non-cycling cells, suggesting a modulation of DNA double-strand break repair. These results indicate a modulation of DNA double-strand break repair to be partially attributed to the radiosensitization effects of SNS-032 observed in hypoxic and quiescent lung tumor cells. Considering the importance of therapy resistance for the radiocurability of solid tumors, our findings may provide the basis for an improvement of the well-established treatment regimens in clinical oncology.

Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts

Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [(3)H]thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity.

A signature-based method for indexing cell cycle phase distribution from microarray profiles

BackgroundThe cell cycle machinery interprets oncogenic signals and reflects the biology of cancers. To date, various methods for cell cycle phase estimation such as mitotic index, S phase fraction, and immunohistochemistry have provided valuable information on cancers (e.g. proliferation rate). However, those methods rely on one or few measurements and the scope of the information is limited. There is a need for more systematic cell cycle analysis methods.ResultsWe developed a signature-based method for indexing cell cycle phase distribution from microarray profiles under consideration of cycling and non-cycling cells. A cell cycle signature masterset, composed of genes which express preferentially in cycling cells and in a cell cycle-regulated manner, was created to index the proportion of cycling cells in the sample. Cell cycle signature subsets, composed of genes whose expressions peak at specific stages of the cell cycle, were also created to index the proportion of cells in the corresponding stages. The method was validated using cell cycle datasets and quiescence-induced cell datasets. Analyses of a mouse tumor model dataset and human breast cancer datasets revealed variations in the proportion of cycling cells. When the influence of non-cycling cells was taken into account, "buried" cell cycle phase distributions were depicted that were oncogenic-event specific in the mouse tumor model dataset and were associated with patients' prognosis in the human breast cancer datasets.ConclusionThe signature-based cell cycle analysis method presented in this report, would potentially be of value for cancer characterization and diagnostics.

P53 and COX-2 Inhibitors Control the Replication and Viability of Non-Transformed and CLL B Lymphocytes

Innate immune system-driven replication of antigen receptor-triggered, non-transformed human B2 cells depends upon BAFF-mediated survival signals. The latter promote a COX-2–PGE2–Mcl-1 pathway within the dividing subpopulation and significantly reduce activation-induced apoptosis within clonal progeny (J. Immunol. 176:6736, 2006). Mcl-1 is known to counter mitochondria-dependent apoptosis induced by Foxo-upregulated Bim and by p53-upregulated Bax, Bak, Bid and Bad. To gain insight into mechanism(s) responsible for this activation-induced apoptosis, levels of the above pro-apoptotic molecules, as well as the transcription factor p53, were monitored in cultures of CFSE-labeled, non-transformed human B cells stimulated by surrogate C3dg-bound antigen, IL-4 and BAFF. The latter typically undergo 5–6 divisions over a week of culture. Based on the preferentially upregulated expression of p53 and its downstream pro-apoptotic molecules, and the reversing effects of p53 siRNA, it was concluded that the activation-induced cell death involves a p53-mediated apoptosis program. The day 2 administration of Nutlin, a pharmacologic inhibitor of p53 interaction with its negative regulator, MdM2, resulted in further augmented p53 expression and activity. This was revealed by diminished cycling and increased apoptosis within the replicating population. To examine whether positive/negative modulation of the COX-2–PGE-2–Mcl-1 pathway can alter the sensitivity to Nutlin (10 mM), activated cultures were additionally supplemented with either exogenous PGE2 (10 nM) or the COX-2 inhibitor, CAY10404 (30 mM). While a pulse with PGE2 reversed the pro-apoptotic effects of Nutlin, a concurrent pulse with COX-2 inhibitor augmented the degree of apoptosis observed. For example, while absolute cell yield (viable + apoptotic) in cultures withDMSO vehicle control,Nutlin,Nutlin + COX-2 inhibitor, orNutlin + PGE2 was 167 ×103, 72 ×103, 41 ×103, and 96 ×103, respectively, the percent viability within the divided fraction was 61%, 48%, 39%, and 71%, respectively.The data suggest that a COX-2–PGE2–Mcl-1 pathway may override the effects of an active p53 pathway in cycling, non-transformed human B lymphocytes. Ongoing studies are exploring whether proliferating CLL clones are likewise reciprocally regulated by p53 and COX-2 pathways. CFSE-labeled CLL B cells were stimulated for 8 days with TLR9 ligand, ODN-2006 (0.2 mM) + IL15 (15 ng/ml), yielding up to 7–8 divisions. A day 5 pulse with Nutlin resulted in significant day 8 apoptosis of both the cycling and few non-cycling cells within activated CLL cultures (e.g. undivided cells = 64% and 10% viability, and divided cells = 45% and 2%, within control and Nutlin cultures, respectively). Preliminary results indicate that COX-2 inhibitor significantly impairs CLL replication and viability in a manner dependent upon the timed addition of inhibitor. A day 2 pulse with CAY10404 reduced the day 8 yield of CLL cells with ≥ 4 divisions, as well as slightly reducing undivided cell viability. A day 5 pulse impaired the yield of cells with ≥ 5 divisions and the viability of both non-cycling and dividing CLL. Present studies are evaluating whether low concentrations of Nutlin and COX-2 inhibitor may exhibit synergy in blocking the growth/viability of CLL clones. A two-pronged therapeutic approach at augmenting p53 levels and diminishing COX-2 levels might be warranted in the treatment of CLL.

Nontelomeric TRF2-REST Interaction Modulates Neuronal Gene Silencing and Fate of Tumor and Stem Cells

Removal of TRF2, a telomere shelterin protein, recapitulates key aspects of telomere attrition including the DNA-damage response and cell-cycle arrest [1]. Distinct from the response of proliferating cells to loss of TRF2 [2, 3], in rodent noncycling cells, TRF2 inhibition promotes differentiation and growth [4, 5]. However, the mechanism that couples telomere gene-silencing features [6-8] to differentiation programs has yet to be elucidated. Here we describe an extratelomeric function of TRF2 in the regulation of neuronal genes mediated by the interaction of TRF2 with repressor element 1-silencing transcription factor (REST), a master repressor of gene networks devoted to neuronal functions [9-12]. TRF2-REST complexes are readily detected by coimmunoprecipitation assays and are localized to aggregated PML-nuclear bodies in undifferentiated pluripotent human NTera2 stem cells. Inhibition of TRF2, either by a dominant-negative mutant or by RNA interference, dissociates TRF2-REST complexes resulting in ubiquitin-proteasomal degradation of REST. Consequentially, REST-targeted neural genes (L1CAM, beta3-tubulin, synaptophysin, and others) are derepressed, resulting in acquisition of neuronal phenotypes. Notably, selective damage to telomeres without affecting TRF2 levels causes neither REST degradation nor cell differentiation. Thus, in addition to protecting telomeres, TRF2 possesses a novel role in stabilization of REST thereby controlling neural tumor and stem cell fate.

Brachytherapy for Oral Tongue Cancer: An Analysis of Treatment Results with Various Biological Markers

Low-dose-rate (LDR) brachytherapy is an effective treatment for tongue cancer. However, little is known about the biological mechanism underlying this therapy, characterized by delivery of continuous exposures of LDR irradiation. It is reported that lower microvessel density (MVD), lower Ki-67 index or higher expression of endogenous hypoxic markers such as carbonic CA IX and Glut-1 are related to the poor control of tumors treated with external irradiation. To elucidate the biological characteristics of LDR brachytherapy, we analyzed our results in cases of tongue cancer treated with LDR brachytherapy by using immunohistochemical stainings with antibodies against Ki-67 and MVD, Glut-1 and CA IX. The prognostic value of Ki-67 index, MVD and the expression of CA IX and Glut-1 was assessed in 68 tongue cancers treated with LDR brachytherapy. The specimens were taken from tongue cancers before radiation therapy and immunohistochemical staining was performed. The local recurrence-free survival rates were significantly different between T1+T2 and T3 (P = 0.00067), but not between low and high Ki-67 indexes (P = 0.54), between low and high MVD (P = 0.071), low and high CA IX indexes (P = 0.062) or low and high Glut-1 indexes (P = 0.107). T stage, the size of the tumor was the only significant factor for local control in multivariate analyses (P = 0.0377). LDR could overcome the radioresistence of non-cycling and hypoxic cells; however, we cannot draw firm conclusions due to the limited number of patients.

The central role of the chemokine receptor, CXCR4, in haemopoietic stem cell transplantation: will CXCR4 antagonists contribute to the treatment of blood disorders?

Recent clinical trials have used CXCR4 antagonists for the rapid mobilization of CD34(+) haemopoietic stem/progenitor cells (HSC/HPC) from the bone marrow to the blood in patients refractory to granulocyte-colony-stimulating factor (G-CSF). These antagonists not only mobilize non-cycling cells with a higher proportion of repopulating cells, but also enhance CD34(+) cell mobilization when used in combination with G-CSF. Here, we review the importance of CXCR4 and its ligand CXCL12 in haemopoiesis, and the potential roles of CXCR4 antagonists in the clinical HSC transplant setting.

The Tyrosine Kinase Inhibitor TK258 Differentially Targets Proliferating Human Hematopoietic Progenitor Cells but Does Not Eliminate the Quiescent Fraction.

NOD-SCID mouse models have shown that, as with normal hematopoietic stem cells, leukemic stem cells (LSC) exist in a quiescent state and are thus capable of sustaining disease in vivo. Targeted therapy of LSC may therefore have a significant impact on disease eradication, relapse rates and toxicity reduction. Inhibition of FLT3 and c-kit is a current therapeutic strategy for AML but the efficacy of this approach on quiescent cells is unknown. Furthermore, although both FLT3 and c-kit play crucial roles in the development of normal human hematopoietic progenitor (HPC) and stem cells, the hematopoietic consequences of FLT3 and c-kit inhibition remain undefined. Here we show that the FLT3, c-kit and PDGF directed tyrosine kinase inhibitor TK258 specifically targets slowly dividing human HPCs, but not rapidly proliferating HPCs or quiescent cells displaying an immature phenotype. For normal CD34+ cells, treatment of short-term cultures with TK258 resulted in a dose dependent inhibition of cell expansion. Moreover, treatment of normal CD34+ cells with TK258 within the clinical dose range [0.2μM to 1.5μM] inhibited myeloid colony growth but had no effect on erythroid colony output, suggesting a differential effect on lineage-committed progenitors. To further elucidate the effect of TK258 on dividing and also quiescent CD34+ cells, normal CD34+ cells were labelled with a non-toxic fluorescent cell membrane dye, PKH26, prior to TK258 treatment in vitro. Coupled with flow cytometry, this dye enabled the discrimination of progenitor cells with different proliferative capacities. Thus, using this technique, we defined a CD34+/CD133+ quiescent population of cells that retained the same PKH fluorescence intensity as from day 0 of culture. TK258 treatment in culture generated a significant increase in the CD34+/CD133+ quiescent fraction (15.8% treated versus 5.9% untreated of total viable cells). In contrast with untreated cells this fraction maintained c-kit expression, suggesting maintenance of an immature population by TK258. Total viable cell recoveries were higher in the TK258 treated quiescent fraction (24% treated versus 13.5% untreated, p=0.037). Rapidly dividing progenitors were unaffected by TK258, while progenitors with a slower division rate showed significant sensitivity to the compound. The anti-proliferative effect of TK258 was mediated via G0/G1 cell cycle arrest, but not by apoptosis of CD34+ cells. We assessed the functional read-out of TK258 pre-treated progenitors in CFU assays, by sorting cells on the basis of their PKH26 fluorescence. These assays demonstrated a higher total colony output and diverse colony type for TK258 pre-treated ‘quiescent' cells compared to untreated controls, in keeping with the preservation of a more immature population by TK258. Rapidly proliferating populations displayed a lower CFU potential, attributable to a reduction in myeloid output, which was not significantly altered by TK258 pre-treatment. CAFC assays and gene expression profiling of PKH26-sorted subsets are in progress. Taken together, these data demonstrate that TK258 has significant inhibitory effects on normal human hematopoietic progenitor cells in vitro and that the sensitivity of progenitor cells to the compound can be predicted from their proliferative index. Importantly, immature hematopoietic quiescent cells are preserved, which may have implications for the activity of tyrosine kinase inhibitors on non-cycling human leukemia cells.

Radiosensitization by the Histone Deacetylase Inhibitor PCI-24781

PCI-24781 is a novel broad spectrum histone deacetylase inhibitor that is currently in phase I clinical trials. The ability of PCI-24781 to act as a radiation sensitizer and the mechanisms of radiosensitization were examined. Exponentially growing human SiHa cervical and WiDr colon carcinoma cells were exposed to 0.1 to 10 micromol/L PCI-24781 in vitro for 2 to 20 h before irradiation and 0 to 4 h after irradiation. Single cells and sorted populations were analyzed for histone acetylation, H2AX phosphorylation, cell cycle distribution, apoptotic fraction, and clonogenic survival. PCI-24781 treatment for 4 h increased histone H3 acetylation and produced a modest increase in gammaH2AX but negligible cell killing or radiosensitization. Treatment for 24 h resulted in up to 80% cell kill and depletion of cells in S phase. Toxicity reached maximum levels at a drug concentration of approximately 1 micromol/L, and cells in G(1) phase at the end of treatment were preferentially spared. A similar dose-modifying factor (DMF(0.1) = 1.5) was observed for SiHa cells exposed for 24 h at 0.1 to 3 micromol/L, and more radioresistant WiDr cells showed less sensitization (DMF(0.1) = 1.2). Limited radiosensitization and less killing were observed in noncycling human fibroblasts. Cell sorting experiments confirmed that depletion of S-phase cells was not a major mechanism of radiosensitization and that inner noncycling cells of SiHa spheroids could be sensitized by nontoxic doses. PCI-24781 pretreatment increased the fraction of cells with gammaH2AX foci 24 h after irradiation but did not affect the initial rate of loss of radiation-induced gammaH2AX or the rate of rejoining of DNA double-strand breaks. PCI-24781 shows promise as a radiosensitizing agent that may compromise the accuracy of repair of radiation damage.

Setting the Stage for S Phase

In a recent issue of Molecular Cell, Tyagi et al. (2007) show that E2F1, a positive regulator of S phase entry, recruits cofactor HCF-1 and associated hSet1/MLL histone H3 lysine 4 methyltransferase complex, facilitating the activation of genes required for proliferation.

APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks.

Aprataxin and polynucleotide kinase (PNK) are DNA end processing factors that are recruited into the DNA single- and double-strand break repair machinery through phosphorylation-specific interactions with XRCC1 and XRCC4, respectively. These interactions are mediated through a divergent class of forkhead-associated (FHA) domain that binds to peptide sequences in XRCC1 and XRCC4 that are phosphorylated by casein kinase 2 (CK2). Here, we identify the product of the uncharacterized open reading frame C2orf13 as a novel member of this FHA domain family of proteins and we denote this protein APLF (aprataxin- and PNK-like factor). We show that APLF interacts with XRCC1 in vivo and in vitro in a manner that is stimulated by CK2. Yeast two-hybrid analyses suggest that APLF also interacts with the double-strand break repair proteins XRCC4 and XRCC5 (Ku86). We also show that endogenous and yellow fluorescent protein-tagged APLF accumulates at sites of H(2)O(2) or UVA laser-induced chromosomal DNA damage and that this is achieved through at least two mechanisms: one that requires the FHA domain-mediated interaction with XRCC1 and a second that is independent of XRCC1 but requires a novel type of zinc finger motif located at the C terminus of APLF. Finally, we demonstrate that APLF is phosphorylated in a DNA damage- and ATM-dependent manner and that the depletion of APLF from noncycling human SH-SY5Y neuroblastoma cells reduces rates of chromosomal DNA strand break repair following ionizing radiation. These data identify APLF as a novel component of the cellular response to DNA strand breaks in human cells.

Mitogenic growth signalling, DNA replication licensing, and survival are linked in prostate cancer

Activation of mitogen/extracellular-signal-regulated kinase kinase 5/extracellular signal-regulated kinase-5 (MEK5/ERK5) growth signalling is coupled to increased cell proliferation in prostate cancer (PCa). Dysregulation of the DNA replication licensing pathway, a critical step in growth control downstream of transduction signalling pathways, is associated with development of PCa. In this study we have investigated linkages between the MEK5/ERK5 pathway and DNA replication licensing during prostate carcinogenesis. The effects of increased MEK5/ERK5 signalling on the expression of replication licensing factors Mcm2 and geminin and the proliferation marker Ki67 were studied in an ecdysone-inducible system expressing a constitutively activated mutant of MEK5 in EcR293 cells and in stable ERK5 over-expressing PC3 clones. In parallel, expression of these biomarkers in PCa biopsy specimens (n=58) was studied and compared to clinicopathological parameters. In both in vitro systems induction of MEK5 expression resulted in increased levels of phosphorylated ERK5 and Mcm2, geminin and Ki67 proteins. In PCa specimens average Mcm2 expression was greater than Ki67 and geminin expression (median labelling index (LI) 36.7, 18.1, and 3.4% respectively), consistent with their differential expression according to growth status (P<0.0001). Mcm2, geminin and Ki67 expression were significantly associated with Gleason grade (P=0.0002, P=0.0003, P=0.004); however there was no link with T or M stage. There was a significant relationship between increasing ERK5 expression and increasing Mcm2 (P=0.003) and Ki67 (P=0.009) expression, with non-significant trends seen with increasing MEK5 expression. There were significant associations between Gleason grade and the number of cells traversing G1 phase (Ki67LI-gemininLI; (P=0.001)), with high ERK5 levels associated with both an increase in replication licensed but non-cycling cells (Mcm2LI-Ki67LI; (P=0.01)) and accelerated cell cycle progression (gemininLI/Ki67LI; (P= 0.005)), all indicative of a shift towards increasing proliferative potential. While Mcm2 and Ki67 were both prognostic factors on univariate analysis, only Mcm2 remained an independent prognostic marker on multivariate analysis. Taken together, our data show that induction of MEK5/ERK5 signalling is linked to activation of the DNA replication licensing pathway in PCa, and that the strong prognostic value of MCM proteins may result from their function as relay stations coupling growth regulatory pathways to genome duplication.

Perturbed gap-filling synthesis in nucleotide excision repair causes histone H2AX phosphorylation in human quiescent cells

Human histone H2AX is rapidly phosphorylated on serine 139 in response to DNA double-strand breaks and plays a crucial role in tethering the factors involved in DNA repair and damage signaling. Replication stress caused by hydroxyurea or UV also initiates H2AX phosphorylation in S-phase cells, although UV-induced H2AX phosphorylation in non-cycling cells has recently been observed. Here we study the UV-induced H2AX phosphorylation in human primary fibroblasts under growth-arrested conditions. This reaction absolutely depends on nucleotide excision repair (NER) and is mechanistically distinct from the replication stress-induced phosphorylation. The treatment of cytosine-beta-D-arabinofuranoside strikingly enhances the NER-dependent H2AX phosphorylation and induces the accumulation of replication protein A (RPA) and ATR-interacting protein (ATRIP) at locally UV-damaged subnuclear regions. Consistently, the phosphorylation appears to be mainly mediated by ataxia-telangiectasia mutated and Rad3-related (ATR), although Chk1 (Ser345) is not phosphorylated by the activated ATR. The cellular levels of DNA polymerases delta and epsilon and proliferating cell nuclear antigen are markedly reduced in quiescent cells. We propose a model that perturbed gap-filling synthesis following dual incision in NER generates single-strand DNA gaps and hence initiates H2AX phosphorylation by ATR with the aid of RPA and ATRIP.

The Novel PARP Inhibitor, AZD2281, Targets Apoptotic Resistant ATM Mutant CLL Tumours for Cellular Killing.

B-CLL is incurable and the development of resistance to standard chemotherapeutics remains an important problem. ATM mutations lead to chemo-resistance in a significant proportion of CLL patients and this is due to impairment in the activation of the DNA-damage induced ATM/p53 apoptotic pathway. Therefore, there is a requirement for novel treatments for ATM mutant CLL tumours, which induce cell death by mechanisms that are independent of this pathway.Breast cancer cells with homozygous BRCA1/2 mutations are deficient in the repair of DNA double strand break (DSB) by homologous recombination (HR). It has recently been shown that inhibition of PARP activity, which is required for repair of DNA single strand breaks (SSB), can lead to selective sensitisation of tumours harbouring BRCA1/2 mutations in replicating cells. The mechanism involves the continual progression of DNA SSBs into DSBs, which in the presence of defective HR, results in accumulation of DSBs and activation of cell death via mitotic catastrophe. ATM regulates the balance between the repair of DNA DSBs and the induction of the DNA DSB apoptotic pathway. Therefore, the phenotype of ATM mutant CLL cells includes a repair defect as well as an apoptotic defect. By analogy with the BRCA study, we investigated whether PARP inhibition can sensitise ATM mutant CLL tumours.We addressed the in vitro cytotoxicity of a similar PARP inhibitor (PARPi), AZD2281 produced by KuDOS, in 20 CLL tumours, including 10 ATM mutant and 10 ATM wild type. We analysed each of the CLL tumours in non-cycling and cycling states in culture. The aim was to mimic the CLL tumour populations in vivo, which are believed to consist of non-cycling peripheral blood tumour cells and cycling lymphoid tissue tumour cells. Given the mechanism of PARP activity, we predicted that its inhibition would preferentially be toxic in the cycling CLL population. In our experiments, CLL cycling was induced using a CD40L/IL4 support system and verified by incorporation of tritiated thymidine or BrdU. Consistent with our expectations, non-cycling CLL cells did not show significant cytotoxicity to increasing doses of AZD2281 (0.5–10μM). By comparison, in cycling CLLs there was increased sensitivity to AZD2281 at concentrations of 1.5μM or greater in ATM mutant compared to ATM wild type tumours. Furthermore, protein analysis revealed that treatment with AZD2281 did not induce the up regulation of p53 or the cleavage of caspases and that the killing in ATM mutant cells did not require induction of Atm/p53 dependent apoptosis. In non-cycling ATM mutant CLL cells, we also investigated whether pre-incubation with the AZD2281 could sensitize cells to DNA damaging chemotherapeutics. Interestingly, we found that 24 hours pre-treatment with AZD2281 rendered ATM mutant non-cycling cells sensitive to Fludarabine.We conclude that the PARP inhibitor AZD2281 is capable of targeting CLL cells with defective ATM function for cellular killing. Furthermore, our results indicate that inhibition of PARP is particularly important in the induction of cell death within proliferating ATM mutant CLL cells, which play a major role in tumour progression. Also, the addition of AZD2281 can sensitise non-cycling ATM mutant tumour cells to killing by Fludarabine. Our results suggest that this compound could be effective in the treatment of CLL patients with apoptotic resistant ATM mutant tumours.

Hepatocytes with extensive telomere deprotection and fusion remain viable and regenerate liver mass through endoreduplication

We report that mouse liver cells are highly resistant to extensive telomere dysfunction. In proliferating cells, telomere dysfunction results in chromosome end fusions, a DNA damage signal, and apoptosis or senescence. To determine the consequences of telomere dysfunction in noncycling cells, we used conditional deletion of the telomeric protein TRF2 in hepatocytes. TRF2 loss resulted in telomeric accumulation of gamma-H2AX and frequent telomere fusions, indicating telomere deprotection. However, there was no induction of p53 or apoptosis, and liver function appeared unaffected. Furthermore, the loss of TRF2 did not compromise liver regeneration after partial hepatectomy. Remarkably, liver regeneration occurred without cell division involving endoreduplication and cell growth, thereby circumventing the chromosome segregation problems associated with telomere fusions. We conclude that nondividing hepatocytes can maintain and regenerate liver function despite substantial loss of telomere integrity.

Developmental Modulation of Nonhomologous End Joining in Caenorhabditis elegans

Homologous recombination and nonhomologous end joining (NHEJ) are important DNA double-strand break repair pathways in many organisms. C. elegans strains harboring mutations in the cku-70, cku-80, or lig-4 NHEJ genes displayed multiple developmental abnormalities in response to radiation-induced DNA damage in noncycling somatic cells. These phenotypes did not result from S-phase, DNA damage, or mitotic checkpoints, apoptosis, or stress response pathways that regulate dauer formation. However, an additional defect in him-10, a kinetochore component, synergized with NHEJ mutations for the radiation-induced developmental phenotypes, suggesting that they may be triggered by mis-segregation of chromosome fragments. Although NHEJ was an important DNA repair pathway for noncycling somatic cells in C. elegans, homologous recombination was used to repair radiation-induced DNA damage in cycling somatic cells and in germ cells at all times. Noncycling germ cells that depended on homologous recombination underwent cell cycle arrest in G2, whereas noncycling somatic cells that depended on NHEJ arrested in G1, suggesting that cell cycle phase may modulate DNA repair during development. We conclude that error-prone NHEJ plays little or no role in DNA repair in C. elegans germ cells, possibly ensuring homology-based double-strand break repair and transmission of a stable genome from one generation to the next.

Senataxin, the yeast Sen1p orthologue: Characterization of a unique protein in which recessive mutations cause ataxia and dominant mutations cause motor neuron disease

A severe recessive cerebellar ataxia, Ataxia-Oculomotor Apraxia 2 (AOA2) and a juvenile onset form of dominant amyotrophic lateral sclerosis (ALS4) result from mutations of the Senataxin (SETX) gene. To begin characterization this disease protein, we developed a specific antibody to the DNA/RNA helicase domain of SETX. In murine brain, SETX concentrates in several regions, including cerebellum, hippocampus and olfactory bulb with a general neuronal expression profile, colocalizing with NeuN. In cultured cells, we found that SETX was cytoplasmically diffuse, but in the nucleus, SETX was punctate, colocalizing with fibrillarin, a marker of the nucleolus. In differentiated non-cycling cells, nuclear SETX was not restricted to the nucleolus but was diffuse within the nucleoplasm, suggesting cell-cycle-dependent localization. SETX missense mutations cluster within the N-terminus and helicase domains. Flag tagging at the N-terminus caused protein mislocation to the nucleoplasm and failure to export to the cytoplasm, suggesting that the N-terminus may be essential for correct SETX localization. We report here the first characterization of SETX protein, which may provide future insights into a new mechanism leading to neuron death.