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Suppl. Table 1: IC50 values for the DNA Ligase inhibitor L67 for three cancer cells lines, a telomerase-immortalized fibroblast and the Rho minus derivatives of each of the cell lines; Suppl. Table 2: DNA sequences of primers used for amplification of specific fragments and nuclear and mitochondrial DNA using the polymerase chain reaction; Suppl. Table 3: Reaction conditions used to specific fragments and nuclear and mitochondrial DNA by the polymerase chain reaction

Panel A. Effect of L67 on the cell cycle distribution of synchronized HCA-Ltrt cells. Panel B. Effect of L67 on mitochondrial superoxide level in HCA-Ltrt cells and HCA-Ltrt Rho minus cells

Panel A. Induction of gammaH2AX foci in HeLa cells, HeLa Rho minus cells and HeLa cells expressing mitochondrially targeted E. coli DNA ligase by L67. Panel B. N-acetyl cysteine inhibits L67-induced autophosphorylation of ATM in HeLa cells.

Supplementary Materials and methods plus legends for six supplementary figures

Panel A. Characterization of HeLA cells expressing mitochondrially-targeted E. coli DNA ligase. DNA encoding the E. coli DNA detected by PCR and expression of GFP-tagged E. coli DNA ligase detected by immunoblotting. Panel B. Effect of L67 on DNA ligation by purified DN Ligase IIIalpha/XRCC1 and E.coli DNA ligase

L67 does not induce cleavage of caspases 7 or 9 in HeLa cells.

Elevated levels of DNA ligase IIIα (LigIIIα) have been identified as a biomarker of an alteration in DNA repair in cancer cells that confers hypersensitivity to a LigIIIα inhibitor, L67, in combination with a poly (ADP-ribose) polymerase inhibitor. Because LigIIIα functions in the nucleus and mitochondria, we examined the effect of L67 on these organelles. Here, we show that, although the DNA ligase inhibitor selectively targets mitochondria, cancer and nonmalignant cells respond differently to disruption of mitochondrial DNA metabolism. Inhibition of mitochondrial LigIIIα in cancer cells resulted in abnormal mitochondrial morphology, reduced levels of mitochondrial DNA, and increased levels of mitochondrially generated reactive oxygen species that caused nuclear DNA damage. In contrast, these effects did not occur in nonmalignant cells. Furthermore, inhibition of mitochondrial LigIIIα activated a caspase 1–dependent apoptotic pathway, which is known to be part of inflammatory responses induced by pathogenic microorganisms in cancer, but not nonmalignant cells. These results demonstrate that the disruption of mitochondrial DNA metabolism elicits different responses in nonmalignant and cancer cells and suggests that the abnormal response in cancer cells may be exploited in the development of novel therapeutic strategies that selectively target cancer cells. Cancer Res; 76(18); 5431–41. ©2016 AACR.

To ensure genome integrity, the joining of breaks in the phosphodiester backbone of duplex DNA is required during DNA replication and to complete the repair of almost all types of DNA damage. In human cells, this task is accomplished by DNA ligases encoded by three genes, LIG1, LIG3 and LIG4. Mutations in LIG1 and LIG4 have been identified as the causative factor in two inherited immunodeficiency syndromes. Moreover, there is emerging evidence that DNA ligases may be good targets for the development of novel anti-cancer agents. In this graphical review, we provide an overview of the roles of the DNA ligases encoded by the three human LIG genes in DNA replication and repair.

Alternative end-joining (a-EJ) pathways, which repair DNA double-strand breaks (DSBs), are initiated by end resection that generates 3' single strands. This reaction is shared, at least in part, with homologous recombination but distinguishes a-EJ from the major nonhomologous end-joining pathway. Although the a-EJ pathways make only a minor and poorly understood contribution to DSB repair in nonmalignant cells, there is growing interest in these pathways, as they generate genomic rearrangements that are hallmarks of cancer cells. Here, we review and discuss the current understanding of the mechanisms and regulation of a-EJ pathways, the role of a-EJ in human disease, and the potential utility of a-EJ as a therapeutic target in cancer.

In human cells, there are three genes that encode DNA ligase polypeptides with distinct but overlapping functions. Previously small molecule inhibitors of human DNA ligases were identified using a structure-based approach. Three of these inhibitors, L82, a DNA ligase I (LigI)-selective inhibitor, and L67, an inhibitor of LigI and DNA ligases III (LigIII), and L189, an inhibitor of all three human DNA ligases, have related structures that are composed of two 6-member aromatic rings separated by different linkers. Here we have performed a structure-activity analysis to identify determinants of activity and selectivity. The majority of the LigI-selective inhibitors had a pyridazine ring whereas the LigI/III- and LigIII-selective inhibitors did not. In addition, the aromatic rings in LigI-selective inhibitors had either arylhydrazone or acylhydrazone, but not vinyl linkers. Among the LigI-selective inhibitors, L82-G17 exhibited increased activity against and selectivity for LigI compared with L82. Notably. L82-G17 is an uncompetitive inhibitor of the third step of the ligation reaction, phosphodiester bond formation. Cells expressing LigI were more sensitive to L82-G17 than isogenic LIG1 null cells. Furthermore, cells lacking nuclear LigIIIα, which can substitute for LigI in DNA replication, were also more sensitive to L82-G17 than isogenic parental cells. Together, our results demonstrate that L82-G17 is a LigI-selective inhibitor with utility as a probe of the catalytic activity and cellular functions of LigI and provide a framework for the future design of DNA ligase inhibitors.

Elevated levels of DNA ligase IIIα (LigIIIα) have been identified as a biomarker of an alteration in DNA repair in cancer cells that confers hypersensitivity to a LigIIIα inhibitor, L67, in combination with a poly (ADP-ribose) polymerase inhibitor. Because LigIIIα functions in the nucleus and mitochondria, we examined the effect of L67 on these organelles. Here, we show that, although the DNA ligase inhibitor selectively targets mitochondria, cancer and nonmalignant cells respond differently to disruption of mitochondrial DNA metabolism. Inhibition of mitochondrial LigIIIα in cancer cells resulted in abnormal mitochondrial morphology, reduced levels of mitochondrial DNA, and increased levels of mitochondrially generated reactive oxygen species that caused nuclear DNA damage. In contrast, these effects did not occur in nonmalignant cells. Furthermore, inhibition of mitochondrial LigIIIα activated a caspase 1–dependent apoptotic pathway, which is known to be part of inflammatory responses induced by pathogenic microorganisms in cancer, but not nonmalignant cells. These results demonstrate that the disruption of mitochondrial DNA metabolism elicits different responses in nonmalignant and cancer cells and suggests that the abnormal response in cancer cells may be exploited in the development of novel therapeutic strategies that selectively target cancer cells. Cancer Res; 76(18); 5431–41. ©2016 AACR.

Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.

Expression of oncogenic BCR-ABL in chronic myeloid leukemia (CML) results in increased reactive oxygen species (ROS) that in turn cause increased DNA damage, including DNA double-strand breaks (DSBs). We have previously shown increased error-prone repair of DSBs by nonhomologous end-joining (NHEJ) in CML cells. Recent reports have identified alternative NHEJ pathways that are highly error prone, prompting us to examine the role of the alternative NHEJ pathways in BCR-ABL–positive CML. Importantly, we show that key proteins in the major NHEJ pathway, Artemis and DNA ligase IV, are down-regulated, whereas DNA ligase IIIα, and the protein deleted in Werner syndrome, WRN, are up-regulated. DNA ligase IIIα and WRN form a complex that is recruited to DSBs in CML cells. Furthermore, “knockdown” of either DNA ligase IIIα or WRN leads to increased accumulation of unrepaired DSBs, demonstrating that they contribute to the repair of DSBs. These results indicate that altered DSB repair in CML cells is caused by the increased activity of an alternative NHEJ repair pathway, involving DNA ligase IIIα and WRN. We suggest that, although the repair of ROS-induced DSBs by this pathway contributes to the survival of CML cells, the resultant genomic instability drives disease progression.

This review seeks to cover the data on primary Sjogren’s syndrome (SS) that has accumulated especially during recent years, and that apparently fits into a common theme of cellular stress abnormalities. This includes demonstrations of hyperactivity of danger-sensing antigen-presenting cells (APC), and of a strong response to DNA damage in many different cell types. The relevance of these findings for the pathogenesis of SS will be addressed by applying them to the danger model of immune activation [1, 2]. This model has been put forward by Matzinger, stating that immune activation, including the innate system as well as the adaptive lymphocyte response, is initiated by danger in a broad sense rather than solely by the presence of infectious non-self material. By means of danger signals, tissue stress is postulated to activate the APC of the innate immune system, in turn influencing also the adaptive immune cells, i.e. T and B lymphocytes. Thus, a consequence of the danger model of immune activation is that danger signals can be assumed to be a primary determinant of autoimmunity. A number of factors that threaten the integrity of a tissue or the host may act as danger signals. These are produced by, for example, necrotic cells, hypoxia, low pH and mechanical damage to blood vessels. Danger signals from necrotic cells that strongly stimulate APC to up-regulate HLA and costimulatory molecule expression include heat shock proteins transferred upon cell death to the cell surface [3], and the release from dying cells of high concentrations of crystalline uric acid [4]. CD40L on activated extravasating platelets, hyaluron modified in traumatized tissue, reactive oxygen species (ROS) and non-self molecules such as bacterial cell wall lipopolysaccharides can also act as potent danger signals, by the mechanism of binding to Tolllike receptors and other surface markers on APC [1, 2, 5–8]. Interestingly, physiological cell death manifested as apoptosis, posing no threat to the tissues, has been found to have a relatively minor influence on APC, in accordance with the lack of inflammation being typical of apoptosis [9].

Human polymorphonuclear leukocytes (PMN) have been reported to completely lack of DNA-dependent protein kinase (DNA-PK) which is composed of Ku protein and the catalytic subunit DNA-PKcs, needed for nonhomologous end-joining (NHEJ) of DNA double-strand breaks. Promyelocytic HL-60 cells express a variant form of Ku resulting in enhanced radiation sensitivity. This raises the question if low efficiency of NHEJ, instrumental for the cellular repair of oxidative damage, is a normal characteristic of myeloid differentiation. Here we confirmed the complete lack of DNA-PK in PMN protein extracts, and the expression of the truncated Ku86 variant form in HL-60. However, this degradation of DNA-PK was shown to be due to a DNA-PK-degrading protease in PMN and HL-60. In addition, by using a protease-resistant whole cell assay, both Ku86 and DNA-PKcs could be demonstrated in PMN, suggesting the previously reported absence in PMN of DNA-PK to be an artefact. The levels of Ku86 and DNA-PKcs were much reduced in PMN, as compared with that of the lymphocytes, whereas HL-60 displayed a markedly elevated DNA-PK concentration. In conclusion, our findings provide evidence of reduced, not depleted expression of DNA-PK during the mature stages of myeloid differentiation.

Objective. To examine the stress response, including the role of DNA-dependent protein kinase (DNA-PK), in B cells from Sjogren's syndrome (SS) patients. Methods. B-cell lines were exposed to gamma radiation and then postincubated to allow inducible stress functions to develop. The magnitude of the DNA damage response was monitored with respect to DNA-PK phosphorylation of a p53 peptide, defence protein levels (Ku, DNA-PK catalytic subunit, ATM, p21 and p53) and flow cytometric determination of cell cycle phases and apoptosis. Results. B cells from SS patients, compared with healthy controls, displayed enhancement of two stress functions in undamaged cells: DNA-PK kinase activity and apoptosis. In addition, SS showed enhanced cell cycle arrest in gamma-irradiated cells. Conclusions. Strong kinase activity of DNA-PK, functioning not only in a DNA damage response but also in immunoglobulin gene rearrangement, may be an important component of the heightened stress response displayed by SS cells. In combination with recent reports, our data indicate that constitutional hyper-reactivity to danger signals is a basic pathogenetic factor in SS.

BackgroundCells from primary Sjogren's syndrome (SS) patients have been reported to show alterations in DNA repair and p53 expression. The DNA-dependent protein kinase (DNA-PK) autoantigen may be involved in both of these alterations in relation to cellular DNA damage responses. We conducted this study of cell-cycle kinetics and p53 to find additional evidence for an abnormal stress response role in the pathogenesis of SS. DesignDNA-dependent protein kinase activity, p53 peptide phosphorylation and p53 protein levels were determined in gamma-irradiated long-term T lymphocyte cultures. Cell-cycle progression of peripheral blood mononuclear cells was analysed with flow cytometry. ResultsNo significant differences in the DNA-PK activities or p53 protein levels appeared between the SS patients and the healthy individuals. However, patients with the SS hallmark Ro/SS-A and La/SS-B autoantibodies showed enhancement of both p53 peptide phosphorylation (P = 0·036) and G1 cell-cycle arrest (P = 0·015) in response to gamma radiation. ConclusionsSjogren's syndrome cells express an enhanced G1 checkpoint function which may be mediated partly by p53 phosphorylation, suggesting that an abnormal stress response in SS is of relevance for the development of this autoimmune disease.

This study of stress-related antinuclear antibody (ANA) reactivity was undertaken with the objective of improving clinical ANA testing. ANA was determined by parallel enzyme-linked immunosorbent assays of crude nuclear protein antigen extracted from HEp-2 cells either grown under optimal conditions (providing nonstress ANA antigen) or exposed to stress (providing stress ANA antigen). The stress stimuli used were gamma radiation (causing DNA damage) and a hypertonic environment (causing apoptosis). Signs of stress-related ANA reactivity were seen among connective tissue disease (CTD) patients (including patients with systemic lupus erythematosus; mixed CTD; calcinosis, Reynaud's phenomenon, esophageal motility disorders, sclerodactyly, and telangiectasia; scleroderma; and Sjögren's syndrome): 11% showed stress-positive ANA (i.e., a significantly stronger ANA reactivity with the extract from stressed cells), whereas 21% showed a markedly weaker reaction with the stress antigen. In contrast, among ANA screening patient sera, with no diagnosis of CTD, the fraction showing stress-positive ANA was higher (7 to 8%, depending on the type of stress) than among those showing a lower reactivity with stress antigen (1.5 to 2.5%). Only one serum among 89 (1%) tested sera from healthy individuals showed a stress-related ANA reaction. This demonstration of stress-related ANA suggests a means to improve the performance of clinical ANA testing.

The aim was to examine tissue expression of Ku protein in lower lip salivary gland (LSG) biopsies from cases of primary Sjogren's syndrome (SS) and from normal subjects. Methods: immunohistochemistry was used with antibodies to Ku70/86 and also Ki67, PCNA and p53. In addition, the Klenow method was applied in order to detect evidence of apoptosis. Sections of hyperplastic tonsil served as additional controls. Results: in normal controls, LSG acinar cells stained negatively whereas LSG excretory duct cell nuclei stained positively with Ku and Klenow and occasionally with PCNA but negatively with Ki67 and p53. In LSG focal sialadenitis of SS cases, some lymphocytic cells showed staining with Ku, Ki67, PCNA, Klenow and p53. In addition to duct cell Ku and Klenow as well as PCNA staining which was not much different from normals, a few ductal epithelial and also mononuclear cells stained with p53. In focal sialadenitis, some acinar cells showed staining with PCNA as well as with Klenow. Conclusions: our findings in LSG biopsies of SS cases added little to an increased understanding about the pathogenetic mechanisms in the development of focal sialadenitis in SS. However. in normal LSG. ductal epithelial but not acinar cells seem to express a constitutively specific Ku protein and Klenow profile, suggestive of DNA strand breaks but not clearly associated with ongoing apoptotic events. It may reflect an enhanced stress response, which may be pathogenetically important in the early events of focal sialadenitis development in primary Sjogren's syndrome. (Less)

DNA-dependent protein kinase (DNA-PK) has been shown to take part in cell cycle regulatory signal transduction and in the repair of X-ray-induced DNA double-strand breaks. Functional DNA-PK is furthermore needed for the generation of antigen specificity during lymphocyte maturation. The Ku86 subunit of DNA-PK has been reported to exist in human B lymphocytes in a truncated form capable of binding to broken DNA but lacking the ability to activate the kinase function of DNA-PK. In the present work the Ku70 and Ku86 dimer proteins in T and B lymphocytes from human blood donors were analysed by immunoblotting and were observed apparently to be of full length. Also, nuclear protein extracted from B and non-B lymphocytes displayed DNA-dependent kinase activity. However, a minor fraction of Ku86 in lymphocytes was observed to be truncated with a molecular mass of approx. 70 kDa.

To investigate the role of beta1 integrin during tumor metastasis, we established a ras-myc transformed fibroblastoid cell line with a disrupted beta1 integrin gene on both alleles (GERM 11). Stable transfection of this cell line with an expression vector encoding beta1A integrin resulted in beta1A integrin-expressing sublines. Tumors were induced by subcutaneous injection of GERM 11 cells and 3 independent beta1 integrin expressing sublines (GERM 116, 1A10, 2F2) into syngeneic mice. After 10 days tumors were surgically removed. While average weights of GERM 11 and GERM 116 tumors were similar, tumors induced by the high expressing clones 1A10 and 2F2 were markedly smaller, suggesting an inverse correlation of tumor growth and beta1 integrin expression. The metastasis potential of all three beta1 integrin-expressing GERM 11 sublines tested was significantly higher than that of the beta1-deficient GERM 11 cells. GERM 116 tumors led in all animals to severe metastasis in lung and liver, while GERM 11 tumors induced only a few metastatic foci in the lung. Stroma of both tumors contained nidogen and high amounts of tenascin C, but only a few very low levels of fibronectin, laminin-1, and collagen type I. Beta1 integrin, therefore, increases but is not essential for metastasis of ras-myc transformed fibroblasts.

Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target.

Disease progression in myeloid malignancies results from the accumulation of "mutations" in genes that control cellular growth and differentiation. Many types of genetic alterations have been identified in myeloid diseases. However, the mechanism(s) by which these cells acquire genetic alterations or "Genomic instability", is less well understood. Increasing evidence suggests that the genetic changes in myeloid malignancies lead to increased production of endogenous sources of DNA damage, such as, reactive oxygen species (ROS). The fusion gene BCR-ABL in chronic myeloid leukemia (CML), FLT3/ITD in acute myeloid leukemia (AML), and RAS mutations in myelodysplastic syndromes (MDS)/myeloproliferative diseases (MPD) result in ROS production. Increased ROS can drive a cycle of genomic instability leading to DNA double strand breaks (DSBs) and altered repair that can lead to acquisition of genomic changes. Evidence is coming to light that defects in a main repair pathway for DSBs, non-homologous end-joining (NHEJ), lead to up-regulation of alternative or "back-up" repair that can create chromosomal deletions and translocations. This article will review evidence for activation of RAS/PI3K/STAT pathways, that lead to increased ROS, DNA damage and defective repair in myeloid diseases, a mechanism for acquisition of additional mutations that can drive disease progression.

Serial sections of lower lip salivary gland (LSG) biopsies were examined by immunohistochemistry, using a battery of B- and partly T-related antibodies (CD5, CD20, CD21, CD27, CD38, CD45RO, CD79a, Bcl-2 and Bcl-6) in different groups of subjects: healthy controls and clinically verified smoking or nonsmoking cases of primary Sjogren's syndrome (SS). The purpose was to characterize the B-cell pattern of the lymphocytic foci and of the tiny perivascular infiltrates preceding the development of foci. Hyperplastic tonsil was used as stain control. In normal LSG, widely dispersed CD38+ and CD79a+ as well as some CD5+ cells are a normal constituent, with lack of staining with the other antibodies. In SS/LSG, the lymphocytic foci showed staining with all the antibodies, with variable degrees of overlapping or nonoverlapping. In SS/LSG of nonsmokers, CD20+ B cells make up a prominent part of the fully developed periductal lymphocytic foci, not overlapping with CD45RO. Also, CD20+ B cells did not overlap in the infiltrates with colocalized CD27+/CD38+ cells. CD20+ B cells and CD45RO+ T cells also occur as minute infiltrates perivascularly in areas of no foci in SS/LSG as well as in SS smokers lacking the typical foci. Smokers lack foci, but tiny infiltrates express CD20 as well CD45R0. Our findings suggest that CD20+ B cells and CD45RO+ T cells are early immigrants in the LSG of SS of smokers as well as nonsmokers and that another subgroup of CD27+/CD38+ B cells gradually mix with the first two to form the characteristic foci in SS/LSG. The simultaneous demonstration of CD20+ and CD27+ B cells in SS/LSG may constitute a significant diagnostic tool. Further, the findings suggest that the early immigrating lymphocytes may have been primed at a site remote from the glands before arriving via the blood to the gland tissue.

BCR-ABL fusion tyrosine kinase in chronic myeloid leukemia (CML), induces high levels of ROS that generate DNA double strand breaks (DSBs). We previously showed that CML cells repair DSBs by aberrant non homologous end-joining (NHEJ) that is characterized by large DNA deletions. The generation of DNA deletions represents a mechanism by which genomic alterations may be acquired in the progression of chronic phase CML to blast crisis. Recently, we demonstrated that a “back-up” or alternative NHEJ pathway is involved in aberrant repair of DSBs in CML. Proteins in this pathway include, DNA ligase IIIα, XRCC1 and poly(-ADP) ribose polymerase (PARP). We have identified that NHEJ proteins, DNA ligase IIIα and WRN are overexpressed in CML. This increased expression appears to be dependent on the presence of BCR-ABL. “Knockdown” of these proteins leads to an accumulation of unrepaired DSBs, demonstrating their essential involvement in DSB repair in CML cells. The goal of the current study is to evaluate the effect of inhibiting “back-up” DNA repair proteins in proliferation and apoptosis of BCR-ABL-positive CML compared with standard Imatinib therapy. To evaluate whether “back-up” repair proteins may be therapeutic targets, we used siRNA down-regulation and small molecule inhibition of DNA ligase IIIα and PARP in BCR-ABL positive cell lines. Importantly, we have recently identified small molecule inhibitors of DNA Ligases by computer aided drug design (CADD). Inhibition of DNA ligases and PARP result in a significant increase in apoptosis of CML cells (K562, Kasumi 4, MEG01 and KU812 and P210 MO7e), comparable with the cell death observed with imatinib treatment. Importantly, CML cell lines resistant to imatinib treatment demonstrate similar apoptotic levels in response to “back-up” repair protein inhibition. These inhibitors are in the process of being tested in CML xenografts and mouse models for therapeutic efficacy in vivo. Our data suggest that the survival of CML cells is at least in part maintained by repair of DSBs using “Back-up” NHEJ. The main proteins involved in this pathway, which include DNA ligase IIIα, XRCCI, DNA Ligase I, PARP and WRN have the potential to be novel therapeutic targets in CML patients that have acquired resistance to imatinib.

The oncogenic BCR-ABL in CML produces increased reactive oxygen species (ROS) leading to DSB and aberrant repair. We have previously shown that CML cells demonstrate an increased frequency of errors of non homologous end-joining (NHEJ). DSB are repaired by two major pathways, homologous recombination (HR) and NHEJ, the dominant pathway in eukaryotic cells, also known as DNA-PK dependent NHEJ (D-NHEJ). Recent reports have identified alternative or “back-up” NHEJ pathways (B-NHEJ) that are highly error-prone, and may explain the altered DSB repair reported in CML. To determine the mechanism for the aberrant NHEJ repair in CML, we examined steady state levels of D-NHEJ proteins, including Ku70/86, DNA-PKcs, Artemis and DNA Ligase IV/XRCC4 in four different BCR-ABL positive CML cell lines compared with three lymphoblastoid cell lines established from normal individuals and one BCR-ABL negative CML cell line. We find that two key components of D-NHEJ, Artemis (4–7 fold) and DNA Ligase IV (2–3 fold) are down-regulated, compared with controls. These data suggest that D-NHEJ repair is compromised in CML. To determine whether alternative NHEJ repair plays a role in the aberrant repair of DSB in CML cells, we next examined expression levels of DNA Ligase IIIα/XRCC1, PARP and other proteins known to be associated with NHEJ repair, such as the protein found to be deleted in Werner’s syndrome, WRN. We find that WRN and DNA Ligase IIIα are increased (3–6 fold) in BCR-ABL-positive CML compared with control cell lines. Importantly, DNA Ligase IIIα/XRCC1 forms a complex with WRN, suggesting that it may be a new member of the alternative repair pathway. To confirm that up-regulation of DNA Ligase IIIα and WRN are elicited by BCR-ABL, we examined the levels of these proteins in primary samples (N=4) from patients with different levels of BCR-ABL, following treatment with the tyrosine kinase inhibitor Gleevec. WRN and DNA Ligase IIIα are down regulated in patient samples where BCR-ABL levels are significantly decreased. Furthermore, we confirmed that these up-regulated proteins are involved in DSB repair in CML cells because they co-localize to induced DSB in BCR-ABL-positive cell lines stably transfected with DSB-containing DRneo plasmid, using fluorescence in situ hybridization (FISH) co-immunostaining. Importantly we show that siRNA down-regulation of WRN and DNA Ligase IIIα leads to elevated levels of unrepaired DSB and a decreased frequency of DSB repair efficiency in CML cells. In addition siRNA down-regulation of WRN leads to large deletions at the site of repair, while siRNA down-regulation of DNA Ligase IIIα results in an increased frequency of misrepair. Finally, we determined whether “correction” of main NHEJ pathway proteins in CML can lead to a decrease in the frequency of errors of end-joining repair. Over-expression of Artemis using pcDNA constructs in CML cells leads to more correct end-joining, compared with vector transfected controls. We conclude that down-regulation of Artemis and DNA Ligase IV leads to compensatory up-regulation of alternative repair pathways in BCR-ABL-positive CML cells, and suggest a role for a new protein complex in CML, in protecting and joining DNA ends, thus ensuring the survival of CML cells. Inhibition of alternative NHEJ repair may be explored in combination with other agents as a therapeutic strategy in CML.

The FMS-like tyrosine kinase (FLT3), which belongs to the class III receptor tyrosine kinase family, is primarily expressed by hematopoietic cells and plays an important role in hematopoiesis. Activating mutations of FLT3 occur in approximately 30% of myeloid malignancies and, at least for the internal tandem duplications (ITDs) of the juxtamembrane region, are an independent predictor of poor clinical outcome. FLT3 signaling in leukemia includes activation of RAS, PI3 kinase/AKT pathways, and signal transducer and activator of transcription-5 (STAT5), leading to increased cellular proliferation and survival. We have previously shown that N-RAS activation can initiate a cycle of genomic instability whereby increased reactive oxygen species (ROS) leads to double strand breaks (DSB) and error-prone repair in myeloid malignancies. Virtually nothing is known about the role of FLT3 mutations in genomic instability. Here we report that FLT3/ITD signaling can lead to genomic instability that may be driven by increased ROS production. We find that mouse myeloid progenitor cells (32D) stably expressing FLT3/ITD (32D/ITD), demonstrate significantly increased ROS production compared with isogenic controls, as measured by flow cytometric analysis of the fuorescent probe, 2’,7’-dichlorofluoresecein (H2DCHF-DA) which is oxidized in the presence of ROS. FLT3/ITD cells treated with small molecule tyrosine kinase inhibitors (TKI) of FLT3 significantly reduced ROS levels. Intriguingly, we find that phosphorylated STAT5 (pSTAT5), which is greatly stimulated by mutant FLT3 signaling, binds RAC1-GTP, an essential component of ROS-producing NADPH oxidase. This novel finding suggests that STAT5 may have unique non-genomic functions involved in regulating RAC1 activation and ROS generation. FLT3 mutant cells treated with siRNA to down-regulate STAT5, show significantly reduced ROS. Inhibition of RAC1 and NADPH oxidase in FLT3 mutant cells also leads to reduced ROS levels, providing further evidence for the involvement of RAC1 in ROS production. While RAC1 activity is unchanged by FLT3 inhibition, significantly less RAC1 binds NADPH oxidase protein GP91phox in the cell membrane. Therefore pSTAT5 binding to RAC1-GTP may be necessary for NADPH oxidase activity and ROS production. Importantly, human leukemia cell lines (MOLM-14 & MV-4-11) and primary samples (N=3) with naturally occurring FLT3/ITD also show significantly decreased ROS in a dose- and time-dependent manner, following treatment with FLT3 TKI, confirming the mouse cell line data. Blocking ROS production by inhibition of FLT3 in AML cells significantly decreased γH2AX foci formation, suggesting reduced DSB. An in vitro DSB repair assay based on plasmid rejoining in nuclear extracts was used to assess repair efficiency and fidelity. Higher repair efficiency and fidelity was observed following inhibition of the FLT3-mediated endogenous ROS production in FLT3/ITD-positive human AML cells. Our data serve as a model system to further study the role of FLT3-mediated increased ROS in propagating genomic instability and for therapeutic targeting in an effort to stabilize the genome and reduce the accumulation of additional mutations in these cases of AML.

(2003). A role of the macrophage in Sjogen's syndrome? Scandinavian Journal of Rheumatology: Vol. 32, No. 4, pp. 255-255.

The Ku autoantigen has been implicated in a number of cellular functions including growth control, immunoglobulin gene rearrangement and DNA repair. A variant truncated form of Ku86, with an apparent molecular weight of 70 kDa, has been reported to be present in many human cell types. We have previously shown that the amount of variant Ku86 is strongly increased in human peripheral blood mononuclear cells (PBMC) by storage of blood prior to isolation of the PBMC. In this study we report that formation of variant Ku86 in protein extracts is mediated by an inducible trypsin-like serine protease with a higher concentration in the nuclear compartment, as compared with the cytoplasm. However, experiments with SDS-PAGE assay of whole cells yielded no evidence of truncated Ku86, suggesting that the protease is not active in intact cells, but is exerting a marked activity during the protein extraction procedure. Interestingly, the protease level became markedly reduced upon transfer of the cells to growth medium. Protease induction did not correlate with apoptosis, necrotic cell death or with signs of general proteolysis or cytotoxicity. Our findings have methodological implications for the interpretation of experimental Ku86 data, and suggest that this protease may play a role for cellular regulation of Ku function.