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Background and objective Liver abscesses are one of the common surgical diseases to be treated as an emergency in any tertiary care hospital in India. The formation of abscesses in the liver is still a major problem and associated with significant morbidity in developing countries. We come across all types of liver abscesses, such as amoebic (most common), pyogenic, mixed, and occasionally fungal. There have been several studies on the percutaneous modality of treatment for pyogenic liver abscesses. Most of the studies suggest that percutaneous catheter drainage (PCD) offers a better approach than aspirations for treating pyogenic liver abscesses. However, a few recent studies suggest that percutaneous aspiration leads to equally good results when compared to percutaneous drainage. In this study, we aimed to review the management of solitary large pyogenic liver abscesses and to assess the effectiveness of ultrasonography (USG)-guided aspiration in the procedure. Methods A retrospective study was carried out at the Department of General Surgery of our institute. In this study, a total of 27 patients treated for solitary pyogenic liver abscess were included. All patients with a large liver abscess greater than 5 cm without the features of frank peritonitis were included. These patients were followed up regularly for six months. Results The single-attempt USG-guided aspiration was successful in 70.3% of patients. Repeat USG-guided aspiration was performed in 18.5% of patients. In 7.4% of patients, a USG-guided percutaneous pigtail catheter was placed. And only 3.7% of cases required exploratory laparotomy. Conclusion Based on our findings, USG-guided aspiration is a fairly efficient method for treating a large solitary pyogenic abscess with acceptable results, shorter hospital stays, and minimal complications.

Background Psoas abscess (PA) is an uncommon disease involving infection of the psoas muscle with abscess formation. The evidence concerning clinical and diagnostic characteristics of PA and its outcomes is limited. The literature is heterogenous, with varying presentations and outcomes in different regions worldwide. We present a retrospective analysis of the clinical, radiological, and laboratory characteristics of PA, its management, and outcomes from a tertiary care center in North India. Methodology We reviewed the clinical records of confirmed cases of PA treated in our institute from January 2016 to December 2020 with a minimum follow-up of one year. Further, we performed a descriptive analysis of demographic characteristics, clinical features, laboratory parameters, radiological investigations, the basis of diagnosis confirmation, causative microorganisms, definitive management, treatment outcomes, and complications. Results We reviewed 33 cases with a mean age of 29.9 ± 16.8 years. Overall, 48.4% of PAs were right-sided, and 24.2% were bilateral. Abdominal discomfort was the most common presenting symptom. Blood laboratory parameters were mostly within the near-normal range except for the elevated erythrocyte sedimentation rate, C-reactive protein, and neutrophil-to-lymphocyte ratio. Ultrasonography was the most commonly performed radiological investigation and was the basis of diagnosis confirmation.

The pathogenesis of inherited genome instability neurodegenerative syndromes remains largely unknown. Here, we report new disease-relevant murine models of genome instability–driven neurodegeneration involving disabled ATM and APTX that develop debilitating ataxia. We show that neurodegeneration and ataxia result from transcriptional interference in the cerebellum via aberrant messenger RNA splicing. Unexpectedly, these splicing defects were restricted to only Purkinje cells, disrupting the expression of critical homeostatic regulators including

There is emerging interest in the role of poly(ADP-ribose) polymerase-1 (PARP-1) in neurodegeneration and potential of its therapeutic targeting in neurodegenerative disorders. New generations of PARP inhibitors exhibit polypharmacological properties; they do not only block enzymatic activity with lower doses, but also alter how PARP-1 interacts with DNA. While these new inhibitors have proven useful in cancer therapy due to their ability to kill cancer cell, their use in neurodegenerative disorders has an opposite goal: cell protection. We hypothesize that newer generation PARP-1 inhibitors jeopardize the viability of dividing CNS cells by promoting DNA damage upon the PARP-DNA interaction. Using enriched murine astrocyte cultures, our study evaluates the effects of a variety of drugs known to inhibit PARP; talazoparib, olaparib, PJ34 and minocycline. Despite similar PARP enzymatic inhibiting activities, we show here that these drugs result in varied cell viability. Talazoparib and olaparib reduce astrocyte growth in a dose-dependent manner, while astrocytes remain unaffected by PJ34 and minocycline. Similarly, PJ34 and minocycline do not jeopardize DNA integrity, while treatment with talazoparib and olaparib promote DNA damage. These two drugs impact astrocytes similarly in basal conditions and upon nitrosative stress, a pathological condition typical for neurodegeneration. Mechanistic assessment revealed that talazoparib and olaparib promote PARP trapping onto DNA in a dose-dependent manner, while PJ34 and minocycline do not induce PARP-DNA trapping. This study provides unique insight into the selective use of PARP inhibitors to treat neurodegenerative disorders whereby inhibition of PARP enzymatic activity must occur without deleteriously trapping PARP onto DNA.

PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of PTEN to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of BRCA-mutated and other HR-deficient (HRD) tumors. Methods: Primary Pten-deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and PTEN-null human tumor cell lines and primary cells were assessed for RAD51 expression (via the Western blot analysis) and DNA damage repair analyses (via alkali comet and &gamma, H2AX foci assays). RAD51 foci analysis was used to measure HR-dependent DNA repair. Xrcc2-deficient MEFs served as an HR-deficient control, while the stable knockdown of RAD51 (shRAD51) served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and shRAD51-expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of PTEN-null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of RAD51 and its paralogs were examined as a function of the PTEN status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs. Results: Pten knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking Xrcc2. Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency (U87, PTEN-mutated, U251 and U373, PTEN-null) show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against RAD51 (shRAD51) display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of PTEN-inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of RAD51 and its paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and DMC1), regardless of the PTEN mutational status. Conclusions: Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural Pten-null cells, PTEN-deficient tumor cell lines, and primary PTEN-mutant GBM patient-derived tissue specimens and BTICs.

One hallmark of cancer is its ability to recruit a vascular supply to support rapid growth. Suppression of angiogenesis holds potential as a second-line or adjuvant therapy to stunt cancer growth, progression, metastasis, and post-resection regeneration. To begin to test the hypothesis that semaphorin 3A and 3F together, will induce endothelial cell apoptosis by inducing DNA damage, mixed primary cultures isolated from normal adult mouse skeletal muscle were treated for 48 hr with Sema3A ± Sema3F (100ng/mL). Changes in surviving-cell density, DNA synthesis, DNA repair (gamma-Histone 2AX, γH2AX, an indirect measure for DNA damage), and apoptotic DNA fragmentation (TUNEL staining) were assayed in cultures of CD31+ endothelial and desmin+ muscle cells. Sema3F increased DNA damage-associated DNA repair in both cell types. Co-treatment with Sema3A+3F increased γH2AX staining ~25-fold over control levels, and further increased apoptosis compared to control and Sema3A alone. Results were negated by treatment with neutralizing anti-semaphorin antibodies and are interpreted as suggesting that Sema3A may sensitize endothelial but not muscle cells to Sema3F-induced DNA damage. These preliminary findings on a complex system of interacting cells may contribute to developing applications that could target angiogenic regulatory mechanisms for their therapeutic potential against cancer progression and metastasis.

The phosphatidyl-inositol 3 kinase (PI3K) &delta, inhibitor, idelalisib (IDE), is a potent inhibitor of the B-cell receptor pathway and a novel and highly effective agent for the treatment of chronic lymphocytic leukemia (CLL). We evaluated the activities of IDE in comparison to bendamusine (BEN), a commonly used alkylating agent, in primary CLL cells ex vivo. In contrast to BEN, IDE was cytotoxic to cells from extensively-treated patients, including those with a deletion (del)17p. Cross-resistance was not observed between BEN and IDE, confirming their different modes of cytotoxicity. Marked synergy was seen between BEN and IDE, even in cases that were resistant to BEN or IDE individually, and those with deletion (del) 17p. CD40L/interleukin 4 (IL4) co-treatment mimicking the CLL microenvironment increased resistance to IDE, but synergy was retained. PI3K&delta, deficient murine splenic B cells were more resistant to IDE and showed reduced synergy with BEN, thus confirming the importance of functional PI3K&delta, protein. Although IDE was observed to induce &gamma, H2AX, IDE did not enhance activation of the DNA damage response nor DNA repair activity. Interestingly, IDE decreased global RNA synthesis and was antagonistic with 5,6-Dichlorobenzimidazole 1-b-D-ribofuranoside (DRB), an inhibitor of transcription. These findings add to the increasingly complex cellular effects of IDE, and B cell receptor (BCR) inhibitors in general, in CLL.

The BH3-only family member BNIP3 has been described as either promoting cell survival or cell death. This depends upon the level of BNIP3 expression and its cellular localization. Increased BNIP3 expression under hypoxia contributes to cell death through increased mitochondrial dysfunction. Furthermore, mice lacking BNIP3 show inhibition of ischemic cardiomyocyte apoptosis. In contrast, nuclear localization of BNIP3 contributes to blockage of apoptosis in glioma cells through repression of pro-apoptotic genes. We have discovered that mouse embryonic fibroblasts (MEFs) lacking BNIP3 expression show increased proliferation and cell number compared to wild-type cells. Furthermore, the cells lacking BNIP3 showed increased MAPK activation. Increased proliferation was not due to decreased cell death as oxidative stress induced cell death in BNIP3 null MEFs. In addition, we isolated astrocytes from wild-type or embryonic mice lacking expression of BNIP3. There was increased density and cell number in the astrocytes lacking BNIP3 expression. To confirm these results in human cells, we inducibly expressed BNIP3 in human embryonic kidney (HEK293) cells and found that induced BNIP3 reduced cell proliferation and failed to change background cell death levels. Transient over-expression of BNIP3 in the nucleus of HEK293 cells also reduced DNA synthesis. Finally, to determine whether this increased proliferation occurs in mice lacking BNIP3, we isolated brains from wild-type mice or those lacking BNIP3 expression. The mice lacking BNIP3 had increased cellularity in the brain of embryonic and adult mice. Taken together, our study describes a new function for BNIP3 in the regulation of cellular proliferation.

DNA damage is considered a prime factor in multiple spinocerebellar neurodegenerative diseases; however, the DNA lesions underpinning disease etiology are unknown. Here we identify the endogenous accumulation of pathogenic topoisomerase-1-DNA cleavage complexes (Top1cc) in murine models of ataxia telangiectasia and spinocerebellar ataxia with axonal neuropathy 1. We also show that the defective DNA damage response factors in these two diseases cooperatively modulate Top1cc turnover in a non-epistatic and ATM kinase-independent manner. Furthermore, coincident neural inactivation of ATM and DNA single strand break repair factors including tyrosyl-DNA phosphodiesterase-1 or XRCC1 result in increased Top1cc formation and excessive DNA damage and neurodevelopmental defects. Importantly, direct topoisomerase-1 poisoning to elevate Top1cc levels phenocopies the neuropathology of the mouse models above. Our study identifies a critical endogenous pathogenic lesion associated with neurodegenerative syndromes arising from DNA repair deficiency, indicating the essential role that genome integrity plays in preventing disease in the nervous system.

Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ0 phenotype. Background: Nonviability of cells lacking mitochondrial DNA ligase suggests essential function of this enzyme. Results: We report the isolation of viable Lig3−/− cells, which lack mtDNA. Conclusion: The lethality of the Lig3 knock-out is mediated by the ρ0 phenotype. Significance: This is definitive proof that the essential function of LIG3 in mitochondria is limited to DNA transactions.

The rapid proliferation of progenitors during neurogenesis requires a stringent genomic maintenance program to ensure transmission of genetic fidelity. However the essential factors that govern neural progenitor genome integrity are unknown. Here we report that conditional inactivation of mouse TopBP1, a protein linked to DNA replication, and a key activator of the DNA damage response kinase ATR (ataxia telangiectasia and rad3-related) is critical for maintenance of early-born neural progenitors. During cortical development TopBP1 prevented replication-associated DNA damage in Emx1-progenitors which otherwise resulted in profound tissue ablation. Notably, disrupted neurogenesis in TopBP1-depleted tissues was substantially rescued by inactivation of p53 but not of ATM. Our data establish that TopBP1 is essential for preventing replication-associated DNA strand breaks, but is not essential per se for DNA replication. Thus, TopBP1 is crucial for maintaining genome integrity in the early progenitors that drive neurogenesis.

The ATR (ATM (ataxia telangiectasia mutated) and rad3related) checkpoint kinase is considered critical for signalling DNA replication stress and its dysfunction can lead to the neurodevelopmental disorder, ATR-Seckel syndrome. To understand how ATR functions during neurogenesis, we conditionally deleted Atr broadly throughout the murine nervous system, or in a restricted manner in the dorsal telencephalon. Unexpectedly, in both scenarios, Atr loss impacted neurogenesis relatively late during neural development involving only certain progenitor populations. Whereas the Atr-deficient embryonic cerebellar external germinal layer underwent p53- (and p16 Ink4a/Arf )-independent proliferation arrest, other brain regions suffered apoptosis that was partially p53 dependent. In contrast to other organs, in the nervous system, p53 loss did not worsen the outcome of Atr inactivation. Coincident inactivation of Atm also did not affect the phenotype after Atr deletion, supporting non-overlapping physiological roles for these related DNA damage-response kinases in the brain. Rather than an essential general role in preventing replication stress, our data indicate that ATR functions to monitor genomic integrity in a selective spatiotemporal manner during neurogenesis.

Mammalian cells contain three different DNA ligase enzymes, each with different properties but all involved in DNA replication and repair. Ligase III (Lig3) is known to form a complex with the nuclear DNA repair protein Xrcc1, and Lig3 null animals cannot be made. This raises the question of whether this nuclear role in base-excision repair (BER) is the critical function of Lig3 that maintains viability. Two groups reporting in this issue of Nature investigate different aspects of Lig3 function in vivo, both concluding that the catalytic activity of Lig3 is critical for mitochondrial DNA maintenance and viability, but unexpectedly, is dispensable for Xrcc1-mediated nuclear BER. These findings suggest that Lig3 mutations might cause some of the human syndromes associated with defects in the replication and/or repair of mitochondrial DNA. In the nucleus, a complex of DNA ligase III (Lig3) and Xrcc1 catalyses the last step of base excision repair. Inactivation of Lig3 in the mouse leads to early embryonic lethality, but the critical role played by Lig3 in viability is unknown. This study shows, using conditional knockouts of Lig3 in the nervous system and cardiac muscle, that its essential function is maintenance of mitochondrial DNA integrity. The results also indicate distinct functional roles for Lig3 and Xrcc1. DNA replication and repair in mammalian cells involves three distinct DNA ligases: ligase I (Lig1), ligase III (Lig3) and ligase IV (Lig4)1. Lig3 is considered a key ligase during base excision repair because its stability depends upon its nuclear binding partner Xrcc1, a critical factor for this DNA repair pathway2,3. Lig3 is also present in the mitochondria, where its role in mitochondrial DNA (mtDNA) maintenance is independent of Xrcc1 (ref. 4). However, the biological role of Lig3 is unclear as inactivation of murine Lig3 results in early embryonic lethality5. Here we report that Lig3 is essential for mtDNA integrity but dispensable for nuclear DNA repair. Inactivation of Lig3 in the mouse nervous system resulted in mtDNA loss leading to profound mitochondrial dysfunction, disruption of cellular homeostasis and incapacitating ataxia. Similarly, inactivation of Lig3 in cardiac muscle resulted in mitochondrial dysfunction and defective heart-pump function leading to heart failure. However, Lig3 inactivation did not result in nuclear DNA repair deficiency, indicating essential DNA repair functions of Xrcc1 can occur in the absence of Lig3. Instead, we found that Lig1 was critical for DNA repair, but acted in a cooperative manner with Lig3. Additionally, Lig3 deficiency did not recapitulate the hallmark features of neural Xrcc1 inactivation such as DNA damage-induced cerebellar interneuron loss6, further underscoring functional separation of these DNA repair factors. Therefore, our data reveal that the critical biological role of Lig3 is to maintain mtDNA integrity and not Xrcc1-dependent DNA repair.

Idelalisib (IDE) is an inhibitor of the δ isoform of PI3 kinase and has shown high activity in chronic lymphocytic leukemia (CLL) either when given alone or in combination with bendamustine (BEN) /rituximab (BR). In the present study, we have determined whether there is cross-resistance between BEN and IDE in primary CLL cells in vitro and whether synergy is observed upon combining these agents. In primary CLL cells in vitro, cross-resistance was not observed between BEN and IDE suggesting different modes of cytotoxicity. In contrast to BEN, sensitivity to IDE was not influenced by prior clinical treatment or the presence of a del 17p. Marked synergy in cytotoxicity was seen between BEN and IDE, which was paralleled by changes in γ-H2AX staining. These findings suggest that the synergistic antitumor effect was related to enhanced DNA damage by the drug combination. The degree of cytotoxic synergy varied and synergy was observed in some cases that were resistant to BEN or IDE, and those with del 17p. Synergy between BEN and IDE was also seen in the B cell lines, BJAB and I83, and in healthy donor peripheral blood mononuclear cells indicating that the phenomenon was not CLL-specific. To simulate the microenvironment, CLL cells were pre-stimulating with CD40L and IL4 prior to drug treatment. Interestingly this treatment caused the cells to become resistant to IDE, but BEN sensitivity was unaffected. However, combining BEN and IDE produced greater synergy than observed when cells were incubated without IL4/CD40. To examine the role of PI3Kδ for synergy, B cells from C57 BL/6 mice, both wild type and with non-functioning PI3Kδ protein, were exposed to BEN and/or IDE. Cells with the non-functioning PI3Kδ protein were more resistant to IDE than the wild-type cells and showed lesser synergy confirming the importance of this protein for the synergistic effect. In summary, these results confirm that IDE can be active against BEN-resistant CLL cells, while BEN may remain active against IDE-resistant samples, demonstrating different modes of anti-tumor activity. However, the unique yet inconsistent synergy seen between these two agents suggest an overlapping mechanism of action. Ongoing studies are evaluating the mechanism for this synergy. Citation Format: Sara E. F. Kost, Ali Saleh, Edgard M. Mejia, Marina Mostafizar, Eric D. J. Bouchard, Versha Banerji, Aaron J. Marshall, Spencer B. Gibson, Sachin Katyal, James B. Johnston. Cross-resistance and synergy between idelalisib and bendamustine in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1891.

Defective responses to DNA single- or double-strand breaks can result in neurological disease, underscoring the critical importance of DNA repair for neural homeostasis. Human DNA repair-deficient syndromes are generally congenital, in which brain pathology reflects the consequences of developmentally incurred DNA damage. Although, it is unclear to what degree DNA strand-break repair defects in mature neural cells contributes to disease pathology. However, DNA single-strand breaks are a relatively common lesion which if not repaired can impact cells via interference with transcription. Thus, this lesion, and probably to a lesser extent DNA double-strand breaks, may be particularly relevant to aging in the neural cell population. In this review we will examine the consequences of defective DNA strand-break repair towards homeostasis in the brain. Further, we also consider the utility of mouse models as reagents to understand the connection between DNA strand breaks and aging in the brain.

Bendamustine (BEN) has structural similarities to an alkylating agent and a nucleoside analog, and effective against tumor cells that are resistant to standard therapy. In this study we compared the activities of BEN against that of the alkylating agent, chlorambucil (CLB), and the nucleoside analogs, fludarabine (FLU) and deoxyadenosine/pentostatin (dADO/PEN), in primary chronic lymphocytic leukemia (CLL) cells in vitro. Cross-resistance was observed between BEN, CLB and FLU, with previously treated patients or those with a deletion 17p being most resistant. In contrast, some resistant CLL cells retained moderate sensitivity to dADO/PEN. Like FLU and CLB, BEN induced apoptosis through both the mitochondrial and death receptor pathways. There was a greater increase in DNA double-strand breaks (DSB) following FLU, as compared to BEN and CLB. Synergistic cytotoxicity was seen on combining BEN or CLB with FLU or dADO/PEN, but not when combining BEN with CLB. These results demonstrate that BEN acts as an alkylating agent, demonstrates cross-resistance to CLB and FLU and resistance to cells with a del 17p. Synergistic cytotoxic activity was seen between BEN and dADO/PEN suggesting that the combination of BEN and PEN should be evaluated in the clinic.

Defects in the response to DNA single-strand or double-strand breaks underpin many human diseases associated with disorders of the nervous system. During nervous system development endogenous DNA damage often results in apoptosis, although cell replacement can occur from germinal zones within this rapidly proliferating tissue. However, if this damage surveillance is faulty, cells with genomic damage may inappropriately become incorporated into the nervous system, and the subsequent demise of these cells may result in neurodegeneration. Ataxia telangiectasia results from defective DNA double strand break signaling, and during development a failure to eliminate damaged neural precursor cells may cause the neurodegeneration present in this disease. In contrast, postmitotic neurons in the mature brain are faced with a less facile option, and in this situation DNA breaks may interfere with transcription required for cellular survival. This scenario may reflect neurodegeneration that occurs in spinocerebellar ataxia with axonal neuropathy, in which single strand break repair is defective. Therefore, the response to DNA damage in the nervous system occurs in a distinct spatiotemporal manner utilizing different DNA repair pathways to ensure genomic stability and to prevent disease.

There are two developmentally regulated alternatively spliced forms of Disabled-1 (Dab1) in the chick retina: an early form (Dab1-E) expressed in retinal precursor cells and a late form (Dab1-L) expressed in neuronal cells. The main difference between these two isoforms is the absence of two Src family kinase (SFK) recognition sites in Dab1-E. Both forms retain two Abl/Crk/Nck recognition sites implicated in the recruitment of SH2 domain-containing signaling proteins. One of the Dab1-L-specific SFK recognition sites, at tyrosine(Y)-198, has been shown to be phosphorylated in Reelin-stimulated neurons. Here, we use Reelin-expressing primary retinal cultures to investigate the role of the four Dab1 tyrosine phosphorylation sites on overall tyrosine phosphorylation, Dab1 phosphorylation, SFK activation and neurite formation. We show that Y198 is essential but not sufficient for maximal Dab1 phosphorylation, SFK activation and neurite formation, with Y232 and Y220 playing particularly important roles in SFK activation and neuritogenesis, and Y185 having modifying effects secondary to Y232 and Y220. Our data support a role for all four Dab1 tyrosine phosphorylation sites in mediating the spectrum of activities associated with Reelin-Dab1 signaling in neurons.

BACKGROUND: Atypical teratoid rhabdoid tumor (ATRTs) constitute 20% of brain tumors in young children (

Ataxia oculomotor apraxia-1 (AOA1) is a neurological disorder caused by mutations in the gene (APTX) encoding aprataxin1, 2. Aprataxin is a member of the histidine triad (HIT) family of nucleotide hydrolases and transferases3, and inactivating mutations are largely confined to this HIT domain. Aprataxin associates with the DNA repair proteins XRCC1 and XRCC4, which are partners of DNA ligase III and ligase IV, respectively4, 5, 6, 7, suggestive of a role in DNA repair. Consistent with this, APTX-defective cell lines are sensitive to agents that cause single-strand breaks and exhibit an increased incidence of induced chromosomal aberrations4, 5, 8. It is not, however, known whether aprataxin has a direct or indirect role in DNA repair, or what the physiological substrate of aprataxin might be. Here we show, using purified aprataxin protein and extracts derived from either APTX-defective chicken DT40 cells or Aptx-/- mouse primary neural cells, that aprataxin resolves abortive DNA ligation intermediates. Specifically, aprataxin catalyses the nucleophilic release of adenylate groups covalently linked to 5'-phosphate termini at single-strand nicks and gaps, resulting in the production of 5'-phosphate termini that can be efficiently rejoined. These data indicate that neurological disorders associated with APTX mutations may be caused by the gradual accumulation of unrepaired DNA strand breaks resulting from abortive DNA ligation events.

DDX1 bodies, cleavage bodies, Cajal bodies (CBs), and gems are nuclear suborganelles that contain factors involved in RNA transcription and/or processing. Although all four nuclear bodies can exist as distinct entities, they often colocalize or overlap with each other. To better understand the relationship between these four nuclear bodies, we examined their spatial distribution as a function of the cell cycle. Here, we report that whereas DDX1 bodies, CBs and gems are present throughout interphase, CPSF-100-containing cleavage bodies are predominantly found during S and G2 phases, whereas CstF-64-containing cleavage bodies are primarily observed during S phase. All four nuclear bodies associate with each other during S phase, with cleavage bodies colocalizing with DDX1 bodies, and cleavage bodies/DDX1 bodies residing adjacent to gems and CBs. Although inhibitors of RNA transcription had no effect on DDX1 bodies or cleavage bodies, inhibitors of DNA replication resulted in loss of CstF-64-containing cleavage bodies. A striking effect on nuclear structures was observed with latrunculin B, an inhibitor of actin polymerization, resulting in the formation of needlelike nuclear spicules made up of CstF-64, CPSF-100, RNA, and RNA polymerase II. Our results suggest that cleavage body components are highly dynamic in nature.

By using a differential screening strategy, we have identified a previously uncharacterised gene that is preferentially expressed in chick retinal precursor cells as well as in the anterior epithelial cells of the lens at early stages of development. The EURL transcript has an open reading frame of 293 amino acids and a 3′ untranslated region of >850 nucleotides. The only recognizable feature in the EURL (early undifferentiated retina and lens) amino acid sequence is a putative coiled-coil domain located at the C-terminus of the protein. The human EURL orthologue maps to chromosome 21q21. Northern blot analysis, in situ hybridization of tissue sections, and whole-mount hybridization indicate elevated levels of EURL mRNA in the embryonic dorsal retina from stage 14 (day 2) to stage 18 (day 3). At day 3.5, when >90% of the retinal cells are in the proliferative stage, EURL transcripts are found primarily in the peripheral dorsal retina, i.e., the most undifferentiated part of the dorsal retina. EURL transcripts are also detected in the lens at stage 18 and remain abundant in the proliferating epithelial cells of the lens until at least day 11. The distribution pattern of EURL in the developing retina and lens suggest a role before the events leading to cell determination and differentiation. Developmental Dynamics 227:409–415, 2003. © 2003 Wiley-Liss, Inc.

Introduction: Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor treatment outcomes and prognoses. Treatment usually consists of surgery, radiation and chemotherapy, which often result in severe systemic side effects frequently accompanied by tumor recurrence of highly radioresistant tumor cell populations. PARP-1 is a nuclear enzyme crucial for DNA damage repair (DDR) response. PARP-1 inhibition is a highly studied adjuvant to many anti-cancer treatment strategies as this arrests DNA damage repair thus enhancing the efficacy of conventional chemoradiotherapy at lower doses. However, PARP inhibition also promotes anti-inflammatory responses. Cancer cells suppress the immune cells ability to recognize and eliminate the tumor and promote their release of cancer-supporting growth factors and promote angiogenesis. Microglia the immune cells of the brain, normally protect the brain from pathogens and help in tissue recovery, but when associated with GBM cells, microglia obtain anti-inflammatory state that can promote tumor growth. We are developing a novel bimodal immunotherapeutic/DNA repair suppressive approach to treating GBM through modulation of microglial PARP-1 activity while concurrently sensitizing GBM to DNA damaging therapeutics. We hypothesize that while PARP-1 inhibition can enhance efficacy of chemoradiotherapy it might further suppress microglial ability to promote tumor elimination. Furthermore PARP-1 inhibitors' efficacy in preventing DNA repair is partially linked to their ability to trap PARP-1 at DNA break-site, which might be detrimental for the viability of non-cancerous brain cells. Objectives: 1) to establish the effects of PARP-1 inhibition alone, and in combination with targeted microglial PARP-1 activation, on microglial ability to eliminate glioblastoma cells in vitro (tumor cells vs. healthy glial cells). 2) to identify how microglial PARP-1 modulation (on/off) impacts glioblastoma elimination in vivo (tumor vs. brain). Methods: We will use GBM cells co-cultured with primary microglial cells in the presence of PARP inhibitors and observe their effect on the proliferation and viability of GBM cells. Efficacy of PARP-DNA trapping and corresponding DNA damage repair activity utilizing a variety of PARP inhibitors will be tested by specific biochemical and genotoxicity assays which directly quantify the degree of PARP-trapping in the glial cells and their impact on tumor cell genotoxcity. These studies take advantage of newly-developed and unique high-throughput DNA damage repair assays that we have been optimized to determine potential synergy of PARP-inhibitors with existing chemotherapeutic agents. Results: DNA damage assays (gamma H2AX foci and alkaline comet analysis) show that co-treatment of GBM cells (U87 and U251) with the PARP-inhibitor Olaparib and the chemotherapeutic agent Camptothecin (CPT) significantly increased the level of DNA damage when compared to CPT treatment alone. In the co-culture experiments, microglia enhanced CPT-induced GBM cell death when treated with Olaparib at 50-100 fold lower doses. Real time co-culture experiments also showed that Olaparib does not jeopardize microglial ability to actively phagocytize GBM cells. Conclusion: Preliminary results show that Olaparib is effective in sensitizing GBM cells to chemotherapy while microglia appear to enhance this effect. Further studies linking this combination therapy with microglia-mediated GBM elimination will help to elucidate the mechanism of this bimodal effect. This study emphasizes the importance of: (1) identifying novel effects of PARP inhibition on microglia in association with GBM and (2) the importance of developing microglia-mediated immunotherapeutic intervention to overcome the limitations of current therapies to significantly improve patient outcome. Citation Format: Asha Sinha, Sachin Katyal, Tiina Kauppinen. PARP-dependent co-modulation of DNA repair and microglial activity as a dual-pronged anti-glioblastoma treatment strategy [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B33.

Purpose: Current methods to treat glioblastoma multiforme (GBM) are highly invasive, lead to poor quality-of-life and an abysmal patient survival rate thus making GBMs one of the most difficult-to-treat and deadliest form of all primary CNS malignancies. The targeted use of Topoisomerase-1 (Top1) poisons/inhibitors to sensitize or augment tumor cell killing is a long-standing and powerful tool in cancer therapy. A number of trials utilizing Top1 inhibitors to manage GBM have shown promise. We have uncovered a novel functional requirement for ATM (Mutated in Ataxia Telangiectasia) in resolving oxidative DNA breaks and Topoisomerase 1-DNA covalent complexes (Top1cc), a DNA-protein intermediate that can generate DNA breaks upon collision with the transcriptional machinery or DNA replication forks. These data have identified Top1cc as the first specific endogenous pathogenic neural DNA lesion associated with loss of ATM and heritable pediatric neurodegenerative disease. Furthermore, biochemical and genetic studies identified collaboration of ATM and TDP1 (Tyrosyl-DNA Phosphodiesterase 1) in the resolution of Top1cc during neurodevelopment. We hypothesize that co-inhibition of ATM and TDP1 will sensitize brain tumors to Top1-dependent chemotherapy by augmenting Top1cc levels and anti-tumor success. Methods: Genetic and biochemical methods to co-inhibit ATM and TDP1 and improve the efficacy of Top1 poisons include novel high-throughput cell-based DNA damage repair and viability assays and in vivo brain tumor regression assays using mouse xenograft models. Results: We have found that brain tumor cell killing via Top1-dependent inhibition is greatly enhanced through an ATM and TDP1 co-inhibition strategy. Induced genotoxic cell death occurs at lower chemotherapeutic drug doses as a result of sensitization to Top1cc. We are presently validating these results and performing in vivo studies. Conclusions: ATM and TDP1 co-inhibition represents an effective two-pronged approach to chemoradiosensitize CNS tumors. As TDP1 antagonizes Top1cc formation and efficacy of Top1 poisons such as the camptothecin (CPT) cohort of drugs, our findings will improve existing Top1-mediated anti-cancer strategies by enhancing tumor cell killing while reducing clinical doses and patient side-effects. Furthermore, these findings also suggest an alternative avenue in the clinical management of GBM. Citation Format: Matthew Packer, Ali Saleh, Sachin Katyal. Co-inhibition of ATM and TDP1 as a novel chemosensitization strategy against malignant brain tumors [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B27.

Introduction: We are targeting DNA repair pathways to enhance existing chemoradiotherapeutic strategies against medulloblastoma (MB) and malignant glioma (MG), two highly invasive tumors of the central nervous system (CNS). Current methods to treat childhood medulloblastoma are highly intrusive and lead to poor quality of life while the three-year survival rate of patients afflicted with malignant glioma remains abysmal ( Methods: I will identify specific differentially-regulated DNA repair enzymes/pathways by gene expression methodology. From these findings, I will inhibit these DNA repair enzymes via shRNA or enzyme-specific inhibitors (if available), to chemosensitize MB and MG cells to DNA damaging therapeutics in an effort to reduce DNA repair in these cells thereby boosting tumor genotoxicity and cell death. Extent of DNA damage will be measured using novel high-throughput DNA repair assays combined with unique cellular DNA damage reporters/sensors to facilitate these studies. Results: In comparing subsets of MB and MG cell lines with existing DNA repair inhibitors, I have found differing DNA repair pathways as potential targets to sensitize these CNS tumors to chemo-radiotherapeutics. MB cells rely on DNA-PK/SSBR (single strand break repair) dependent pathways to resolve induced genotoxicity, while MG utilizes the ATM/SSBR pathways. My focus is now to expand this dataset in additional pertinent tumors and to perform a detailed comparative analysis to identify specific highly active tumor-specific DNA repair enzymes with which to target in order to enhance tumor cell death. These include the use of DNA damage repair response PCR arrays to compare the expression levels of individual DNA repair enzyme/pathway members amongst these CNS tumors. Conclusion: I have identified unique DNA repair enzymes, which may mediate specific differential chemo-radioresistant phenotypes in MB and MG. An expanded analysis is currently underway to further differentiate DNA repair mechanisms and therapeutic responses between these two brain tumor types. I hope to translate these findings into pre-clinical models whereby my data may lead to identifying next-generation brain cancer treatments with improved patient survival and quality-of-life. Citation Format: Marina Mostafizar, Sachin Katyal. Enhancing chemotherapeutic responses in CNS malignancy through suppression of hyperactive DNA damage repair pathways [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A21.

The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. In tumor cells, the ability to repair DSBs predicts response to radiation and many cytotoxic anti-cancer drugs. DSB repair pathways include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggests that NHEJ includes mechanisms to minimize error. Proteins required for mammalian NHEJ include Ku70/80, the DNA-dependent protein kinase (DNA-PKcs), XLF/Cernunnos and the XRCC4:DNA ligase IV complex. NHEJ also utilizes accessory proteins that include DNA polymerases, nucleases, and other end-processing factors. In yeast, mutations of tyrosyl-DNA phosphodiesterase (TDP1) reduced NHEJ fidelity. TDP1 plays an important role in repair of topoisomerase-mediated DNA damage and 3′-blocking DNA lesions, and mutation of the human TDP1 gene results in an inherited human neuropathy termed SCAN1. We found that human TDP1 stimulated DNA binding by XLF and physically interacted with XLF to form TDP1:XLF:DNA complexes. TDP1:XLF interactions preferentially stimulated TDP1 activity on dsDNA as compared to ssDNA. TDP1 also promoted DNA binding by Ku70/80 and stimulated DNA-PK activity. Because Ku70/80 and XLF are the first factors recruited to the DSB at the onset of NHEJ, our data suggest a role for TDP1 during the early stages of mammalian NHEJ.

Bendamustine (BEN) is a promising new treatment for chronic lymphocytic leukemia (CLL) and may function as an alkylating agent (eg, chlorambucil, CLB) while sharing structural similarities to a nucleoside analogue (eg, fludarabine, FLU). Enhanced cell death has been observed with the combination of BEN and FLU in primary CLL cells in vitro, but both BEN and FLU are marrow-toxic, potentially limiting the clinical value of this combination. While the nucleoside analogue pentostatin (PEN) is active in CLL and less marrow-toxic than FLU, it is unknown whether it produces synergy when combined with BEN. PEN acts by inhibiting adenosine deaminase, resulting in the accumulation of deoxyadenosine (dADO), which is toxic to CLL cells. In the present study, we evaluated the activity of BEN against CLL cells in vitro, as compared to CLB, FLU or dADO/PEN, alone and in combination. Cell death was assessed using annexin V/7-ADD staining and the MTT assay. We observed that BEN, CLB, FLU, or dADO/PEN induced apoptosis, the extent of which was time- and concentration-dependent. In general, cross-resistance was observed to all agents, with previously treated patients or those with a del 17p being most resistant. Enhanced cell killing was seen when combining BEN or CLB with FLU or dADO/PEN, with the extent of synergy being similar for FLU or dADO/PEN. To correlate mechanism of cell death as a result of treatment with these agents, we analyzed the expression of death receptors (DR4 and DR5) and mitochondrial stress (DICO6 and DHE). An increase in DR5 (but not DR4) surface expression, loss of mitochondrial membrane potential, and increased reactive oxygen species production was observed following treatment indicating that all agents induced death through the death receptor and mitochondrial apoptotic pathways. However, using γH2AX and the alkaline comet assay, FLU produced a greater number of DNA double-strand breaks (DSB) than BEN or CLB. Thus, all these agents induce apoptosis through the mitochondrial and death receptor pathways with cross-resistance being observed in most cases. BEN is more similar to CLB in producing fewer DNA DSBs than FLU and demonstrating synergy when combined with FLU or dADO/PEN. The latter observation suggests that combining BEN with PEN might be useful clinically. Citation Format: Sara EF Kost, Eric DJ Bouchard, Élise LaBossière, Xibiao Ye, Michelle L. Queau, William S. Liang, Versha Banerji, Spencer B. Gibson, Sachin Katyal, James B. Johnston. Bendamustine has the biochemical properties of an alkylating agent that synergizes with nucleoside analogues in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 281.

DNA strand break repair is essential for the prevention of multiple human diseases, particularly those which feature neuropathology. To further understand the pathogenesis of these syndromes, we recently developed animal models in which the DNA single-strand break repair (SSBR) components, XRCC1 and DNA Ligase III (LIG3), were inactivated in the developing nervous system. Although biochemical evidence suggests that inactivation of XRCC1 and LIG3 should share common biological defects, we found profound phenotypic differences between these two models, implying distinct biological roles for XRCC1 and LIG3 during DNA repair. Rather than a key role in nuclear DNA repair, we found LIG3 function was central to mitochondrial DNA maintenance. Instead, our data indicate that DNA Ligase 1 is the main DNA ligase for XRCC1-mediated DNA repair. These studies refine our understanding of DNA SSBR and the etiology of neurological disease.

Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1., Author Summary Chromosomal rearrangements are associated with many tumor types, as they are one way in which genes affecting cancer initiation and progression become mutated. One type of rearrangement is a chromosomal translocation, in which parts of two different chromosomes join together. Although infrequent, translocations occur when both chromosomes undergo breakage and the ends from different chromosomes join rather than the two ends from the same chromosome. Human and mouse cells have three known DNA ligases which catalyze the joining of DNA ends (Lig1, Lig3, and Lig4). Lig4 is important for joining the correct ends together, thereby suppressing translocations. In this report, the role of the other two DNA ligases is examined in a novel mouse cell system. Lig3 is found to be required for efficient chromosomal translocation formation, but in its absence Lig1 can substitute, although less efficiently and although the joining characteristics of the two DNA ligases differ. These studies define the hierarchy of the three DNA ligases in this type of genomic rearrangement.

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1.

Defective responses to DNA single strand breaks underlie various neurodegenerative diseases. However, the exact role of this repair pathway during the development and maintenance of the nervous system is unclear. Using murine neural-specific inactivation of Xrcc1, a factor that is critical for the repair of DNA single strand breaks, we found a profound neuropathology that is characterized by the loss of cerebellar interneurons. This cell loss was linked to p53-dependent cell cycle arrest and occurred as interneuron progenitors commenced differentiation. Loss of Xrcc1 also led to the persistence of DNA strand breaks throughout the nervous system and abnormal hippocampal function. Collectively, these data detail the in vivo link between DNA single strand break repair and neurogenesis and highlight the diverse consequences of specific types of genotoxic stress in the nervous system.

Understanding the physiological requirements for DNA repair is of paramount importance in establishing the links between genotoxic stress, development and disease. Defective DNA repair can lead to a variety of systemic pathology including cancer, neurodegeneration and immune defects. The genetically modified mouse is a critical resource for analysis of the effects of genotoxic stress and the tissue-specific requirements for DNA repair pathways. In the following we will outline how mouse models have aided our understanding of the connections between genotoxic stress and cancer and how they will contribute to therapeutic approaches for cancer treatment.

Ataxia oculomotor apraxia 1 (AOA1) results from mutations in aprataxin, a component of DNA strand break repair that removes AMP from 5' termini. Despite this, global rates of chromosomal strand break repair are normal in a variety of AOA1 and other aprataxin-defective cells. Here we show that short-patch single-strand break repair (SSBR) in AOA1 cell extracts bypasses the point of aprataxin action at oxidative breaks and stalls at the final step of DNA ligation, resulting in the accumulation of adenylated DNA nicks. Strikingly, this defect results from insufficient levels of nonadenylated DNA ligase, and short-patch SSBR can be restored in AOA1 extracts, independently of aprataxin, by the addition of recombinant DNA ligase. Since adenylated nicks are substrates for long-patch SSBR, we reasoned that this pathway might in part explain the apparent absence of a chromosomal SSBR defect in aprataxin-defective cells. Indeed, whereas chemical inhibition of long-patch repair did not affect SSBR rates in wild-type mouse neural astrocytes, it uncovered a significant defect in Aptx(-/-) neural astrocytes. These data demonstrate that aprataxin participates in chromosomal SSBR in vivo and suggest that short-patch SSBR arrests in AOA1 because of insufficient nonadenylated DNA ligase.

Alternative splicing represents a source of great diversity for regulating protein expression and function. It has been estimated that one-third to two-thirds of mammalian genes are alternatively spliced. With the sequencing of the chicken genome and analysis of transcripts expressed in chicken tissues, we are now in a position to address evolutionary conservation of alternative splicing events in chicken and mammals. Here, we compare chicken and mammalian transcript sequences of 41 alternatively-spliced genes and 50 frequently accessed genes. Our results support a high frequency of splicing events in chicken, similar to that observed in mammals.

Bendamustine (BEN) is a promising new treatment for chronic lymphocytic leukemia (CLL) and is believed to function as an alkylating agent (eg, chlorambucil, CLB) while sharing structural similarities to a nucleoside analogue (eg, fludarabine, FLU). Enhanced cell death has been observed with the combination of BEN and FLU in primary CLL cells in vitro, likely due to the inhibition of repair of BEN DNA cross-linking by FLU. However, both BEN and FLU are marrow-toxic, limiting the use of this combination in the clinic. The nucleoside analog, pentostatin (PEN), is an adenosine deaminase inhibitor that causes the accumulation of deoxyadenosine (dADO) and is less marrow-suppressive than FLU. However, the cytotoxic effectiveness of dADO/PEN in combination with BEN is not known. Flow cytometry analysis using annexin V/7-ADD and the MTT assay were performed to determine the effectiveness of BEN treatment in primary CLL cells as compared to or in combination with CLB, FLU, or dADO/PEN at varying drug concentrations and time points. Using the MTT assay, there was a linear relationship between cytotoxicity and drug concentration, similar to that seen with CLB. In vitro treatment of CLL cells with BEN, CLB, FLU, or dADO/PEN induced apoptosis, the degree being time- and concentration-dependent. However, the sensitivity of CLL cells to BEN or CLB varied, suggesting different mechanisms of action. Enhanced cell kill was seen with the combination of BEN or CLB with FLU or dADO/PEN, with the extent of increased cytotoxicity being similar for FLU or dADO/PEN. Cell death mechanisms were analyzed via staining for surface expression of death receptors (DR4 and DR5) and mitochondrial stress (DICO6 and DHE). γ-H2AX staining was used to measure DNA double-strand breaks (DSBs) and the alkaline comet assay was used to measure both DNA DSBs and single-strand breaks (SSBs). An increase in DR4 and DR5 surface expression, loss of mitochondrial membrane potential, and increased reactive oxygen species production was observed with all agents. There was an increase in DNA SSB and DSB following FLU, as compared to BEN and CLB, while a combination of BEN and FLU further increased the number of breaks (especially SSBs) compared to FLU alone. Thus, BEN has the properties of an alkylating agent, rather than a nucleoside analog, but has a different spectrum of antitumor activity to CLB. BEN induces apoptosis through both the death receptor and mitochondrial pathways. Increased cell kill is seen on combining BEN with dADO/PEN suggesting that this might be a useful combination in the clinic. Citation Format: Sara E.F. Kost, Eric D.J. Bouchard, William S. Liang, Versha Banerji, Spencer B. Gibson, Sachin Katyal, James B. Johnston. The mechanism of action of bendamustine alone or in combination with nucleoside analogs in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2555. doi:10.1158/1538-7445.AM2015-2555

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The Reelin–Disabled 1 (Dab1)‐signaling pathway plays a critical role in neuronal cell positioning in the brain. We have isolated two alternatively spliced variants of Dab1 from chick retina, an early form (chDab1‐E) expressed in undifferentiated cells and a late form (chDab1‐L) expressed in amacrine and ganglion cells. A key difference between the two forms is the exclusion in chDab1‐E of two Src‐related tyrosine kinase recognition sites implicated in Reelin‐mediated Dab1 tyrosine phosphorylation. Retinal cultures transfected with a chDab1‐L expression construct undergo a dramatic change in morphology, accompanied by the formation of numerous thin elongated processes, increased tyrosine phosphorylation, activation of Src family kinase(s) and increased levels of the axonal outgrowth protein growth‐associated protein‐43. In contrast, chDab1‐E transfectants retain an undifferentiated morphology. Mutational analysis implicates a specific tyrosine (tyr‐198) in the morphological and biochemical alterations associated with chDab1‐L expression. We propose that alternative splicing of chDab1 represents an effective and flexible way of regulating the Reelin–Dab1‐signaling pathway in a mixed cell population, by ensuring that secreted Reelin activates the signaling cascade only in target neuronal cells.

DEAD box proteins are putative RNA unwinding proteins found in organisms ranging from mammals to bacteria. While some DEAD box genes expressed in higher eukaryotes are ubiquitous, others have distribution profiles that suggest a cell-, tissue-, or developmental-specific role. The DEAD box gene, DDX1, was identified by differential screening of a subtracted retinoblastoma cDNA library. A limited survey of human fetal tissues indicated that DDX1 mRNA has a widespread distribution but is not uniformly expressed in all tissues. To further document the spatial and temporal distribution of DDX1 during embryonic development, we cloned the chicken DDX1 cDNA. The predicted amino acid sequence of chicken DDX1 was 93% identical to that of human DDX1. All DEAD box motifs, as well as a SPRY domain, were present in chicken DDX1. Northern and Western blot analyses showed highest levels of DDX1 at early stages of development. Tissue maturation was generally accompanied by a decrease in expression, although DDX1 levels remained elevated in late embryonic retina and brain. In situ hybridization of retinal tissue sections revealed widespread distribution of DDX1 mRNA at early developmental stages with preferential expression in amacrine and ganglion cells of the differentiated tissue. Preferential expression of DDX1 was also observed in specific areas of the brain in older embryos, such as the external granule layer of the cerebellum. These results suggest a specific role for DDX1 in subsets of differentiated cells as well as a more general role in undifferentiated cells.