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ObjectiveThe efficacy of intra-arterial vasodilators (IADs) for the treatment of vasospasm following aneurysmal subarachnoid hemorrhage (aSAH) remains debatable. The objective of this meta-analysis was to pool estimates of angiographic and neurological response, clinical outcome, and mortality following treatment of vasospasm with IADs.MethodsWe searched PubMed, Embase, Scopus, Clinicaltrials.gov, Cochrane database, and CINAHL in December 2015 and August 2016. Studies reporting angiographic and neurological response, clinical outcome, and mortality following IAD treatment of vasospasm in 10 or more adults with aSAH were included. All established IADs were allowed. Two authors independently selected studies and abstracted the data. Mean weighted probabilities (MWP) were calculated using random effects model.ResultsInclusion criteria were met by 55 studies (n=1571). MWP for immediate angiographic response to IAD treatment was 89% (95% CI 83% to 94%), post-IAD neurological improvement 57% (95% CI 49% to 65%), good outcome 66% (95% CI 60% to 71%), and mortality was 9% (95% CI 7% to 12%). After adjusting for publication bias, MWP for mortality was 5% (95% CI 4% to 7%). When transcranial Doppler (TCD) was used along with clinical deterioration for patient selection, rates of neurological response (64%) and good outcome (72%) were better. IADs were not superior to controls (balloon angioplasty or medical management).ConclusionIAD treatment leads to a robust angiographic response and fair (but lower) rates of neurological response and good clinical outcome. Mortality was lower than the average reported in the literature. Rates of neurological response and good outcome were better when TCD was used for patient selection. Carefully designed studies are needed to compare IADs against medical management and balloon angioplasty.

Aim. Glutamic acid decarboxylase antibodies (GAD‐Abs) have been implicated in refractory epilepsy. The association with refractory status epilepticus in adults has been rarely described. We discuss our experience in managing three adult patients who presented with refractory status epilepticus associated with GAD‐Abs. Methods. Case series with retrospective chart and literature review. Results. Three patients without pre‐existing epilepsy who presented to our institution with generalized seizures between 2013 and 2014 were identified. Seizures proved refractory to first and second‐line therapies and persisted beyond 24 hours. Patient 1 was a 22‐year‐old female who had elevated serum GAD‐Ab titres at 0.49 mmol/l (normal: <0.02) and was treated with multiple immuno‐ and chemotherapies, with eventual partial seizure control. Patient 2 was a 61‐year‐old black female whose serum GAD‐Ab titre was 0.08 mmol/l. EEG showed persistent generalized periodic discharges despite maximized therapy with anticonvulsants but no immunotherapy, resulting in withdrawal of care and discharge to nursing home. Patient 3 was a 50‐year‐old black female whose serum GAD‐Ab titre was 0.08 mmol/l, and was discovered to have pulmonary sarcoidosis. Treatment with steroids and intravenous immunoglobulin resulted in seizure resolution. Conclusion. Due to the responsiveness to immunotherapy, there may be an association between GAD‐Abs and refractory seizures, including refractory status epilepticus. Causation cannot be established since GAD‐Abs may be elevated secondary to concurrent autoimmune diseases or formed de novoin response to GAD antigen exposure by neuronal injury. Based on this report and available literature, there may be a role for immuno‐ and chemotherapy in the management of refractory status epilepticus associated with GAD‐Abs.

Dysembryoplastic neuroepithelial tumours (DNET) are a common cause of tumour‐associated epilepsy, and are usually located in the temporal lobes. We present a case of multifocal DNETs in both infra‐ and supra‐tentorial locations, in a 23‐year‐old man with a coincident Type I Chiari malformation, presenting with medically refractory focal seizures. The extensive anatomical distribution of the lesions suggests a genetic component in their tumourigenesis.

Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)–driven chemistry effort, we describe compound 1(CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.

The overexpression of P-glycoprotein, encoded by the ATP Binding Cassette B1 (ABCB1) gene, contributes to multidrug resistance (MDR) and is considered one of the major obstacles to successful cancer chemotherapy. The authors previously developed a T-lineage acute lymphoblastic leukemia (T-ALL) cell line that overexpresses ABCB1 and exhibits MDR to daunorubicin (DNR), prednisolone, and vincristine. Using this cell line and the fluorescent probe JC-1, they developed a flow cytometry-based, high-throughput screening (HTS) assay that quantifies ABCB1 efflux. They screened a library of 880 off-patent drugs for their ability to inhibit ABCB1 efflux and then measured the ability of 11 lead compounds to reverse in vitro DNR-mediated drug resistance and the toxic doses for each agent. Seven of the 11 drugs were able to reverse drug resistance at a concentration significantly below its toxic dose. Of the remaining 7, only 1 compound, mometasone furoate, has not been previously described as an ABCB1 antagonist to DNR-mediated drug resistance. On the basis of its high ABC modulator activity and relatively large in vitro therapeutic window, this drug warrants further investigation. In addition, the approach used in this study is useful for identifying off-patent drugs that may be repurposed for novel clinical indications.

The clinical and cytogenetic features associated with T-cell acute lymphoblastic leukemia (T-ALL) are not predictive of early treatment failure. Based on the hypothesis that microarrays might identify patients who fail therapy, we used the Affymetrix U133 Plus 2.0 chip and prediction analysis of microarrays (PAM) to profile 50 newly diagnosed patients who were treated in the Children's Oncology Group (COG) T-ALL Study 9404. We identified a 116-member genomic classifier that could accurately distinguish all 6 induction failure (IF) cases from 44 patients who achieved remission; network analyses suggest a prominent role for genes mediating cellular quiescence. Seven genes were similarly upregulated in both the genomic classifier for IF patients and T-ALL cell lines having acquired resistance to neoplastic agents, identifying potential target genes for further study in drug resistance. We tested whether our classifier could predict IF within 42 patient samples obtained from COG 8704 and, using PAM to define a smaller classifier for the U133A chip, correctly identified the single IF case and patients with persistently circulating blasts. Genetic profiling may identify T-ALL patients who are likely to fail induction and for whom alternate treatment strategies might be beneficial.

The clinical and cytogenetic features associated with T-cell acute lymphoblastic leukemia (T-ALL) are not predictive of early treatment failure. Based on the hypothesis that microarrays might identify patients who fail therapy, we used the Affymetrix U133 Plus 2.0 chip and prediction analysis of microarrays (PAM) to profile 50 newly diagnosed patients who were treated in the Children's Oncology Group (COG) T-ALL Study 9404. We identified a 116-member genomic classifier that could accurately distinguish all 6 induction failure (IF) cases from 44 patients who achieved remission; network analyses suggest a prominent role for genes mediating cellular quiescence. Seven genes were similarly upregulated in both the genomic classifier for IF patients and T-ALL cell lines having acquired resistance to neoplastic agents, identifying potential target genes for further study in drug resistance. We tested whether our classifier could predict IF within 42 patient samples obtained from COG 8704 and, using PAM to define a smaller classifier for the U133A chip, correctly identified the single IF case and patients with persistently circulating blasts. Genetic profiling may identify T-ALL patients who are likely to fail induction and for whom alternate treatment strategies might be beneficial.

Objective: To assesses the frequency and degree of pituitary hyperplasia in patients with primary hypothyroidism, the association of pituitary enlargement with disease severity, and the response to treatment. Methods: Between April 2002 and August 2004 at the National Center for Diabetes, Endocrinology and Genetics in Amman, Jordan, 53 patients (49 female and 4 male subjects) with primary hypothyroidism and serum thyrotropin (thyroid-stimulating hormone or TSH) levels of ³ 50 ¼ IU/mL were encountered. Initial and follow-up investigations included thyroid function tests, serum prolactin levels, and magnetic resonance imaging (MRI) of the pituitary. Visual field examination was requested for all patients with pituitary enlargement (and adequately completed in 24). Results: Pituitary enlargement on MRI was found in 37 of the 53 patients (70%), with 31 of the 37 patients (84%) having TSH levels of ³ 100 ¼ IU/mL. After thyroxine treatment, 85% of the patients with pituitary enlargement who underwent a follow-up MRI showed a decrease in size of the gland. About half of the patients were referred to our facility with the diagnosis of hypothyroidism; presenting features in the rest of the patients included galactorrhea, menstrual irregularities, learning disability, short stature, precocious puberty, ovarian hyperstimulation syndrome, headaches, visual field defects, and dry ichthyotic skin. Conclusion: The association between pituitary gland enlargement and primary hypothyroidism should be kept in mind when pituitary hyperplasia is detected on MRI, before unwarranted and drastic interventions are initiated. (Endocr Pract. 2006;12:29-34)

We investigated the effect of MEN 11467 ((1R,2S)‐2‐N[1(H)indol‐3‐yl‐carbonyl]‐1‐N‐{Nα(p‐tolylacetyl)‐Nα(methyl)‐D‐3‐(2‐naphthyl)alanyl}diaminocyclohexane) on tachykinin‐induced mucus secretion in ferret trachea in vitroand determined its effect on secretion by tracheae from allergic ferrets in response to allergen challenge.Repeated administration of [Sar9,Met(O2)11]‐substance P ([Sar9]SP, 1 μM) maintained mucus output above control values for at least 1.75 h. MEN 11467 inhibited secretion in a concentration‐dependent manner with maximal inhibition at 10 μMand an approximate IC50of 0.3 μM. Inhibition by MEN 11467 (0.1 – 10 μM) was maintained, to varying degree, for at least 1.75 h after washout in the continued presence of [Sar9]SP.In electrically stimulated tracheae, tachykininergic neural secretion was virtually abolished by 1 μMMEN 11467.In tracheae from ovalbumin‐sensitised animals, repeated administration of ovalbumin maintained mucus output above controls for 1.5 h. MEN 11467 inhibited ovalbumin‐induced secretion in a concentration‐dependent manner, with complete inhibition at 1 μM. Inhibition by MEN 11467 (1 and 10 μM) was maintained, to varying degree, after drug washout for the 1.5 h of ovalbumin stimulation.MEN 11467 1 μMdid not affect secretion induced by either acetylcholine or histamine, whereas 10 μMMEN 11467 did inhibit agonist‐induced secretion.We conclude that, in ferret trachea in vitro, MEN 11467 at concentrations of 0.1 – 1 μMis a long acting and selective inhibitor of tachykininergic‐induced mucus secretion, and may have therapeutic potential for bronchial hypersecretion associated with allergic conditions, for example in asthma.

Diabetes mellitus (DM) is a well-known risk factor for Non-alcoholic fatty liver disease (NAFLD). Patients with type 2 DM (T2DM) who have NAFLD are at a higher risk of developing advanced stages of liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma compared to non-diabetic patients. This study aimed to estimate the prevalence of NAFLD among patients with T2DM, using hepatic ultrasonographic changes combined with derangement of hepatic transaminases level.

We investigated whether vasoactive intestinal peptide (VIP) and its related peptides, pituitary adenylate cyclase activating peptide (PACAP) and secretin, regulate cholinergic neural mucus secretion in ferret trachea in vitro, using 35SO4as a mucus marker. We also studied the interaction between VIP and secretin on cholinergic mucus output.VIP (1 and 10 μM) increased secretion, whereas neither PACAP1–27, PACAP1–38nor secretin (up to 10 μM) increased mucus output. In contrast, VIP, PACAP1–27and PACAP1–38concentration‐dependently inhibited cholinergic neural secretion, with an order of potency of VIP>PACAP 1–38>PACAP1–27. Neither PACAP1–27nor PACAP1–38altered the secretion induced by acetylcholine (ACh).Secretin increased cholinergic neural secretion with a maximal increase of 190% at 1 μM. This potentiation was blocked by VIP or atropine. Similarly, secretin (1 μM) potentiated VIP (1 μM)‐induced mucus output by 160%. Secretin did not alter exogenous ACh‐induced secretion. VIP vssecretin competition curves suggested these two peptides were competing reversibly for the same receptor.We conclude that, in ferret trachea in vitro, VIP and PACAPs inhibit cholinergic neural secretion viapre‐junctional modulation of cholinergic neurotransmission. VIP and secretin compete for the same receptor, possibly a VIP1receptor, at which secretin may be a receptor antagonist.

The aims of this study were to determine: (1) whether vasoactive intestinal peptide (VIP) regulates cholinergic and ‘sensory‐efferent’ (tachykininergic) 35SO4labelled mucus output in ferret trachea in vitro, using a VIP antibody, (2) the class of potassium (K+) channel involved in VIP‐regulation of cholinergic neural secretion using glibenclamide (an ATP‐sensitive K+(KATP) channel inhibitor), iberiotoxin (a large conductance calcium activated K+(BKca) channel blocker), and apamin (a small conductance Kca(SKca) channel blocker), and (3) the effect of VIP on cholinergic neurotransmission using [3H]‐choline overflow as a marker for acetylcholine (ACh) release.Exogenous VIP (1 and 10 μM) alone increased 35SO4output by up to 53% above baseline, but suppressed (by up to 80% at 1 μM) cholinergic and tachykininergic neural secretion without altering secretion induced by ACh or substance P (1 μMeach). Endogenous VIP accounted for the minor increase in non‐adrenergic, non‐cholinergic (NANC), non‐tachykininergic neural secretion, which was compatible with the secretory response of exogenous VIP.Iberiotoxin (3 μM), but not apamin (1 μM) or glibenclamide (0.1 μM), reversed the inhibition by VIP (10 nM) of cholinergic neural secretion.Both endogenous VIP (by use of the VIP antibody; 1 : 500 dilution) and exogenous VIP (0.1 μM), the latter by 34%, inhibited ACh release from cholinergic nerve terminals and this suppression was completely reversed by iberiotoxin (0.1 μM).We conclude that, in ferret trachea in vitro, endogenous VIP has dual activity whereby its small direct stimulatory action on mucus secretion is secondary to its marked regulation of cholinergic and tachykininergic neurogenic mucus secretion. Regulation is viainhibition of neurotransmitter release, consequent upon opening of BKCachannels. In the context of neurogenic mucus secretion, we propose that VIP joins NO as a neurotransmitter of i‐NANC nerves in ferret trachea.

We investigated the inhibitory effects of the cysteinyl leukotriene (CysLT1) receptor antagonists, pranlukast and zafirlukast, on 35SO4labelled mucus output, in vitro, in guinea‐pig trachea, induced by leukotriene D4(LTD4) or by antigen challenge of sensitized animals. Agonists and antagonists were administered mucosally, except in selected comparative experiments where drugs were administered both mucosally and serosally to assess the influence of the epithelium on evoked‐secretion.LTD4increased 35SO4output in a concentration‐related manner with a maximal increase of 23 fold above controls at 100 μMand an approximate EC50of 2 μM. Combined mucosal and serosal addition of LTD4did not significantly affect the secretory response compared with mucosal addition alone. Neither LTC4nor LTE4(10 μMeach) affected 35SO4output. Pranlukast or zafirlukast significantly inhibited 10 μMLTD4‐evoked 35SO4output in a concentration‐dependent fashion, with maximal inhibitions of 83% at 10 μMpranlukast and 78% at 10 μMzafirlukast, and IC50values of 0.3 μMfor pranlukast and 0.6 μMfor zafirlukast. Combined mucosal and serosal administration of the antagonists (5 μMeach) gave degrees of inhibition of mucosal‐serosal 10 μMLTD4‐evoked 35SO4output similar to those of the drugs given mucosally. Pranlukast (0.5 μM) caused a parallel rightward shift of the LTD4concentration‐response curve with a pKBof 7. Pranlukast did not inhibit ATP‐induced 35SO4output.Ovalbumin (10–500 μg ml−1) challenge of tracheae from guinea‐pigs actively sensitized with ovalbumin caused a concentration‐related increase in 35SO4output with a maximal increase of 20 fold above vehicle controls at 200 μg ml−1. The combination of the antihistamines pyrilamine and cimetidine (0.1 mMeach) did not inhibit ovalbumin‐induced 35SO4output in sensitized guinea‐pigs. Neither mucosal (10 μMor 100 μM) nor mucosal‐serosal (100 μM) histamine had any significant effect on 35SO4output.Pranlukast or zafirlukast (5 μMeach) significantly suppressed ovalbumin‐induced secretion in tracheae from sensitized guinea‐pigs by 70% and 65%, respectively.We conclude that LTD4or ovalbumin challenge of sensitized animals provokes mucus secretion from guinea‐pig trachea in vitroand this effect is inhibited by the CysLT1receptor antagonists pranlukast and zafirlukast. These antagonists may be beneficial in the treatment of allergic airway diseases in which mucus hypersecretion is a clinical symptom, for example asthma and allergic rhinitis.

1. Muscarinic receptor subtypes mediating neurogenic mucus secretion in ferret trachea were characterized in vitro and in vivo using 35SO4 as a label for secreted mucus, and the muscarinic receptor antagonists telenzepine for the M1 receptor subtype, methoctramine for the M2 subtype and 4‐diphenylacetoxy‐N‐methylpiperidine methobromide (4‐DAMP) for the M3 receptor. We also performed receptor binding and mapping studies. 2. Each muscarinic antagonist displaced [N‐methyl‐3H]scopolamine binding with high‐affinity binding constant (KH) values of 1.9, 2.7 and 5.0 nM for telenzepine, methoctramine and 4‐DAMP, respectively. Muscarinic M1 and M3 receptors localized to submucosal glands, whereas M2 receptors did not. 3. In vitro, electrical stimulation (50 V, 10 Hz, 0.5 ms for 5 min) increased 35SO4 output by 160%. Telenzepine did not inhibit the neurogenic secretory response at concentrations two‐or twentyfold its KH value, nor did it inhibit secretion induced by acetylcholine (ACh). 4‐DAMP inhibited neurogenic secretion by 80 and 95%, respectively, at concentrations two‐and twentyfold its KH value, and also inhibited ACh‐induced secretion. Methoctramine potentiated neurogenic secretion induced at 2.5 Hz (50 V, 0.5 ms for 5 min) in a dose‐related (5.4‐100 nM) manner with increases of 33‐451% above electrically stimulated values. Methoctramine did not potentiate secretion induced at 10 Hz and did not have any effect on ACh‐induced secretion. 4. In vivo, vagal stimulation (10 V, 10 Hz, 2 ms for 8 min) increased output of 35SO4 by approximately 120%. Telenzepine had no significant effect on neurogenic secretion. Methoctramine approximately doubled the stimulated response, whereas 4‐DAMP abolished the stimulated secretory response. 5. We conclude that in ferret trachea, cholinergic nerve stimulation increases mucus secretion via muscarinic M3 receptors on the submucosal glands. The magnitude of the secretory response is regulated by neuronal M2 muscarinic receptors. The muscarinic M1 receptors localized to the submucosal glands do not appear to be involved with mucus secretion.

At the 67th annual meeting of the American Academy of Neurology (AAN), there was a roundtable discussion with representatives from the International Working Group of Young Neurologist and Trainees–World Federation of Neurology (IWGYNT-WFN), Asia and Oceanian Association of Neurology, Association of South East Asian Nations Neurological Association, European Association of Young Neurologists and Trainees (EAYNT), and the AAN's Consortium of Neurology Residents and Fellows (AAN-CNRF). The purpose of the meeting was to provide updates on the developments affecting young neurologists throughout the world, as well as to discuss current initiatives focused on the improvement of neurology training on the international stage.

Interviews were carried out with 322 children, ages 5 to 16 years, to examine the association of war experience and their anxiety. Body language was used as indicator of anxiety. Girls showed significantly more discrete body movements than boys.

Acquired drug resistance eventually leads to treatment failure in T-cell acute lymphoblastic leukemia (T-ALL). To define mechanisms of vincristine (VCR)-resistance, and to address whether inducible resistance in T-ALL might be mechanistically-dependent on ABC drug transporter expression, we developed VCR-resistant Sup T1 and Jurkat by incrementally growing cells in increasing drug concentrations. Jurkat (CD1a−, sCD3+) and Sup T1 (CD1a+, sCD3−) are arrested at “mature” and “cortical” stages of T-cell development and, respectively, showed 1190-fold (0.0006 μM to 0.714 μM) and 790-fold (0.0006 μM to 0.474 μM) increased resistance to VCR. Microarray analysis showed that expression of the ATP Binding Cassette C1 (ABCC1; Multidrug Resistance Protein, MDR2) was increased more than 29-fold in VCR-resistant cells as compared to the parental controls; fold increases in mRNA of ABCC1 were confirmed by RT-PCR, and cell surface expression by flow cytometric analysis. We then employed a flow cytometry-based assay that measures the ability of ABC pumps to extrude fluorescent dyes (Calcein AM). We confirmed that VCR-resistant Jurkat and Sup T1 actively extrude Calcein AM; drug extrusion was also effectively blocked by verapamil and other known ABCB1 antagonists. In contrast to our previous observations for ABCB1-mediated multidrug resistance (Estes, et al., British Journal of Haematology, in press), ABCC1-mediated drug resistance conferred a more narrow spectrum of multidrug resistance for the chemotherapeutic agents commonly used in T-ALL induction. To investigate whether ABC expression might impact patient outcome, we next examined ABCC1 and ABCB1 expression (>1.5-fold) in 80 T-ALL patients treated on COG studies 8704 and 9404. Different from our earlier findings that ABCB1 expression in diagnostic marrows conferred a 100% risk for on-therapy relapse, we found that ABCC1 expression only predicted treatment failure in approximately 25% of affected patients. These results suggest that ABC transporters are differentially regulated in Sup T1 and Jurkat, and that substrate-specific ABC protein regulation results in unique multidrug resistance phenotypes in T-ALL. This study contributes to the repertoire of substrate-specific, drug resistant T-ALL cells lines that are available for modeling treatment failure, provides mechanistically-based insights into relapse, and establishes an experimental means for investigating compounds that reverse ABCC1 mediated drug resistance.

Acquired drug resistance eventually leads to treatment failure in T-cell acute lymphoblastic leukemia (T-ALL). To define mechanisms of vincristine (VCR)-resistance, and to address whether inducible resistance in T-ALL might be mechanistically-dependent on ABC drug transporter expression, we developed VCR-resistant Sup T1 and Jurkat by incrementally growing cells in increasing drug concentrations. Jurkat (CD1a−, sCD3+) and Sup T1 (CD1a+, sCD3−) are arrested at “mature” and “cortical” stages of T-cell development and, respectively, showed 1190-fold (0.0006 μM to 0.714 μM) and 790-fold (0.0006 μM to 0.474 μM) increased resistance to VCR. Microarray analysis showed that expression of the ATP Binding Cassette C1 (ABCC1; Multidrug Resistance Protein, MDR2) was increased more than 29-fold in VCR-resistant cells as compared to the parental controls; fold increases in mRNA of ABCC1 were confirmed by RT-PCR, and cell surface expression by flow cytometric analysis. We then employed a flow cytometry-based assay that measures the ability of ABC pumps to extrude fluorescent dyes (Calcein AM). We confirmed that VCR-resistant Jurkat and Sup T1 actively extrude Calcein AM; drug extrusion was also effectively blocked by verapamil and other known ABCB1 antagonists. In contrast to our previous observations for ABCB1-mediated multidrug resistance (Estes, et al., British Journal of Haematology , in press), ABCC1-mediated drug resistance conferred a more narrow spectrum of multidrug resistance for the chemotherapeutic agents commonly used in T-ALL induction. To investigate whether ABC expression might impact patient outcome, we next examined ABCC1 and ABCB1 expression (>1.5-fold) in 80 T-ALL patients treated on COG studies 8704 and 9404. Different from our earlier findings that ABCB1 expression in diagnostic marrows conferred a 100% risk for on-therapy relapse, we found that ABCC1 expression only predicted treatment failure in approximately 25% of affected patients. These results suggest that ABC transporters are differentially regulated in Sup T1 and Jurkat, and that substrate-specific ABC protein regulation results in unique multidrug resistance phenotypes in T-ALL. This study contributes to the repertoire of substrate-specific, drug resistant T-ALL cells lines that are available for modeling treatment failure, provides mechanistically-based insights into relapse, and establishes an experimental means for investigating compounds that reverse ABCC1 mediated drug resistance.

Acquisition of drug resistance in childhood T-cell acute lymphoblastic leukemia (T-ALL) is a major cause of treatment failure. Drug resistant cell lines are effective tools for elucidating mechanisms of acquired drug resistance. In this study, we developed novel drug resistant cell lines that could be employed to identify mechanisms of drug resistance. We established three new drug resistant T-ALL cell lines: 2 resistant to L-asparaginase (L-asp), and one resistant to daunorubicin (DNR). The DNR-resistant line (Jurkat) acquired a 117-fold increase in EC50 to DNR, from 20.6 nM to 2.4 μM, while the L-asp resistant cells (Jurkat and Sup T1) showed respective increases in resistance of 320-fold (0.003 IU/mL to 0.962 IU/mL) and 29-fold (0.042 IU/mL to 1.22 IU/mL). The DNR resistant cell line acquired a multidrug resistant phenotype, showing 310 and 120-fold increase in resistance to vincristine and prednisolone, respectively. Resistance to L-asp was unchanged. Microarray analysis showed that ABCB1 (MDR1, P-glycoprotein) was significantly upregulated (567-fold) in DNR resistant cells. siRNA experiments that reduced ABCB1 mRNA levels by 74% restored DNR sensitivity. In L-asp resistant T-ALL cells (Jurkat and Sup T1), two notable genes were upregulated, asparagine synthetase (ASNS) which catalyzes synthesis of asparagine (41-fold and 1.5-fold) and argininosuccinate synthase (ASS)(32-fold and 6.5-fold), respectively. Reduction of ASNS with siRNA restored drug sensitivity in both cell lines. Interestingly, siRNA suppression of ASS in conjunction with ASNS achieved an exaggerated restoration of drug sensitivity compared to ASNS alone. We next examined the microarray profiles of drug resistant cells with those of 86 T-ALL patients; of which 8 failed induction (IF). Interestingly, these 3 key genes are upregulated in 25–62% of IF cases. Although ABCB1 overexpression has been shown to be a mechanism of DNR resistance in many cancers, there is a paucity of resistant T-ALL cell lines to adequately model the effect of stage of differentiation and genetic heterogeneity underlying drug resistance in T-ALL. With the establishment of 3 new T-ALL cell lines in this report, there exist 5 T-ALL drug resistant cell lines, representing the spectrum of T-cell differentiation (pre-T, cortical T, and mature T cells). Finally, this is the first report of the potential contribution of ASS in addition to ASNS to L-asp resistance in leukemia cells.

In acute leukemias, the overexpression of P-glycoprotein, encoded by the ATP Binding Cassette B1 (ABCB1) gene contributes to multidrug resistance (MDR), and is considered one of the major obstacles to successful cancer chemotherapy. The identification of small molecules that reverse ABCB1 efflux activity is critical for the successful treatment of cancers, including relapsed T-lineage acute lymphoblastic leukemia (T-ALL). However, the dose-limiting toxicities of many MDR reversal agents have restricted their use in clinical trials, and more effective and clinically applicable reversal agents remain to be identified. We have recently developed a T-ALL cell line that overexpresses ABCB1 and exhibits multiple drug resistance (MDR) to daunorubicin, prednisolone, and vincristine, but not L-asparaginase. The MDR can be reversed by suppression of ABCB1 expresssion with siRNA or 5 μM cyclosporine (CSA). Using this cell line, we developed a flow cytometry based, high-throughput screening assay that quantifies ABCB1 efflux using the fluorescent probe JC-1 (Swerts, et al Leuk Lymphoma 45:2221–8, 2004). We screened a library of 880 off-patent drugs for their ability to inhibit ABCB1 efflux at concentrations of 4 μM, and 19 compounds were identified, including CSA. Based on a published record of safe internal use in humans, 12 compounds were retained for further analysis (3 compounds were originally described as calcium channel blockers, 1 as sodium channel blocker, 1 as ACE inhibitor, 1 as dopamine uptake inhibitor, 1 as Topoisomerase II inhibitor, 2 as steroids, 2 as antifungal and 1 as immunosuppressant). We determined the 50% inhibitory concentration (IC50) of drug for ABCB1 efflux, the efflux reversal concentration of drug that rescued DNR-induced T-ALL cell death (ECrev50), and the corresponding in vitro toxic dose in 50% of treated T-ALL cells (TD50). Due to space limitations, detailed data could not be presented in the abstract. To our knowledge, 8 of the 12 compounds have not been previously described as ABCB1 inhibitors. These compounds may be useful as chemosensitizers or as lead compounds for development of improved ABCB1 inhibitors in T-ALL as well as other cancers.

Acquisition of drug resistance in childhood T-cell acute lymphoblastic leukemia (T-ALL) is a major cause of treatment failure. Drug resistant cell lines are effective tools for elucidating mechanisms of acquired drug resistance. In this study, we developed novel drug resistant cell lines that could be employed to identify mechanisms of drug resistance. We established three new drug resistant T-ALL cell lines: 2 resistant to L-asparaginase (L-asp), and one resistant to daunorubicin (DNR). The DNR-resistant line (Jurkat) acquired a 117-fold increase in EC50to DNR, from 20.6 nM to 2.4 μM, while the L-asp resistant cells (Jurkat and Sup T1) showed respective increases in resistance of 320-fold (0.003 IU/mL to 0.962 IU/mL) and 29-fold (0.042 IU/mL to 1.22 IU/mL). The DNR resistant cell line acquired a multidrug resistant phenotype, showing 310 and 120-fold increase in resistance to vincristine and prednisolone, respectively. Resistance to L-asp was unchanged. Microarray analysis showed that ABCB1 (MDR1, P-glycoprotein) was significantly upregulated (567-fold) in DNR resistant cells. siRNA experiments that reduced ABCB1 mRNA levels by 74% restored DNR sensitivity. In L-asp resistant T-ALL cells (Jurkat and Sup T1), two notable genes were upregulated, asparagine synthetase (ASNS) which catalyzes synthesis of asparagine (41-fold and 1.5-fold) and argininosuccinate synthase (ASS)(32-fold and 6.5-fold), respectively. Reduction of ASNS with siRNA restored drug sensitivity in both cell lines. Interestingly, siRNA suppression of ASS in conjunction with ASNS achieved an exaggerated restoration of drug sensitivity compared to ASNS alone. We next examined the microarray profiles of drug resistant cells with those of 86 T-ALL patients; of which 8 failed induction (IF). Interestingly, these 3 key genes are upregulated in 25–62% of IF cases. Although ABCB1 overexpression has been shown to be a mechanism of DNR resistance in many cancers, there is a paucity of resistant T-ALL cell lines to adequately model the effect of stage of differentiation and genetic heterogeneity underlying drug resistance in T-ALL. With the establishment of 3 new T-ALL cell lines in this report, there exist 5 T-ALL drug resistant cell lines, representing the spectrum of T-cell differentiation (pre-T, cortical T, and mature T cells). Finally, this is the first report of the potential contribution of ASS in addition to ASNS to L-asp resistance in leukemia cells.

In acute leukemias, the overexpression of P-glycoprotein, encoded by the ATP Binding Cassette B1 (ABCB1) gene contributes to multidrug resistance (MDR), and is considered one of the major obstacles to successful cancer chemotherapy. The identification of small molecules that reverse ABCB1 efflux activity is critical for the successful treatment of cancers, including relapsed T-lineage acute lymphoblastic leukemia (T-ALL). However, the dose-limiting toxicities of many MDR reversal agents have restricted their use in clinical trials, and more effective and clinically applicable reversal agents remain to be identified. We have recently developed a T-ALL cell line that overexpresses ABCB1 and exhibits multiple drug resistance (MDR) to daunorubicin, prednisolone, and vincristine, but not L-asparaginase. The MDR can be reversed by suppression of ABCB1 expresssion with siRNA or 5 μM cyclosporine (CSA). Using this cell line, we developed a flow cytometry based, high-throughput screening assay that quantifies ABCB1 efflux using the fluorescent probe JC-1 (Swerts, et al Leuk Lymphoma 45:2221–8, 2004). We screened a library of 880 off-patent drugs for their ability to inhibit ABCB1 efflux at concentrations of 4 μM, and 19 compounds were identified, including CSA. Based on a published record of safe internal use in humans, 12 compounds were retained for further analysis (3 compounds were originally described as calcium channel blockers, 1 as sodium channel blocker, 1 as ACE inhibitor, 1 as dopamine uptake inhibitor, 1 as Topoisomerase II inhibitor, 2 as steroids, 2 as antifungal and 1 as immunosuppressant). We determined the 50% inhibitory concentration (IC50) of drug for ABCB1 efflux, the efflux reversal concentration of drug that rescued DNR-induced T-ALL cell death (ECrev50), and the corresponding in vitro toxic dose in 50% of treated T-ALL cells (TD50). Due to space limitations, detailed data could not be presented in the abstract. To our knowledge, 8 of the 12 compounds have not been previously described as ABCB1 inhibitors. These compounds may be useful as chemosensitizers or as lead compounds for development of improved ABCB1 inhibitors in T-ALL as well as other cancers.

Acquisition of drug resistance in tumor cells in children with T-cell acute lymphoblastic leukemia (T-ALL) during chemotherapy results in relapse and poor outcome. T-ALL cell lines that have acquired resistance to chemotherapeutics are therefore critical tools for the study of acquired resistance, yet there is a paucity of cell lines available for study. In this study, we hypothesize that drug resistant T-ALL cells can be produced by prolonged exposure to chemotherapeutics and that microarray analysis can be employed to identify the gene products responsible for acquired drug resistance. By incrementally increasing the drug concentration in growth media, we have produced T-ALL cell lines (Jurkat and Sup T1) that grow well in the presence of therapeutic concentrations of L-asparaginase (ASNase) and daunorubicin (DNR). The genetic profiles of the drug-resistant cell lines were compared to their parental progenitors using the Affymetrix HG-U133Plus2 GeneChip platform, capable of hybridizing ~54,000 genes and ESTs/chip. Signal intensity was normalized using the robust multi-array average (RMA) technique in GeneSpring 7.2. The Sup T1 and Jurkat ASNase-resistant cell lines increased their IC50s 26-fold (0.044 IU/mL to 1.14 IU/mL) and 320-fold (0.003 IU/mL to 0.96 IU/mL), respectively. The IC50 of the Jurkat DNR resistant cell line increased 77-fold (30 nM to 2300 nM), and 4.0-fold, (0.46 nM to 1.85 nM), respectively. Notably, DNR resistant Jurkat cells were also resistant to therapeutic concentrations of vincristine and prednisolone, but not ASNase. In contrast, the ASNase resistant cell lines remained sensitive to DNR, vincristine, and prednisolone. Microarray data comparing DNR-resistant and parental cell lines showed 288 genes upregulated >1.5-fold in the resistant line. Two sets of genes were the most upregulated in the drug resistant cells in comparison to parental cells. ABCB1 (ABC transporter P-glycoprotein) was upregulated ~940-fold and genes coding for 6 different killer-cell immunoglobulin-like receptors (KIRs) were upregulated >6-fold. In the case of the ASNase-resistant cell lines, 96 genes were found to be upregulated >1.5-fold in both Jurkat and Sup T1 lines. The most highly upregulated gene in both cell lines was argininosuccinate synthase (ASS), 32-fold upregulated in Jurkat and 6.5-fold in Sup T1. All expression results were confirmed by qRT-PCR. These genes have previously been implicated in the acquisition of drug resistance: ASS is critical for responding to asparagine depletion caused by ASNase. ABCB1 acts as a molecular pump capable of lowering intracellular concentrations of substrate chemotherapeutics such as DNR, vincristine, and prednisolone, consistent with our observation of multi-drug resistance in that cell line. To our knowledge, this is the first description of DNR and ASNase resistant Jurkat and Sup T1 T-ALL cell lines. In addition, our results suggest that microarray technology is a valid method for elucidating the genetic nature of drug resistance in T-ALL cell lines, making it a productive approach to identify mechanisms of chemotherapy resistance. Finally, these cell lines will serve as useful tools for studying mechanisms of chemotherapeutic resistance in T-ALL.

Acquisition of drug resistance in tumor cells in children with T-cell acute lymphoblastic leukemia (T-ALL) during chemotherapy results in relapse and poor outcome. T-ALL cell lines that have acquired resistance to chemotherapeutics are therefore critical tools for the study of acquired resistance, yet there is a paucity of cell lines available for study. In this study, we hypothesize that drug resistant T-ALL cells can be produced by prolonged exposure to chemotherapeutics and that microarray analysis can be employed to identify the gene products responsible for acquired drug resistance. By incrementally increasing the drug concentration in growth media, we have produced T-ALL cell lines (Jurkat and Sup T1) that grow well in the presence of therapeutic concentrations of L-asparaginase (ASNase) and daunorubicin (DNR). The genetic profiles of the drug-resistant cell lines were compared to their parental progenitors using the Affymetrix HG-U133Plus2 GeneChip platform, capable of hybridizing ~54,000 genes and ESTs/chip. Signal intensity was normalized using the robust multi-array average (RMA) technique in GeneSpring 7.2. The Sup T1 and Jurkat ASNase-resistant cell lines increased their IC50s 26-fold (0.044 IU/mL to 1.14 IU/mL) and 320-fold (0.003 IU/mL to 0.96 IU/mL), respectively. The IC50of the Jurkat DNR resistant cell line increased 77-fold (30 nM to 2300 nM), and 4.0-fold, (0.46 nM to 1.85 nM), respectively. Notably, DNR resistant Jurkat cells were also resistant to therapeutic concentrations of vincristine and prednisolone, but not ASNase. In contrast, the ASNase resistant cell lines remained sensitive to DNR, vincristine, and prednisolone. Microarray data comparing DNR-resistant and parental cell lines showed 288 genes upregulated >1.5-fold in the resistant line. Two sets of genes were the most upregulated in the drug resistant cells in comparison to parental cells. ABCB1 (ABC transporter P-glycoprotein) was upregulated ~940-fold and genes coding for 6 different killer-cell immunoglobulin-like receptors (KIRs) were upregulated >6-fold. In the case of the ASNase-resistant cell lines, 96 genes were found to be upregulated >1.5-fold in both Jurkat and Sup T1 lines. The most highly upregulated gene in both cell lines was argininosuccinate synthase (ASS), 32-fold upregulated in Jurkat and 6.5-fold in Sup T1. All expression results were confirmed by qRT-PCR. These genes have previously been implicated in the acquisition of drug resistance: ASS is critical for responding to asparagine depletion caused by ASNase. ABCB1 acts as a molecular pump capable of lowering intracellular concentrations of substrate chemotherapeutics such as DNR, vincristine, and prednisolone, consistent with our observation of multi-drug resistance in that cell line. To our knowledge, this is the first description of DNR and ASNase resistant Jurkat and Sup T1 T-ALL cell lines. In addition, our results suggest that microarray technology is a valid method for elucidating the genetic nature of drug resistance in T-ALL cell lines, making it a productive approach to identify mechanisms of chemotherapy resistance. Finally, these cell lines will serve as useful tools for studying mechanisms of chemotherapeutic resistance in T-ALL.