Publications by authors named "John R Silber"

33 Publications

siRNA nanoparticle suppresses drug-resistant gene and prolongs survival in an orthotopic glioblastoma xenograft mouse model.

Adv Funct Mater 2021 Feb 6;31(6). Epub 2020 Nov 6.

Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, United States; Department of Neurological Surgery, University of Washington, Seattle, WA 98195, United States.

Temozolomide (TMZ) is the standard of care chemotherapy drug for treating glioblastomas (GBMs), the most aggressive cancer that affects people of all ages. However, its therapeutic efficacy is limited by the drug resistance mediated by a DNA repair protein, O-methylguanine-DNA methyltransferase (MGMT), which eliminates the TMZ-induced DNA lesions. Here we report the development of an iron oxide nanoparticle (NP) system for targeted delivery of siRNAs to suppress the TMZ-resistance gene (MGMT). We show that our NP is able to overcome biological barriers, bind specifically to tumor cells, and reduce MGMT expression in tumors of mice bearing orthotopic GBM serially-passaged patient-derived xenografts. The treatment with sequential administration of this NP and TMZ resulted in increased apoptosis of GBM stem-like cells, reduced tumor growth, and significantly-prolonged survival as compared to mice treated with TMZ alone. This study introduces an approach that holds great promise to improve the outcomes of GBM patients.
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http://dx.doi.org/10.1002/adfm.202007166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7942690PMC
February 2021

A kinase-deficient NTRK2 splice variant predominates in glioma and amplifies several oncogenic signaling pathways.

Nat Commun 2020 06 12;11(1):2977. Epub 2020 Jun 12.

Human Biology Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Mailstop C3-168, Seattle, WA, 98109, USA.

Independent scientific achievements have led to the discovery of aberrant splicing patterns in oncogenesis, while more recent advances have uncovered novel gene fusions involving neurotrophic tyrosine receptor kinases (NTRKs) in gliomas. The exploration of NTRK splice variants in normal and neoplastic brain provides an intersection of these two rapidly evolving fields. Tropomyosin receptor kinase B (TrkB), encoded NTRK2, is known for critical roles in neuronal survival, differentiation, molecular properties associated with memory, and exhibits intricate splicing patterns and post-translational modifications. Here, we show a role for a truncated NTRK2 splice variant, TrkB.T1, in human glioma. TrkB.T1 enhances PDGF-driven gliomas in vivo, augments PDGF-induced Akt and STAT3 signaling in vitro, while next generation sequencing broadly implicates TrkB.T1 in the PI3K signaling cascades in a ligand-independent fashion. These TrkB.T1 findings highlight the importance of expanding upon whole gene and gene fusion analyses to include splice variants in basic and translational neuro-oncology research.
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http://dx.doi.org/10.1038/s41467-020-16786-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293284PMC
June 2020

Nanoparticle-mediated knockdown of DNA repair sensitizes cells to radiotherapy and extends survival in a genetic mouse model of glioblastoma.

Nanomedicine 2017 Oct 11;13(7):2131-2139. Epub 2017 Jun 11.

Department of Neurological Surgery, University of Washington, Seattle, WA, United States; Department of Materials Science and Engineering, University of Washington, Seattle, WA, United States; Department of Radiology, University of Washington, Seattle, WA, United States. Electronic address:

Glioblastoma (GBM) remains incurable, and recurrent tumors rarely respond to standard-of-care radiation and chemo-therapies. Therefore, strategies that enhance the effects of these therapies should provide significant benefits to GBM patients. We have developed a nanoparticle delivery vehicle that can stably bind and protect nucleic acids for specific delivery into brain tumor cells. These nanoparticles can deliver therapeutic siRNAs to sensitize GBM cells to radiotherapy and improve GBM treatment via systemic administration. We show that nanoparticle-mediated knockdown of the DNA repair protein apurinic endonuclease 1 (Ape1) sensitizes GBM cells to radiotherapy and extend survival in a genetic mouse model of GBM. Specific knockdown of Ape1 activity by 30% in brain tumor tissue doubled the extended survival achieved with radiotherapy alone. Ape1 is a promising target for increasing the effectiveness of radiotherapy, and nanoparticle-mediated delivery of siRNA is a promising strategy for tumor specific knockdown of Ape1.
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http://dx.doi.org/10.1016/j.nano.2017.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002851PMC
October 2017

pH-Sensitive O6-Benzylguanosine Polymer Modified Magnetic Nanoparticles for Treatment of Glioblastomas.

Bioconjug Chem 2017 01 12;28(1):194-202. Epub 2016 Dec 12.

Department of Materials Science and Engineering, ‡Department of Chemistry, §Department of Biochemistry, ∥Department of Neurological Surgery, and ⊥Department of Radiology, University of Washington , Seattle, Washington 98195, United States.

Nanoparticle-mediated delivery of chemotherapeutics has demonstrated potential in improving anticancer efficacy by increasing serum half-life and providing tissue specificity and controlled drug release to improve biodistribution of hydrophobic chemotherapeutics. However, suboptimal drug loading, particularly for solid core nanoparticles (NPs), remains a challenge that limits their clinical application. In this study we formulated a NP coated with a pH-sensitive polymer of O-methylguanine-DNA methyltransferase (MGMT) inhibitor analog, dialdehyde modified O-benzylguanosine (DABGS) to achieve high drug loading, and polyethylene glycol (PEG) to ameliorate water solubility and maintain NP stability. The base nanovector consists of an iron oxide core (9 nm) coated with hydrazide functionalized PEG (IOPH). DABGS and PEG-dihydrazide were polymerized on the iron oxide nanoparticle surface (IOPH-pBGS) through acid-labile hydrazone bonds utilizing a rapid, freeze-thaw catalysis approach. DABGS polymerization was confirmed by FTIR and quantitated by UV-vis spectroscopy. IOPH-pBGS demonstrated excellent drug loading of 33.4 ± 5.1% by weight while maintaining small size (36.5 ± 1.8 nm). Drug release was monitored at biologically relevant pHs and demonstrated pH dependent release with maximum release at pH 5.5 (intracellular conditions), and minimal release at physiological pH (7.4). IOPH-pBGS significantly suppressed activity of MGMT and potentiated Temozolomide (TMZ) toxicity in vitro, demonstrating potential as a new treatment option for glioblastomas (GBMs).
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http://dx.doi.org/10.1021/acs.bioconjchem.6b00545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5510587PMC
January 2017

Culture on 3D Chitosan-Hyaluronic Acid Scaffolds Enhances Stem Cell Marker Expression and Drug Resistance in Human Glioblastoma Cancer Stem Cells.

Adv Healthc Mater 2016 12 2;5(24):3173-3181. Epub 2016 Nov 2.

Department of Materials Science and Engineering, University of Washington, Seattle, WA, 98195, USA.

The lack of in vitro models that support the growth of glioblastoma (GBM) stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient-derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly, and frequently unsuccessful endeavor. To address these limitations, a 3D porous scaffold composed of chitosan and hyaluronic acid (CHA) is synthesized. Growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers is compared. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self-adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial-mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold-grown cells also display higher expression of genes that promote resistance to hypoxia-induced oxidative stress. In accord, scaffold-grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments.
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http://dx.doi.org/10.1002/adhm.201600684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5253135PMC
December 2016

Towards use of MRI-guided ultrasound for treating cerebral vasospasm.

J Ther Ultrasound 2016 29;4. Epub 2016 Feb 29.

Department of Neurological Surgery, University of Washington, 325 9th Ave, Box 359924, Seattle, WA 98104 USA ; Department of Radiology, University of Washington, RR-218 Health Science Building, 1959 NE Pacific St, Seattle, WA 98195 USA ; Division of Engineering, University of Washington, 18115 Campus Way NE, Bothell, WA 98011 USA.

Cerebral vasospasm is a major cause of morbidity and mortality in patients with subarachnoid hemorrhage (SAH), causing delayed neurological deficits in as many as one third of cases. Existing therapy targets induction of cerebral vasodilation through use of various drugs and mechanical means, with a range of observed efficacy. Here, we perform a literature review supporting our hypothesis that transcranially delivered ultrasound may have the ability to induce therapeutic cerebral vasodilation and, thus, may one day be used therapeutically in the context of SAH. Prior studies demonstrate that ultrasound can induce vasodilation in both normal and vasoconstricted blood vessels in peripheral tissues, leading to reduced ischemia and cell damage. Among the proposed mechanisms is alteration of several nitric oxide (NO) pathways, where NO is a known vasodilator. While in vivo studies do not point to a specific physical mechanism, results of in vitro studies favor cavitation induction by ultrasound, where the associated shear stresses likely induce NO production. Two papers discussed the effects of ultrasound on the cerebral vasculature. One study applied clinical transcranial Doppler ultrasound to a rodent complete middle cerebral artery occlusion model and found reduced infarct size. A second involved the application of pulsed ultrasound in vitro to murine brain endothelial cells and showed production of a variety of vasodilatory chemicals, including by-products of arachidonic acid metabolism. In sum, nine reviewed studies demonstrated evidence of either cerebrovascular dilation or elaboration of vasodilatory compounds. Of particular interest, all of the reviewed studies used ultrasound capable of transcranial application: pulsed ultrasound, with carrier frequencies ranging between 0.5 and 2.0 MHz, and intensities not substantially above FDA-approved intensity values. We close by discussing potential specific treatment paradigms of SAH and other cerebral ischemic disorders based on MRI-guided transcranial ultrasound.
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http://dx.doi.org/10.1186/s40349-016-0050-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770693PMC
March 2016

Nanoparticle-Mediated Target Delivery of TRAIL as Gene Therapy for Glioblastoma.

Adv Healthc Mater 2015 Dec 26;4(17):2719-26. Epub 2015 Oct 26.

Department of Materials Science and Engineering, University of Washington, Seattle, WA, 98195, USA.

Human tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is an attractive cancer therapeutic because of its ability to induce apoptosis in tumor cells while having a negligible effect on normal cells. However, the short serum half-life of TRAIL and lack of efficient in vivo administration approaches have largely hindered its clinical use. Using nanoparticles (NPs) as carriers in gene therapy is considered as an alternative approach to increase TRAIL delivery to tumors as transfected cells would be induced to secrete TRAIL into the tumor microenvironment. To enable effective delivery of plasmid DNA encoding TRAIL into glioblastoma (GBM), we developed a targeted iron oxide NP coated with chitosan-polyethylene glycol-polyethyleneimine copolymer and chlorotoxin (CTX) and evaluated its effect in delivering TRAIL in vitro and in vivo. NP-TRAIL successfully delivers TRAIL into human T98G GBM cells and induces secretion of 40 pg mL(-1) of TRAIL in vitro. Transfected cells show threefold increased apoptosis as compared to the control DNA bound NPs. Systemic administration of NP-TRAIL-CTX to mice bearing T98G-derived flank xenografts results in near-zero tumor growth and induces apoptosis in tumor tissue. Our results suggest that NP-TRAIL-CTX can potentially serve as a targeted anticancer therapeutic for more efficient TRAIL delivery to GBM.
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http://dx.doi.org/10.1002/adhm.201500563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715716PMC
December 2015

Nanoparticle mediated silencing of DNA repair sensitizes pediatric brain tumor cells to γ-irradiation.

Mol Oncol 2015 Jun 29;9(6):1071-80. Epub 2015 Jan 29.

Department of Neurological Surgery, University of Washington, Seattle, WA 98195, USA; Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA; Department of Radiology, University of Washington, Seattle, WA 98195, USA. Electronic address:

Medulloblastoma (MB) and ependymoma (EP) are the most common pediatric brain tumors, afflicting 3000 children annually. Radiotherapy (RT) is an integral component in the treatment of these tumors; however, the improvement in survival is often accompanied by radiation-induced adverse developmental and psychosocial sequelae. Therefore, there is an urgent need to develop strategies that can increase the sensitivity of brain tumors cells to RT while sparing adjacent healthy brain tissue. Apurinic endonuclease 1 (Ape1), an enzyme in the base excision repair pathway, has been implicated in radiation resistance in cancer. Pharmacological and specificity limitations inherent to small molecule inhibitors of Ape1 have hindered their clinical development. Here we report on a nanoparticle (NP) based siRNA delivery vehicle for knocking down Ape1 expression and sensitizing pediatric brain tumor cells to RT. The NP comprises a superparamagnetic iron oxide core coated with a biocompatible, biodegradable coating of chitosan, polyethylene glycol (PEG), and polyethyleneimine (PEI) that is able to bind and protect siRNA from degradation and to deliver siRNA to the perinuclear region of target cells. NPs loaded with siRNA against Ape1 (NP:siApe1) knocked down Ape1 expression over 75% in MB and EP cells, and reduced Ape1 activity by 80%. This reduction in Ape1 activity correlated with increased DNA damage post-irradiation, which resulted in decreased cell survival in clonogenic assays. The sensitization was specific to therapies generating abasic lesions as evidenced by NP:siRNA not increasing sensitivity to paclitaxel, a microtubule disrupting agent. Our results indicate NP-mediated delivery of siApe1 is a promising strategy for circumventing pediatric brain tumor resistance to RT.
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http://dx.doi.org/10.1016/j.molonc.2015.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4439369PMC
June 2015

Deep sequencing of multiple regions of glial tumors reveals spatial heterogeneity for mutations in clinically relevant genes.

Genome Biol 2014 Dec 3;15(12):530. Epub 2014 Dec 3.

Background: The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors.

Results: We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations.

Conclusions: Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.
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http://dx.doi.org/10.1186/s13059-014-0530-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272528PMC
December 2014

O-methylguanine-DNA methyltransferase activity is associated with response to alkylating agent therapy and with promoter methylation in glioblastoma and anaplastic glioma.

BBA Clin 2015 Jun;3:1-10

Department of Neurological Surgery, University of Washington, Seattle, WA.

Background: CpG methylation in the O-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies.

Methods: We examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP).

Results: Cox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous ( ≤ 0.001) and continuous ( ≤ 0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors had significantly lower activity ( ≤ 0.005) and longer PFS ( ≤ 0.036) compared to unmethylated tumors, despite overlapping activities. PFS was also significantly greater in methylated . unmethylated GBMs with comparable activity ( ≤ 0.005), and among unmethylated tumors with less than median activity ( ≤ 0.026), suggesting that mechanisms in addition to MGMT promote alkylator resistance. Similar associations of MGMT activity with PFS and promoter methylation status were observed for AGs.

Conclusions: Our results provide strong support for the hypotheses that MGMT activity promotes alkylator resistance and reflects promoter methylation status in malignant gliomas.

General Significance: MGMT activity is an attractive target for anti-resistance therapy regardless of methylation status.
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http://dx.doi.org/10.1016/j.bbacli.2014.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280839PMC
June 2015

Redox-responsive magnetic nanoparticle for targeted convection-enhanced delivery of O6-benzylguanine to brain tumors.

ACS Nano 2014 Oct 29;8(10):10383-95. Epub 2014 Sep 29.

Department of Materials Science and Engineering, University of Washington , Seattle, Washington 98195, United States.

Resistance to temozolomide (TMZ) based chemotherapy in glioblastoma multiforme (GBM) has been attributed to the upregulation of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Inhibition of MGMT using O(6)-benzylguanine (BG) has shown promise in these patients, but its clinical use is hindered by poor pharmacokinetics that leads to unacceptable toxicity. To improve BG biodistribution and efficacy, we developed superparamagnetic iron oxide nanoparticles (NP) for targeted convection-enhanced delivery (CED) of BG to GBM. The nanoparticles (NPCP-BG-CTX) consist of a magnetic core coated with a redox-responsive, cross-linked, biocompatible chitosan-PEG copolymer surface coating (NPCP). NPCP was modified through covalent attachment of BG and tumor targeting peptide chlorotoxin (CTX). Controlled, localized BG release was achieved under reductive intracellular conditions and NPCP-BG-CTX demonstrated proper trafficking of BG in human GBM cells in vitro. NPCP-BG-CTX treated cells showed a significant reduction in MGMT activity and the potentiation of TMZ toxicity. In vivo, CED of NPCP-BG-CTX produced an excellent volume of distribution (Vd) within the brain of mice bearing orthotopic human primary GBM xenografts. Significantly, concurrent treatment with NPCP-BG-CTX and TMZ showed a 3-fold increase in median overall survival in comparison to NPCP-CTX/TMZ treated and untreated animals. Furthermore, NPCP-BG-CTX mitigated the myelosuppression observed with free BG in wild-type mice when administered concurrently with TMZ. The combination of favorable physicochemical properties, tumor cell specific BG delivery, controlled BG release, and improved in vivo efficacy demonstrates the great potential of these NPs as a treatment option that could lead to improved clinical outcomes.
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http://dx.doi.org/10.1021/nn503735wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4212796PMC
October 2014

Periostin is a novel therapeutic target that predicts and regulates glioma malignancy.

Neuro Oncol 2015 Mar 19;17(3):372-82. Epub 2014 Aug 19.

Department of Neurological Surgery, University of Washington School of Medicine, Seattle, Washington (A.M.M., S.A.M, S.N.E., C.A.P., R.G.O., J.R.S., R.C.R.); Department of Radiation Oncology, University of Washington School of Medicine, Seattle, Washington (A.D.T.); Division of Medical Genetics, Department of Internal Medicine, University of Washington School of Medicine, Seattle, Washington (M.J.T); Department of Bioengineering, University of Washington School of Medicine, Seattle, Washington (S.F., D.-H.K.); Department of Radiology, University of Washington School of Medicine, Seattle, Washington (K.R.T.-F.); Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington (A.M.M., S.A.M., S.F., D.-H.K., R.C.R.); Sage Bionetworks, Seattle, Washington (A.D.T.); Neuropathology Service, Department of Pathology, Stanford University School of Medicine, Stanford, California (D.E.B.); Laboratory of Immunology, IRCCS San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy (B.C.); Case Western Reserve School of Medicine, Cleveland, Ohio (Y.K.); Neuroscience Associates, Boise, Idaho (T.C.M.).

Background: Periostin is a secreted matricellular protein critical for epithelial-mesenchymal transition and carcinoma metastasis. In glioblastoma, it is highly upregulated compared with normal brain, and existing reports indicate potential prognostic and functional importance in glioma. However, the clinical implications of periostin expression and function related to its therapeutic potential have not been fully explored.

Methods: Periostin expression levels and patterns were examined in human glioma cells and tissues by quantitative real-time PCR and immunohistochemistry and correlated with glioma grade, type, recurrence, and survival. Functional assays determined the impact of altering periostin expression and function on cell invasion, migration, adhesion, and glioma stem cell activity and tumorigenicity. The prognostic and functional relevance of periostin and its associated genes were analyzed using the TCGA and REMBRANDT databases and paired recurrent glioma samples.

Results: Periostin expression levels correlated directly with tumor grade and recurrence, and inversely with survival, in all grades of adult human glioma. Stromal deposition of periostin was detected only in grade IV gliomas. Secreted periostin promoted glioma cell invasion and adhesion, and periostin knockdown markedly impaired survival of xenografted glioma stem cells. Interactions with αvβ3 and αvβ5 integrins promoted adhesion and migration, and periostin abrogated cytotoxicity of the αvβ3/β5 specific inhibitor cilengitide. Periostin-associated gene signatures, predominated by matrix and secreted proteins, corresponded to patient prognosis and functional motifs related to increased malignancy.

Conclusion: Periostin is a robust marker of glioma malignancy and potential tumor recurrence. Abrogation of glioma stem cell tumorigenicity after periostin inhibition provides support for exploring the therapeutic impact of targeting periostin.
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http://dx.doi.org/10.1093/neuonc/nou161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4483094PMC
March 2015

Proliferation and enrichment of CD133(+) glioblastoma cancer stem cells on 3D chitosan-alginate scaffolds.

Biomaterials 2014 Nov 7;35(33):9137-43. Epub 2014 Aug 7.

Department of Neurological Surgery, University of Washington, Seattle, WA 98195, USA; Department of Materials Science & Engineering, University of Washington, Seattle, WA 98195, USA. Electronic address:

Emerging evidence implicates cancer stem cells (CSCs) as primary determinants of the clinical behavior of human cancers, representing an ideal target for next-generation anti-cancer therapies. However CSCs are difficult to propagate in vitro, severely limiting the study of CSC biology and drug development. Here we report that growing cells from glioblastoma (GBM) cell lines on three dimensional (3D) porous chitosan-alginate (CA) scaffolds dramatically promotes the proliferation and enrichment of cells possessing the hallmarks of CSCs. CA scaffold-grown cells were found more tumorigenic in nude mouse xenografts than cells grown from monolayers. Growing in CA scaffolds rapidly promoted expression of genes involved in the epithelial-to-mesenchymal transition that has been implicated in the genesis of CSCs. Our results indicate that CA scaffolds have utility as a simple and inexpensive means to cultivate CSCs in vitro in support of studies to understand CSC biology and develop more effective anti-cancer therapies.
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http://dx.doi.org/10.1016/j.biomaterials.2014.07.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4167581PMC
November 2014

Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain.

Aging Cell 2014 Feb 11;13(1):29-38. Epub 2013 Sep 11.

Translational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave, Seattle, WA, 98109, USA.

Due largely to the inability to accurately quantify and characterize de novo deletion events, the mechanisms underpinning the pathogenic expansion of mtDNA deletions in aging and neuromuscular disorders remain poorly understood. Here, we outline and validate a new tool termed 'Digital Deletion Detection' (3D) that allows for high-resolution analysis of rare deletions occurring at frequencies as low as 1 × 10(-8) . 3D is a three-step process that includes targeted enrichment for deletion-bearing molecules, single-molecule partitioning of genomes into thousands of droplets for direct quantification via droplet digital PCR, and breakpoint characterization using massively parallel sequencing. Using 3D, we interrogated over 8 billion mitochondrial genomes to analyze the age-related dynamics of mtDNA deletions in human brain tissue. We demonstrate that the total deletion load increases with age, while the total number and diversity of unique deletions remain constant. Our data provide support for the hypothesis that expansion of pre-existing mutations is the primary factor contributing to age-related accumulation of mtDNA deletions.
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http://dx.doi.org/10.1111/acel.12146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068027PMC
February 2014

Synthesis and characterization of DNA minor groove binding alkylating agents.

Chem Res Toxicol 2013 Jan 26;26(1):156-68. Epub 2012 Dec 26.

Department of Pharmaceutical Sciences, University of Pittsburgh , Pittsburgh, Pennsylvania 15261, United States.

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.
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http://dx.doi.org/10.1021/tx300437xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3618862PMC
January 2013

Repair of 3-methyladenine and abasic sites by base excision repair mediates glioblastoma resistance to temozolomide.

Front Oncol 2012 30;2:176. Epub 2012 Nov 30.

Department of Neurological Surgery, University of Washington Medical Center Seattle, WA, USA.

Alkylating agents have long played a central role in the adjuvant therapy of glioblastoma (GBM). More recently, inclusion of temozolomide (TMZ), an orally administered methylating agent with low systemic toxicity, during and after radiotherapy has markedly improved survival. Extensive in vitro and in vivo evidence has shown that TMZ-induced O(6)-methylguanine (O(6)-meG) mediates GBM cell killing. Moreover, low or absent expression of O(6)-methylguanine-DNA methyltransferase (MGMT), the sole human repair protein that removes O(6)-meG from DNA, is frequently associated with longer survival in GBMs treated with TMZ, promoting interest in developing inhibitors of MGMT to counter resistance. However, the clinical efficacy of TMZ is unlikely to be due solely to O(6)-meG, as the agent produces approximately a dozen additional DNA adducts, including cytotoxic N3-methyladenine (3-meA) and abasic sites. Repair of 3-meA and abasic sites, both of which are produced in greater abundance than O(6)-meG, is mediated by the base excision repair (BER) pathway, and occurs independently of removal of O(6)-meG. These observations indicate that BER activities are also potential targets for strategies to potentiate TMZ cytotoxicity. Here we review the evidence that 3-meA and abasic sites mediate killing of GBM cells. We also present in vitro and in vivo evidence that alkyladenine-DNA glycosylase, the sole repair activity that excises 3-meA from DNA, and Ape1, the major human abasic site endonuclease, mediate TMZ resistance in GBMs and represent potential anti-resistance targets.
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http://dx.doi.org/10.3389/fonc.2012.00176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515961PMC
December 2012

Increased age of transformed mouse neural progenitor/stem cells recapitulates age-dependent clinical features of human glioma malignancy.

Aging Cell 2012 Dec 11;11(6):1027-35. Epub 2012 Oct 11.

Department of Neurological Surgery, University of Washington School of Medicine, Seattle, WA 98195, USA.

Increasing age is the most robust predictor of greater malignancy and treatment resistance in human gliomas. However, the adverse association of clinical course with aging is rarely considered in animal glioma models, impeding delineation of the relative importance of organismal versus progenitor cell aging in the genesis of glioma malignancy. To address this limitation, we implanted transformed neural stem/progenitor cells (NSPCs), the presumed cells of glioma origin, from 3- and 18-month-old mice into 3- and 20-month host animals. Transplantation with progenitors from older animals resulted in significantly shorter (P ≤ 0.0001) median survival in both 3-month (37.5 vs. 83 days) and 20-month (38 vs. 67 days) hosts, indicating that age-dependent changes intrinsic to NSPCs rather than host animal age accounted for greater malignancy. Subsequent analyses revealed that increased invasiveness, genomic instability, resistance to therapeutic agents, and tolerance to hypoxic stress accompanied aging in transformed NSPCs. Greater tolerance to hypoxia in older progenitor cells, as evidenced by elevated HIF-1 promoter reporter activity and hypoxia response gene (HRG) expression, mirrors the upregulation of HRGs in cohorts of older vs. younger glioma patients revealed by analysis of gene expression databases, suggesting that differential response to hypoxic stress may underlie age-dependent differences in invasion, genomic instability, and treatment resistance. Our study provides strong evidence that progenitor cell aging is responsible for promoting the hallmarks of age-dependent glioma malignancy and that consideration of progenitor aging will facilitate development of physiologically and clinically relevant animal models of human gliomas.
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http://dx.doi.org/10.1111/acel.12004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504614PMC
December 2012

O(6)-methylguanine-DNA methyltransferase in glioma therapy: promise and problems.

Biochim Biophys Acta 2012 Aug 8;1826(1):71-82. Epub 2012 Jan 8.

Department of Neurological Surgery, University of Washington, Seattle, WA 98195, USA.

Gliomas are the most frequent adult primary brain tumor, and are invariably fatal. The most common diagnosis glioblastoma multiforme (GBM) afflicts 12,500 new patients in the U.S. annually, and has a median survival of approximately one year when treated with the current standard of care. Alkylating agents have long been central in the chemotherapy of GBM and other gliomas. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), the principal human activity that removes cytotoxic O(6)-alkylguanine adducts from DNA, promotes resistance to anti-glioma alkylators, including temozolomide and BCNU, in GBM cell lines and xenografts. Moreover, MGMT expression assessed by immunohistochemistry, biochemical activity or promoter CpG methylation status is associated with the response of GBM to alkylator-based therapies, providing evidence that MGMT promotes clinical resistance to alkylating agents. These observations suggest a role for MGMT in directing adjuvant therapy of GBM and other gliomas. Promoter methylation status is the most clinically tractable measure of MGMT, and there is considerable enthusiasm for exploring its utility as a marker to assign therapy to individual patients. Here, we provide an overview of the biochemical, genetic and biological characteristics of MGMT as they relate to glioma therapy. We consider current methods to assess MGMT expression and discuss their utility as predictors of treatment response. Particular emphasis is given to promoter methylation status and the methodological and conceptual impediments that limit its use to direct treatment. We conclude by considering approaches that may improve the utility of MGMT methylation status in planning optimal therapies tailored to individual patients.
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http://dx.doi.org/10.1016/j.bbcan.2011.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340514PMC
August 2012

Doxorubicin loaded iron oxide nanoparticles overcome multidrug resistance in cancer in vitro.

J Control Release 2011 May 26;152(1):76-83. Epub 2011 Jan 26.

Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA.

Multidrug resistance (MDR) is characterized by the overexpression of ATP-binding cassette (ABC) transporters that actively pump a broad class of hydrophobic chemotherapeutic drugs out of cancer cells. MDR is a major mechanism of treatment resistance in a variety of human tumors, and clinically applicable strategies to circumvent MDR remain to be characterized. Here we describe the fabrication and characterization of a drug-loaded iron oxide nanoparticle designed to circumvent MDR. Doxorubicin (DOX), an anthracycline antibiotic commonly used in cancer chemotherapy and substrate for ABC-mediated drug efflux, was covalently bound to polyethylenimine via a pH sensitive hydrazone linkage and conjugated to an iron oxide nanoparticle coated with amine terminated polyethylene glycol. Drug loading, physiochemical properties and pH lability of the DOX-hydrazone linkage were evaluated in vitro. Nanoparticle uptake, retention, and dose-dependent effects on viability were compared in wild-type and DOX-resistant ABC transporter over-expressing rat glioma C6 cells. We found that DOX release from nanoparticles was greatest at acidic pH, indicative of cleavage of the hydrazone linkage. DOX-conjugated nanoparticles were readily taken up by wild-type and drug-resistant cells. In contrast to free drug, DOX-conjugated nanoparticles persisted in drug-resistant cells, indicating that they were not subject to drug efflux. Greater retention of DOX-conjugated nanoparticles was accompanied by reduction of viability relative to cells treated with free drug. Our results suggest that DOX-conjugated nanoparticles could improve the efficacy of chemotherapy by circumventing MDR.
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http://dx.doi.org/10.1016/j.jconrel.2011.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3110619PMC
May 2011

Apurinic/apyrimidinic endonuclease is inversely associated with response to radiotherapy in pediatric ependymoma.

Int J Cancer 2011 Nov 5;129(10):2370-9. Epub 2011 May 5.

Department of Neurological Surgery, University of Washington, Seattle, WA 98195-6470, USA.

Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair activity that confers radiation resistance in human cells. Here we examined the association between Ap endo activity and response to radiotherapy in pediatric ependymomas, tumors for which treatment options are limited and survival rates are only about 50%. We assayed Ap endo activity in 36 ependymomas and expression of Ape1/Ref-1, the predominant Ap endo activity in humans, in 44 tumors by immunostaining. Cox proportional hazards regression models were used to analyze the association of activity or expression with progression-free survival or with overall survival. Activity varied 13-fold and was not associated with tumor or patient characteristics. In univariate models with Ap endo activity entered as a continuous variable, the hazard ratio for progression increased by a factor of 2.18 for every 0.01 unit increase in activity (p ≤ 0.003) in 24 grade II ependymomas. Risk for death increased by a factor of 1.89 (p ≤ 0.02) in the same population. The fraction of Ape1/Ref-1 immunopositive cells varied widely within individual tumors and was not associated with either progression-free or with overall survival. Suppressing Ap endo activity in pediatric ependymoma cells significantly increased radiation sensitivity, suggesting that the association of activity with radiation response reflected, at least in part, repair of radiation-induced DNA lesions. Our data indicate that Ap endo activity is predictive of outcome following radiotherapy, and suggest that Ape1/Ref-1 promotes radiation resistance in pediatric ependymomas. Our findings support the use of inhibitors of Ap endo activity to overcome resistance.
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http://dx.doi.org/10.1002/ijc.25900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285472PMC
November 2011

TWIST1 promotes invasion through mesenchymal change in human glioblastoma.

Mol Cancer 2010 Jul 20;9:194. Epub 2010 Jul 20.

Department of Neurological Surgery, University of Washington School of Medicine, Seattle, WA 98195, USA.

Background: Tumor cell invasion into adjacent normal brain is a mesenchymal feature of GBM and a major factor contributing to their dismal outcomes. Therefore, better understandings of mechanisms that promote mesenchymal change in GBM are of great clinical importance to address invasion. We previously showed that the bHLH transcription factor TWIST1 which orchestrates carcinoma metastasis through an epithelial mesenchymal transition (EMT) is upregulated in GBM and promotes invasion of the SF767 GBM cell line in vitro.

Results: To further define TWIST1 functions in GBM we tested the impact of TWIST1 over-expression on invasion in vivo and its impact on gene expression. We found that TWIST1 significantly increased SNB19 and T98G cell line invasion in orthotopic xenotransplants and increased expression of genes in functional categories associated with adhesion, extracellular matrix proteins, cell motility and locomotion, cell migration and actin cytoskeleton organization. Consistent with this TWIST1 reduced cell aggregation, promoted actin cytoskeletal re-organization and enhanced migration and adhesion to fibronectin substrates. Individual genes upregulated by TWIST1 known to promote EMT and/or GBM invasion included SNAI2, MMP2, HGF, FAP and FN1. Distinct from carcinoma EMT, TWIST1 did not generate an E- to N-cadherin "switch" in GBM cell lines. The clinical relevance of putative TWIST target genes SNAI2 and fibroblast activation protein alpha (FAP) identified in vitro was confirmed by their highly correlated expression with TWIST1 in 39 human tumors. The potential therapeutic importance of inhibiting TWIST1 was also shown through a decrease in cell invasion in vitro and growth of GBM stem cells.

Conclusions: Together these studies demonstrated that TWIST1 enhances GBM invasion in concert with mesenchymal change not involving the canonical cadherin switch of carcinoma EMT. Given the recent recognition that mesenchymal change in GBMs is associated with increased malignancy, these findings support the potential therapeutic importance of strategies to subvert TWIST1-mediated mesenchymal change.
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http://dx.doi.org/10.1186/1476-4598-9-194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920263PMC
July 2010

Minimally cytotoxic doses of temozolomide produce radiosensitization in human glioblastoma cells regardless of MGMT expression.

Mol Cancer Ther 2010 May;9(5):1208-18

Department of Neurological Surgery, University of Washington, Seattle, WA 98195-6470, USA.

Concurrent treatment with the methylating agent temozolomide during radiotherapy has yielded the first significant improvement in the survival of adult glioblastomas (GBM) in the last three decades. However, improved survival is observed in a minority of patients, most frequently those whose tumors display CpG methylation of the O(6)-methylguanine (O(6)-meG)-DNA methyltransferase (MGMT) promoter, and adult GBMs remain invariably fatal. Some, although not all, preclinical studies have shown that temozolomide can increase radiosensitivity in GBM cells that lack MGMT, the sole activity in human cells that removes O(6)-meG from DNA. Here, we systematically examined the temozolomide dose dependence of radiation killing in established GBM cell lines that differ in ability to remove O(6)-meG or tolerate its lethality. Our results show that minimally cytotoxic doses of temozolomide can produce dose-dependent radiosensitization in MGMT-deficient cells, MGMT-proficient cells, and MGMT-deficient cells that lack mismatch repair, a process that renders cells tolerant of the lethality of O(6)-meG. In cells that either possess or lack MGMT activity, radiosensitization requires exposure to temozolomide before but not after radiation and is accompanied by formation of double-strand breaks within 45 minutes of radiation. Moreover, suppressing alkyladenine-DNA glycosylase, the only activity in human cells that excises 3-methyladenine from DNA, reduces the temozolomide dose dependence of radiosensitization, indicating that radiosensitization is mediated by 3-methyladenine as well as by O(6)-meG. These results provide novel information on which to base further mechanistic study of radiosensitization by temozolomide in human GBM cells and to develop strategies to improve the outcome of concurrent temozolomide radiotherapy.
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http://dx.doi.org/10.1158/1535-7163.MCT-10-0010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2869471PMC
May 2010

O6-methylguanine-DNA methyltransferase deficiency in developing brain: implications for brain tumorigenesis.

DNA Repair (Amst) 2007 Aug 17;6(8):1127-33. Epub 2007 May 17.

Department of Neurological Surgery, Box 356470, University of Washington, Seattle, WA 98195-6470, USA.

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a cardinal defense against the mutagenic and carcinogenic effects of alkylating agents. We have reported evidence that absence of detectable MGMT activity (MGMT(-) phenotype) in human brain is a predisposing factor for primary brain tumors that affects ca. 12% of individuals [J.R. Silber, A. Blank, M.S. Bobola, B.A. Mueller, D.D. Kolstoe, G.A. Ojemann, M.S. Berger, Lack of the DNA repair protein O(6)-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary brain tumors, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 6941-6946]. We report here that MGMT(-) phenotype in the brain of children and adults, and the apparent increase in risk of neurocarcinogenesis, may arise during gestation. We found that MGMT activity in 71 brain specimens at 6-19 weeks post-conception was positively correlated with gestational age (P
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http://dx.doi.org/10.1016/j.dnarep.2007.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692685PMC
August 2007

Human glioma cell sensitivity to the sequence-specific alkylating agent methyl-lexitropsin.

Clin Cancer Res 2007 Jan;13(2 Pt 1):612-20

Department of Neurological Surgery, University of Washington, Seattle, WA 98105, USA.

Purpose: Defining the cytotoxicity of individual adducts in DNA is necessary for mechanistic understanding of human brain tumor resistance to therapeutic alkylating agents and for design of DNA repair-related antiresistance strategies. Our purpose is to characterize the sensitivity of human glioma cells to methyl-lexitropsin (Me-lex), a sequence-specific alkylator that produces 3-methyladenine (3-meA) as the predominant (>90%) DNA lesion.

Experimental Design: We quantitated the Me-lex cytotoxicity of 10 human glioma cell lines that differ in O(6)-methylguanine (O(6)-meG)-DNA methyltransferase (MGMT) and mismatch repair activity. We used antisense suppression of alkyladenine DNA glycosylase (AAG) and Ape1 to assess the contribution of 3-meA and abasic sites to lethality and measured abasic sites.

Results: (a) The LD(10) for Me-lex varied widely among the cell lines. (b) MGMT-proficient lines were more resistant than MGMT-deficient lines, an unexpected finding because Me-lex produces very little O(6)-meG. (c) Suppression of AAG increased Me-lex killing and reduced abasic site content. (d) Suppression of Ape1 increased Me-lex killing and increased abasic site content. (e) Ablation of MGMT had no effect on Me-lex cytotoxicity.

Conclusions: (a) Me-lex is cytotoxic in human glioma cells and AAG promotes resistance, indicating that 3-meA is a lethal lesion in these cells. (b) Abasic sites resulting from 3-meA repair are cytotoxic and Ape1 promotes resistance to these derivative lesions. (c) A factor(s) associated with MGMT expression, other than repair of O(6)-meG, contributes to Me-lex resistance. (d) Me-lex may have clinical utility in the adjuvant therapy of gliomas. (e) AAG and Ape1 inhibitors may be useful in targeting alkylating agent resistance.
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http://dx.doi.org/10.1158/1078-0432.CCR-06-1127DOI Listing
January 2007

Complications of a temozolomide overdose: a case report.

J Neurooncol 2006 Oct 28;80(1):57-61. Epub 2006 Apr 28.

Department of Neurology, University of Washington School of Medicine, Room RR650, 1959 NE Pacific Street, Seattle, WA 98195, USA.

Purpose And Background: This is a report of a 53 year-old man with a glioblastoma multiforme (GBM) treated with an excessive dose of temozolomide (TMZ).

Methods: This is a single case review of all clinically relevant records. O6-methylguanine-DNA methyltransferase activity was determined by a biochemical assay.

Results: Following conventional radiotherapy (RT) without concurrent chemotherapy, the patient received 5,500 mg of TMZ over 2 days. At the standard dose of 200 mg/m2/day his total 5-day dose should have been 1,940 mg. Acutely he had nausea, vomiting and diarrhea for 2 days which cleared. The dominant severe toxicity was pancytopenia between one and four weeks after TMZ which was complicated by secondary infections that were successfully managed. Transient transaminitis occurred but there were no significant pulmonary, renal or other systemic toxicities. His progression free survival was 22 months and overall survival 24 months.

Conclusion: His outcome suggests that TMZ may prove to be a good agent for dose-escalation trials with hematopoietic stem cell rescue.
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http://dx.doi.org/10.1007/s11060-006-9152-yDOI Listing
October 2006

Apurinic/apyrimidinic endonuclease activity is associated with response to radiation and chemotherapy in medulloblastoma and primitive neuroectodermal tumors.

Clin Cancer Res 2005 Oct;11(20):7405-14

Division of Neurosurgery, Department of Surgery, Children's Hospital and Regional Medical Center, Seattle, WA 98105, USA.

Purpose: Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair activity that confers resistance to radiation- and alkylator-induced cytotoxic abasic sites in human cells. We assayed apurinic/apyrimidinic endonuclease activity in medulloblastomas and primitive neuroectodermal tumors (PNET) to establish correlates with tumor and patient characteristics and with response to adjuvant radiation plus multiagent chemotherapy.

Experimental Design: Ap endo activity was assayed in 52 medulloblastomas and 10 PNETs from patients 0.4 to 21 years old. Ape1/Ref-1, the predominant human Ap endo activity, was measured in 42 medulloblastomas by immunostaining. Cox proportional hazards regression models were used to analyze the association of activity with time to tumor progression (TTP).

Results: Tumor Ap endo activity varied 180-fold and was significantly associated with age and gender. Tumor Ape1/Ref-1 was detected almost exclusively in nuclei. In a multivariate model, with Ap endo activity entered as a continuous variable, the hazard ratio for progression after adjuvant treatment in 46 medulloblastomas and four PNETs increased by a factor of 1.073 for every 0.01 unit increase in activity (P < or = 0.001) and was independent of age and gender. Suppressing Ap endo activity in a human medulloblastoma cell line significantly increased sensitivity to 1,3-bis(2-chlororethyl)-1-nitrosourea and temozolomide, suggesting that the association of tumor activity with TTP reflected, at least in part, abasic site repair.

Conclusions: Our data (a) suggest that Ap endo activity promotes resistance to radiation plus chemotherapy in medulloblastomas/PNETs, (b) provide a potential marker of treatment outcome, and (c) suggest clinical use of Ap endo inhibitors to overcome resistance.
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http://dx.doi.org/10.1158/1078-0432.CCR-05-1068DOI Listing
October 2005

TWIST is expressed in human gliomas and promotes invasion.

Neoplasia 2005 Sep;7(9):824-37

Department of Neurological Surgery, University of Washington, Seattle, WA 98195, USA.

TWIST, a basic helix-loop-helix (bHLH) transcription factor that regulates mesodermal development, has been shown to promote tumor cell metastasis and to enhance survival in response to cytotoxic stress. Our analysis of rat C6 glioma cell-derived cDNA revealed TWIST expression, suggesting that the gene may play a role in the genesis and physiology of primary brain tumors. To further delineate a possible oncogenic role for TWIST in the central nervous system (CNS), we analyzed TWIST expression in human gliomas and normal brain by using reverse transcription polymerase chain reaction, Northern blot analysis, in situ hybridization, and immunohistochemistry. TWIST expression was detected in the large majority of human glioma-derived cell lines and human gliomas examined. Levels of TWIST mRNA were associated with the highest grade gliomas, and increased TWIST expression accompanied transition from low grade to high grade in vivo, suggesting a role for TWIST in promoting malignant progression. In accord, elevated TWIST mRNA abundance preceded the spontaneous malignant transformation of cultured mouse astrocytes hemizygous for p53. Overexpression of TWIST protein in a human glioma cell line significantly enhanced tumor cell invasion, a hallmark of high-grade gliomas. These findings support roles for TWIST both in early glial tumorigenesis and subsequent malignant progression. TWIST was also expressed in embryonic and fetal human brain, and in neurons, but not glia, of mature brain, indicating that, in gliomas, TWIST may promote the functions also critical for CNS development or normal neuronal physiology.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1501937PMC
http://dx.doi.org/10.1593/neo.04352DOI Listing
September 2005

O6-methylguanine-DNA methyltransferase, O6-benzylguanine, and resistance to clinical alkylators in pediatric primary brain tumor cell lines.

Clin Cancer Res 2005 Apr;11(7):2747-55

Division of Neurosurgery, Department of Surgery and Hematology/Oncology, Children's Hospital and Regional Medical Center, Seattle, Washington 98105, USA.

Purpose: Primary brain tumors are the leading cause of cancer death in children. Our purpose is (a) to assess the contribution of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) to the resistance of pediatric brain tumor cell lines to clinical alkylating agents and (b) to evaluate variables for maximal potentiation of cell killing by the MGMT inhibitor O6-benzylguanine, currently in clinical trials. Few such data for pediatric glioma lines, particularly those from low-grade tumors, are currently available.

Experimental Design: We used clonogenic assays of proliferative survival to quantitate cytoxicity of the chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and the methylating agent temozolomide in 11 glioma and five medulloblastoma lines. Twelve lines are newly established and characterized here, nine of them from low-grade gliomas including pilocytic astrocytomas.

Results: (a) MGMT is a major determinant of BCNU resistance and the predominant determinant of temozolomide resistance in both our glioma and medulloblastoma lines. On average, O(6)-benzylguanine reduced LD10 for BCNU and temozolomide, 2.6- and 26-fold, respectively, in 15 MGMT-expressing lines. (b) O6-Benzylguanine reduced DT (the threshold dose for killing) for BCNU and temozolomide, 3.3- and 138-fold, respectively. DT was decreased from levels higher than, to levels below, clinically achievable plasma doses for both alkylators. (c) Maximal potentiation by O6-benzylguanine required complete and prolonged suppression of MGMT.

Conclusions: Our results support the use of O6-benzylguanine to achieve full benefit of alkylating agents, particularly temozolomide, in the chemotherapy of pediatric brain tumors.
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http://dx.doi.org/10.1158/1078-0432.CCR-04-2045DOI Listing
April 2005

Apurinic endonuclease activity in adult gliomas and time to tumor progression after alkylating agent-based chemotherapy and after radiotherapy.

Clin Cancer Res 2004 Dec;10(23):7875-83

Department of Neurological Surgery, University of Washington, 1959 N.E Pacific Street, Seattle, WA 98195-6470, USA.

Purpose: Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair enzyme that cleaves DNA at cytotoxic abasic sites caused by alkylating agents and radiation. We have observed that human glioma cells deficient in Ap endo activity are hypersensitive to clinically used alkylators (Silber et al., Clin Cancer Res 2002;8:3008.). Here we examine the association of glioma Ap endo activity with clinical response after alkylating agent-based chemotherapy or after radiotherapy.

Experimental Design: Cox proportional hazards regression models were used to analyze the relationship of Ap endo activity with time to tumor progression (TTP).

Results: In a univariate model with Ap endo activity entered as a continuous variable, the hazard ratio (HR) for progression after alkylator therapy in 30 grade III gliomas increased by a factor of 1.061 for every 0.01 increase in activity (P = 0.013). Adjusting for age, gender, extent of resection, and prior treatment strengthened slightly the association (HR = 1.094; P = 0.003). Similarly, the HR for progression after radiotherapy in 44 grade II and III tumors increased by a factor of 1.069 (P = 0.008). Adjusting for the aforementioned variables had little effect on the association. In contrast, we observed no association between activity and TTP in grade IV gliomas after either alkylator therapy in 34 tumors or radiotherapy in 26 tumors.

Conclusions: Our data suggest that Ap endo activity mediates resistance to alkylating agents and radiation and may be a useful predictor of progression after adjuvant therapy in a subset of gliomas.
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http://dx.doi.org/10.1158/1078-0432.CCR-04-1161DOI Listing
December 2004

The Werner syndrome protein confers resistance to the DNA lesions N3-methyladenine and O6-methylguanine: implications for WRN function.

DNA Repair (Amst) 2004 Jun;3(6):629-38

Department of Pathology, University of Washington, Seattle, WA 98195-7705, USA.

The Werner syndrome (WS) protein (WRN), a DNA helicase/exonuclease, is required for genomic stability and avoidance of cancer. Current evidence suggests that WRN is involved in the resolution of stalled and/or collapsed replication forks. This function is indicated, in part, by replication defects in WS cells and by hypersensitivity to agents causing major structural aberrations in DNA that block replication. We show here that antisense suppression of WRN in two human glioma cell lines reproduces hallmarks of the drug cytotoxicity profile of WS cells, namely, hypersensitivity to 4-nitroquinoline 1-oxide, camptothecin and hydroxyurea. We also show that antisense-treated cells are hypersensitive to methyl-lexitropsin, a site-specific alkylating agent that produces mainly N3-methyladenine, a cytotoxic and replication-blocking lesion. Antisense-treated cells are hypersensitive to O(6)-methylguanine adducts as well, but only when repair by O(6)-methylguanine-DNA methyltransferase is lacking. Our results illustrate the drug sensitivity caused by deficiency of WRN in a uniform genetic background. They extend the WRN DNA damage sensitivity spectrum to methyl base adducts that can result in blocked replication, and suggest that WRN may be required for resumption of processive replication when incomplete repair of DNA damage leaves blocking lesions at forks. The evidence that highly disparate lesions fall within the purview of WRN, and that abrogating DNA repair can reveal dependence on WRN, suggests that WRN may protect the genome from the lethal, mutagenic and carcinogenic effects of widely diverse DNA damage arising from endogenous processes and environmental agents.
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http://dx.doi.org/10.1016/j.dnarep.2004.02.003DOI Listing
June 2004
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