David Nguyen

Molecular properties of CD133+ glioblastoma stem cells derived from treatment-refractory recurrent brain tumors

Glioblastoma multiforme (GBM) remains refractory to conventional therapy. CD133+ GBM cells have been recently isolated and characterized as chemo-/radio-resistant tumor-initiating cells and are hypothesized to be responsible for post-treatment recurrence. In order to explore the molecular properties of tumorigenic CD133+ GBM cells that resist treatment, we isolated CD133+ GBM cells from tumors that are recurrent and have previously received chemo-/radio-therapy. We found that the purified CD133+ GBM cells sorted from the CD133+ GBM spheres express SOX2 and CD44 and are capable of clonal self-renewal and dividing to produce fast-growing CD133− progeny, which form the major cell population within GBM spheres. Intracranial injection of purified CD133+, not CD133− GBM daughter cells, can lead to the development of YKL-40+ infiltrating tumors that display hypervascularity and pseudopalisading necrosis-like features in mouse brain. The molecular profile of purified CD133+ GBM cells revealed characteristics of neuroectoderm-like cells, expressing both radial glial and neural crest cell developmental genes, and portraying a slow-growing, non-differentiated, polarized/migratory, astrogliogenic, and chondrogenic phenotype. These data suggest that at least a subset of treated and recurrent GBM tumors may be seeded by CD133+ GBM cells with neural and mesenchymal properties. The data also imply that CD133+ GBM cells may be clinically indolent/quiescent prior to undergoing proliferative cell division (PCD) to produce CD133− GBM effector progeny. Identifying intrinsic and extrinsic cues, which promote CD133+ GBM cell self-renewal and PCD to support ongoing tumor regeneration may highlight novel therapeutic strategies to greatly diminish the recurrence rate of GBM.

Depolarization and CaM Kinase IV Modulate NMDA Receptor Splicing through Two Essential RNA Elements

Alternative splicing controls the activity of many proteins important for neuronal excitation, but the signal-transduction pathways that affect spliced isoform expression are not well understood. One particularly interesting system of alternative splicing is exon 21 (E21) of the NMDA receptor 1 (NMDAR1 E21), which controls the trafficking of NMDA receptors to the plasma membrane and is repressed by Ca++/calmodulin-dependent protein kinase (CaMK) IV signaling. Here, we characterize the splicing of NMDAR1 E21. We find that E21 splicing is reversibly repressed by neuronal depolarization, and we identify two RNA elements within the exon that function together to mediate the inducible repression. One of these exonic elements is similar to an intronic CaMK IV–responsive RNA element (CaRRE) originally identified in the 3′ splice site of the BK channel STREX exon, but not previously observed within an exon. The other element is a new RNA motif. Introduction of either of these two motifs, called CaRRE type 1 and CaRRE type 2, into a heterologous constitutive exon can confer CaMK IV–dependent repression on the new exon. Thus, either exonic CaRRE can be sufficient for CaMK IV–induced repression. Single nucleotide scanning mutagenesis defined consensus sequences for these two CaRRE motifs. A genome-wide motif search and subsequent RT-PCR validation identified a group of depolarization-regulated alternative exons carrying CaRRE consensus sequences. Many of these exons are likely to alter neuronal function. Thus, these two RNA elements define a group of co-regulated splicing events that respond to a common stimulus in neurons to alter their activity.

Optimizing Prostate Cancer Suicide Gene Therapy Using Herpes Simplex Virus Thymidine Kinase Active Site Variants

The herpes simplex virus (HSV) thymidine kinase gene (tk) forms the basis of a widely used strategy for suicide gene therapy. A library of HSV thymidine kinase enzyme (TK) active site mutants having different affinities for guanosine analog prodrugs was developed. We sought to determine the optimal combination of tk variant and prodrug specifically for prostate cancer gene therapy, using in vitro and in vivo studies of adenovirally infected CL1, DU-145, and LNCaP tumor lines carrying wild-type tk, tk30, tk75, and sr39tk mutants expressed by a strong, constitutive cytomegalovirus promoter and treated with ganciclovir and acyclovir. In vitro experiments involving prostate cancer (CaP) cell line infection were carried out with a broad range of prodrug concentrations, and cell killing was determined by limiting dilution (colony-forming), MTT, and propidium iodide assays. In vivo studies based on CL1-GFP xenograft experiments were carried out to examine the ability of each TK variant to prevent tumor formation and to inhibit tumor growth and development of metastases in established orthotopic and subcutaneous tumors in SCID mice. Both in vitro and in vivo studies suggest improved killing with the sr39tk variant. Thus, the results suggest that the use of sr39tk in future trials of prostate cancer tk suicide gene therapy may be beneficial.

Autocrine Endothelin-3/Endothelin Receptor B Signaling Maintains Cellular and Molecular Properties of Glioblastoma Stem Cells

Glioblastoma stem cells (GSC) express both radial glial cell and neural crest cell (NCC)-associated genes. We report that endothelin 3 (EDN3), an essential mitogen for NCC development and migration, is highly produced by GSCs. Serum-induced proliferative differentiation rapidly decreased EDN3 production and downregulated the expression of stemness-associated genes, and reciprocally, two glioblastoma markers, EDN1 and YKL-40 transcripts, were induced. Correspondingly, patient glioblastoma tissues express low levels of EDN3 mRNA and high levels of EDN1 and YKL-40 mRNA. Blocking EDN3/EDN receptor B (EDNRB) signaling by an EDNRB antagonist (BQ788), or EDN3 RNA interference (siRNA), leads to cell apoptosis and functional impairment of tumor sphere formation and cell spreading/migration in culture and loss of tumorigenic capacity in animals. Using exogenous EDN3 as the sole mitogen in culture does not support GSC propagation, but it can rescue GSCs from undergoing cell apoptosis. Molecular analysis by gene expression profiling revealed that most genes downregulated by EDN3/EDNRB blockade were those involved in cytoskeleton organization, pause of growth and differentiation, and DNA damage response, implicating the involvement of EDN3/EDNRB signaling in maintaining GSC migration, undifferentiation, and survival. These data suggest that autocrine EDN3/EDNRB signaling is essential for maintaining GSCs. Incorporating END3/EDNRB-targeted therapies into conventional cancer treatments may have clinical implication for the prevention of tumor recurrence. Mol Cancer Res; 9(12); 1668–85. ©2011 AACR.

CL1-SR39: A noninvasive molecular imaging model of prostate cancer suicide gene therapy using positron emission tomography.

PURPOSE We developed a prostate cancer tumor model capable of being noninvasively imaged using positron emission tomography (PET) based on expression of the herpes simplex virus thymidine kinase (HSV1-tk) reporter gene. MATERIALS AND METHODS The androgen independent, metastatic prostate cancer cell lines CL1 and CL1-GFP were stably transfected with the mutant HSV1-tk gene pcDNA3.1/pCMV-sr39tk, which has increased ability to phosphorylate penciclovir. The presence of the sr39tk gene product was analyzed by Western blot analysis and relative thymidine kinase enzyme activity was assessed by a functional thymidine kinase enzyme activity assay. Subcutaneous and orthotopic CL1 and CL1-SR39 tumor xenografts were established in SCID mice. The ability to image CL1-SR39 was assessed using fluorodeoxyglucose and F-penciclovir ( F-FHBG) micro-PET (a rodent PET scanner). To investigate the systemic distribution of intratumoral sr39tk injections established CL1 tumors were transiently injected with first generation adenoviral vectors carrying the sr39tk gene under control of the strong cytomegalovirus promoter Ad-CMV-HSV1-sr39tk and imaged using micro-PET. RESULTS Transfection of sr39tk into CL1 cells was successful. CL1-SR39 thymidine kinase enzyme activity was greater than twice the activity of the glioma cell line C6-SR39 control and above the threshold necessary for micro-PET detection. Fluorodeoxyglucose micro-PET in SCID mice was positive for CL1 and CL1-SR39 tumors. Selective micro-PET of subcutaneous CL1-SR39 tumors was done using F-FHBG. Micro-PET imaging after systemic and intratumoral injection of Ad-CMV-HSV1-sr39tk revealed significant systemic transgene leakage with significant hepatic expression of sr39TK protein. CONCLUSIONS Molecular based imaging of sr39tk transfected prostate cancer tumors and adenoviral delivered HSV1-tk suicide gene therapy based on the selective conversion and intracellular trapping of F-FHBG by sr39tk is feasible. Potential applications include noninvasive monitoring of the location, duration and intensity of gene constructs, which may contribute to the safety of clinical gene therapy protocols, and noninvasive imaging of the prostate cancer xenograft response to experimental therapy.

Ethnic differences in arm vein diameter and arteriovenous fistula creation rates in men undergoing hemodialysis access

OBJECTIVE The National Kidney Foundation recommends that arteriovenous fistulas (AVFs) be placed in at least 65% of hemodialysis patients. Some studies suggest that African American patients are less likely to receive a first-time AVF than patients of other ethnicities, although the reason for this disparity is unclear. The purpose of our study is to determine (1) whether there are ethnic differences in AVF creation, (2) whether this may be related to differences in vein diameters, and (3) whether AVF patency rates are similar between African American and non-African American male patients. METHODS Consecutive male patients undergoing first-time hemodialysis access from 2006 to 2010 at two institutions were retrospectively reviewed. Data collected included age, ethnicity, weight, height, body mass index, diabetes, hypertension, congestive heart failure, smoking history, intravenous drug abuse, need for temporary access placement, and preoperative venous ultrasound measurements. Categoric variables were compared using χ(2) analysis, and the Wilcoxon rank-sum test was used to compare continuous variables. RESULTS Of 249 male patients identified, 95 were African American. Median age in African American and non-African American patients was 63 years. Hypertension and hyperlipidemia were statistically significantly greater in African American patients. The need for temporary access before hemoaccess was similar between the cohorts. African American patients demonstrated significantly smaller median basilic and cephalic vein diameters at most measured sites. Overall, 221 of 249 (88.8%) underwent AVF first. An AV graft was created in 17.9% of African American patients vs in only 7.1% of non-African Americans (odds ratio, 2.8; 95% confidence interval, 1.3-6.4; P = .009). The difference between median vein diameters used for autologous fistula creation in African American and non-African American patients was not significant. There was no significant difference in the primary patency (80.8% vs 76.2%; P = .4), primary functional patency (73.1% vs 69.2%; P = .5), or secondary functional patency rates (91.0% vs 96.5%; P = .1). Average primary fistula survival time was 257 days in African American and 256 in non-African American patients (P = .2). CONCLUSIONS African American patients are less likely than non-African American patients to undergo AVF during first-time hemodialysis access surgery. This ethnic discrepancy appears to be due to smaller arm vein diameters in African American patients. In African American patients with appropriate vein diameters who do undergo AVF, primary and functional patencies are equivalent to non-African American patients.

Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples

Background Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA. Methods Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3’ UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing. Results Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%). Conclusion Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.

Poster 393 C5 Palsy After Cervical Laminectomy and Fusion: 2 Case Reports

Disclosures: Alexander Tucker: I Have No Relevant Financial Relationships To Disclose Case/Program Description: Proximal upper extremity weakness is a poorly understood complication of cervical spine surgery. The cause of this complication is unknown, although most patients recover completely over weeks to months with conservative treatment. We present two cases of postoperative, transient C5 palsy after laminectomy and fusion. Electrodiagnostic (EDX) studies performed after surgery revealed active denervation of the deltoid and biceps muscles on the symptomatic side, however the rhomboid muscles appeared unaffected. Setting: Tertiary care hospital. Results: By 6 months postoperatively, both patients had clinically improved strength in all previously affected muscle groups. We suggest that the current models of postoperative C5 palsy pathogenesis may be inadequate to explain our findings and propose an alternative theory of watershed ischemia distal to the origin of the dorsal scapular nerve as a contributing factor to the etiology of this surgical complication. Discussion: The cases described above illustrate postoperative C5 palsy, a notorious and under-recognized complication of cervical decompressive surgery, that was diagnosed clinically and correlated with EDX evidence. This description is the first to present the results of preoperative and postoperative electrophysiological studies; confirming deltoid and biceps weakness with rhomboid sparing. Conclusions: Serial EDX studies, as were performed on the patients presented here, may be used to assist with diagnosis, confirmation of involved root levels, prognostication, to follow recovery or to offer clues into the pathophysiology of the condition. Level of Evidence: Level V