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In contrast to Aβ plaques, the spatiotemporal distribution of neurofibrillary tangles of hyperphosphorylated tau (p-tau) predicts cognitive impairment in Alzheimer’s disease (AD), underscoring the key pathological role of p-tau and the utmost need to develop AD therapeutics centering upon the control of p-tau aggregation and cytotoxicity. Our drug discovery program is focused on compounds that prevent the aggregation and cytotoxicity of p-tau moieties of the tau isoform 1N4R due to its prevalence (1 N) and long-distance trans-synaptic propagation (4R). We prepared and tested twenty-four newly synthesized small molecules representing the urea (1, 2, 3), sulfonylurea (4), and sulfonamide (5–24) series and evaluated their anti-aggregation effects with biophysical methods (thioflavin T and S fluorescence assays, transmission electron microscopy) and intracellular inclusion cell-based assays. Pre-evaluation was performed on alpha-synuclein (α-syn) to identify molecules to be challenged with p-tau. The sulfonamide derivatives 18 and 20 exhibited an anti-fribrillization activity on α-syn and p-tau. Sulfonamide compounds 18 and 20 reduced inclusion formation in M17D neuroblastoma cells that express inclusion-prone αSynuclein3K::YFP. This project advances new concepts in targeting prone-to-aggregate proteins such as α-syn and p-tau, and provides a molecular scaffold for further optimization and pre-clinical studies focused on AD drug development.

The University of Missouri (MU) has established a colony of dystrophin‐deficient dogs with a mixed breed background to mirror the variable pathologic effects of dystrophinopathies between persons of a given kindred to further the understanding of the genetic and molecular basis of the variable phenotype; thus to facilitate discovery of an effective therapeutic strategy. Herein we report the phenotype and genotype of a normal‐appearing 10‐month‐old colony female that died suddenly. At necropsy examination, there were reduced skeletal and laryngeal muscle volume and mild dilatation of the oesophagus. Microscopic findings consisted of extensive degeneration and regeneration of the axial skeletal, tongue, oesophageal, and laryngeal muscles that were characterized by considerable central nucleation, individual fibre mineralization and interstitial fibrosis. The myocardial findings were limited to infiltration of adipose cells in the interstitium. The female dog was a compound heterozygote with one X chromosome carrying a point mutation in intron 6 of the dystrophin gene and the other X chromosome carrying a repetitive element insertion in intron 13 of the dystrophin gene. Although the direct cause of death was uncertain, it might likely be due to sudden cardiac death as has been seen in Duchenne muscular dystrophy patients. This case demonstrated dystrophinopathy in female dogs that have no ameliorating normal X chromosome., A 10‐month‐old mixed breed female dog in a muscular dystrophy research colony at the University of Missouri died suddenly without premonitory symptoms. At necropsy examination, there were reduced skeletal and laryngeal muscle volume and mild dilatation of the esophagus. Microscopic findings consisted of extensive degeneration and regeneration of the axial skeletal, tongue, esophageal, and laryngeal muscles that were characterized by considerable central nucleation, individual fiber mineralization and interstitial fibrosis.

Digital image analysis (DIA) is impacted by the quality of tissue staining. This study examined the influence of preanalytical variables—staining protocol design, reagent quality, section attributes, and instrumentation—on the performance of automated DIA software. Our hypotheses were that (1) staining intensity is impacted by subtle differences in protocol design, reagent quality, and section composition and that (2) identically programmed and loaded stainers will produce equivalent immunohistochemical (IHC) staining. We tested these propositions by using 1 hematoxylin and eosin stainer to process 13 formalin-fixed, paraffin-embedded (FFPE) mouse tissues and by using 3 identically programmed and loaded immunostainers to process 5 FFPE mouse tissues for 4 cell biomarkers. Digital images of stained sections acquired with a commercial whole slide scanner were analyzed by customizable algorithms incorporated into commercially available DIA software. Staining intensity as viewed qualitatively by an observer and/or quantitatively by DIA was affected by staining conditions and tissue attributes. Intrarun and inter-run IHC staining intensities were equivalent for each tissue when processed on a given stainer but varied measurably across stainers. Our data indicate that staining quality must be monitored for each method and stainer to ensure that preanalytical factors do not impact digital pathology data quality.

Background Pituitary pars intermedia dysfunction (PPID) develops slowly in aged horses as degeneration of hypothalamic dopaminergic neurons leads to proliferation of pars intermedia (PI) melanotropes through hyperplasia and adenoma formation. Dopamine (DA) concentrations and tyrosine hydroxylase (TH) immunoreactivity are markedly reduced in PI tissue of PPID-affected equids and treatment with the DA receptor agonist pergolide results in notable clinical improvement. Thus, we hypothesized that pergolide treatment of PPID-affected horses would result in greater DA and TH levels in PI tissue collected from PPID-affected horses versus untreated PPID-affected horses. To test this hypothesis, pituitary glands were removed from 18 horses: four untreated PPID-affected horses, four aged and four young horses without signs of PPID, and six PPID-affected horses that had been treated with pergolide at 2 µg/kg orally once daily for 6 months. DA concentrations and TH expression levels in PI tissues were determined by high performance liquid chromatography with electrochemical detection and Western blot analyses, respectively. Results DA and TH levels were lowest in PI collected from untreated PPID-affected horses while levels in the pergolide treated horses were similar to those of aged horses without signs of PPID. Conclusions These findings provide evidence of restoration of DA and TH levels following treatment with pergolide. Equine PPID is a potential animal model of dopaminergic neurodegeneration, which could provide insight into human neurodegenerative diseases.

In humans with type 2 diabetes, at least 70% of patients exhibit islet amyloid plaques formed by misfolding islet amyloid polypeptides (IAPP). The oligomeric conformation and accumulation of the IAPP plaques lead to a panoply of cytotoxic effects on the islet β-cells. Currently, no marketed therapies for the prevention or elimination of these amyloid deposits exist, and therefore significant efforts are required to address this gap. To date, most of the experimental treatments are limited to only in vitro stages of testing. In general, the proposed therapeutics use various targeting strategies, such as binding to the N-terminal region of islet amyloid polypeptide on residues 1-19 or the hydrophobic region of IAPP. Other strategies include targeting the peptide self-assembly through π-stacking. These methods are realized by using several different families of compounds, four of which are highlighted in this review: naturally occurring products, small molecules, organometallic compounds, and nanoparticles. Each of these categories holds immense potential to optimize and develop inhibitor(s) of pancreatic amyloidosis in the near future.

Equine pituitary pars intermedia dysfunction (PPID) is a common endocrine disease of aged horses that shows a similar pathophysiology as Parkinson’s Disease (PD) with increased levels of α-synuclein (α-syn). While α-syn is thought to play a pathogenic role in horses with PPID, it is unclear if α-syn is also misfolded in the pars intermedia and could similarly promote self-aggregation and propagation. Consequently, α-syn was isolated from the pars intermedia from groups of healthy young and aged horses, and aged PPID-afflicted horses. Seeding experiments confirmed the prion-like properties of α-syn isolated from PPID-afflicted horses. Next, detection of α-syn fibrils in pars intermedia via transmission electron microscopy (TEM) was exclusive to PPID-afflicted horses. A bank of fragment peptides was designed to further characterize equine α-syn misfolding. Region 62–87 of equine and human α-syn peptides was found to be most prone to aggregation according to Tango bioinformatic program and kinetics of aggregation via a thioflavin T fluorescence assay. In both species, fragment peptide 62–87 is capable of generating mature fibrils as demonstrated by TEM. The combined animal, bioinformatic, and biophysical studies provide evidence that equine α-syn is misfolded in PPID horses.

BackgroundAlzheimer’s disease (AD) is an irreversible neurodegenerative disease without a cure or prevention to date. The defining features of AD include neurofibrillary tangles (NFTs) of hyperphosphorylated tau fibrils, and the senile plaques of β amyloid (Aβ) aggregates. In addition to AD, aggregation of hyperphosphorylated tau underlies approximately 20 neurodegenerative disorders collectively known as tauopathies, suggesting a pathogenic role of tau fibrils. The majority of AD disease-modifying drug trials targeted Aβ control, which have yet to produce satisfactory outcomes. The interest of developing anti-NFT drugs has been increasing. However, an effective method that produces hyperphosphorylated tau with disease relevant characters to support drug discovery is lacking.MethodsWe previously reported the PIMAX system for the production of recombinant proteins bearing a desired post-translational modification. Here we use PIMAX to express hyperphosphorylated tau (p-tau) in E. coli, and subjected this p-tau to mass spectrometry mapping of phosphorylation, to biochemical and biophysical assays of fibrillogenesis, and to cell culture treatment for the effects of hyperphosphorylation on tau molecular characters.ResultsMass spectrometry mapped p-tau phosphorylation to phosphoepitopes linked to AD pathological progression. In stark contrast to the unmodified tau that required an inducer for efficient aggregation, and which had only mild effects on cell functions, p-tau formed fibrils in an inducer-free and redox-independent manner. Immediately after the purification, a disease-specific conformation recognized by the MC-1 monoclonal antibody was readily detectable, which continued to increase during the fibrillization reaction. When applied to cultured cells, p-tau triggered a spike of mitochondrial superoxide, induced apoptosis, and caused cell death at sub-micromolar concentrations. In both aggregation and cytotoxicity assays, p-tau exhibited seeding activities that converted the unmodified tau into a cytotoxic species with increased propensity for fibrillization, consistent with the emerging view that hyperphosphorylated tau spreads in a prion-like fashion.ConclusionsHyperphosphorylation potentiates tau fibrillization and cytotoxicity. These characters are consistent with the model that abnormally phosphorylated tau plays a direct role in neurodegeneration in tauopathies. We suggest that p-tau produced by PIMAX affords a feasible tool for drug discovery and disease mechanistic studies for Alzheimer's disease and other tauopathies.

Phenyl-3-(2-chloroethyl)ureas (CEU) are alkylating agents exhibiting potent antiproliferative properties both in vitro and in vivo. CEUs' cytoxicity is not influenced by hypoxia or other clinically relevant mechanisms of chemoresistance, such as alteration of topoisomerase II activity and increased P-glycoprotein over-expression, DNA repair, intracellular gluthatione-S-transferase activity. A first subset of CEU exemplified by the prototypical molecules 4-tert-butyl-CEU (tBCEU) and 4-iodo-CEU (ICEU) have been shown to alkylate the colchicine-binding site on βII-tubulin. In parallel, these molecules disrupted the cytoskeleton and arrested the cell cycle in G2/M leading to anoikis. So far we have conducted several structure-activity relationship and molecular pharmacology studies in an attempt to refine the specificity of CEU for β-tubulin and the colchicine-binding site. Unexpectedly, a prototypical molecule designated as 1-(2-chloroethyl)-3-(4-cyclohexylphenyl)urea (cHCEU) emerged as arresting the cell cycle in G0/G1 phase instead of G2/M. Experiments using [14C-urea]-cHCEU showed its binding to thioredoxin isoform-1 (Trx-1) and prohibitin (PHB), but not β-tubulin. Complementary immunocytofluorometry experiments clearly showed the abrogation of the nuclear translocation of Trx-1 from the cytosol. We report herein that in addition to Trx-1 and PHB, cHCEU also alkylates galectin-1 and -3 isoforms. The cellular localization of thioredoxin-1, prohibitin, and galectin-3 (Gal-3) is changed following cHCEU treatment. In addition, the expression of Trx-1, PHB, and Gal-3 seems unaffected by CEU and its analogues. Interestingly, these proteins are all known to play an important role in the cell cycle regulation through various mechanisms. The mechanism(s) underlying the effect of cHCEU on protein translocation is unknow and might be relevant to design new potent anticancer drugs that will target specific and lethal biological pathways essential to tumor growth. Citation Format: Jessica S. Fortin, Thomas E. Wineland, Kelsey E. Duggan, René C.-Gaudreault. Revisiting small molecules that influence the nuclear translocation of thioredoxin-1, prohibitin, and galectin-3 for cancer chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 288.

Recent “best practice” recommendations for peripheral nervous system sampling and processing provide guidance regarding nerve preparation for animal toxicity studies. This study explored the impact of delayed fixation, type of fixative, processing cycle times, starting ethanol concentration, and water bath temperature to improve nerve preservation in routinely prepared (paraffin-embedded, hematoxylin and eosin [H&E]-stained) sections. Sciatic nerves from adult Wistar rats (diameter, 1.04 ± 0.1 mm) and young domestic pigs (diameter 5.9 ± 1.2 mm) fixed at necropsy (“0” hours) or 3, 6, 12, or 24 hours after death were immersed in neutral-buffered 10% formalin containing 1.2% methanol (NBF) or methanol-free 4% formaldehyde (MFF) at room temperature. After fixation for 24 hours (rat) or 48 hours (pig), specimens were processed into paraffin, and ∼5-μm-thick sections were flattened on water baths set at 35°C, 40°C, or 45°C before H&E staining. Large-diameter nerves (pig) required longer processing cycles to ensure sufficient paraffin infiltration. For both small-diameter (rat) and large-diameter nerves, structural integrity was optimal if fixation by NBF or MFF occurred within 3 hours and the initial ethanol concentration for tissue processing was lowered to 50%. At all time points, structural preservation of nerve fibers was acceptable using NBF but was better with MFF. Use of a water bath at 35°C reduced processing-related nerve fiber separation within sections.

A 21‐year‐old American Saddlebred mare died with a history of weight loss and breathing difficulties of 1 month duration. Post‐mortem examination revealed a copious pleural effusion with multifocal to coalescing numerous white to grey nodular masses on the serosal surface of the pericardium, lungs and thoracic cavity. In addition, the left thyroid gland was markedly enlarged. A thoracic mesothelioma and C‐cell adenoma with amyloid deposits of the left thyroid gland were diagnosed by histopathology and confirmed by immunohistochemistry employing antibodies against cytokeratin (CK), vimentin and calcitonin. Amyloid deposits in the thyroid tumour were confirmed by Congo red staining with apple‐green birefringence under polarized light. Mesothelioma remains an uncommon neoplasm encountered in aged horses. Discussion includes the diagnostic challenge of differentiating carcinomatosis from mesothelioma by histology and differentiating reactive and neoplastic mesothelial cells by cytology.

Background Pythiosis is a potentially fatal but non-contagious disease affecting humans and animals living in tropical and subtropical climates, but is also reasonably widespread in temperate climates, throughout the world. The most commonly reported affected animal species with pythiosis are equine and canine, with fewer cases in bovine and feline. Extracutaneous infections caused by Pythium insidiosum have been rarely described in the cat. Case presentation Sublingual pythiosis was diagnosed in a 2-year-old, male, Domestic Shorthair cat. The cat had a multilobulated, sublingual mass present for 3 months. Histopathological examination revealed severe multifocal coalescing eosinophilic granulomatous inflammation. Centers of the inflammation contained hyphae that were 3–7 μm-wide, non-parallel, uncommonly septate and rarely branching. The fungal-like organism was identified as P. insidiosum by polymerase chain reaction and subsequent amplicon sequencing. Conclusions Only a few feline pythiosis cases have been reported and, when encountered, it usually causes granulomatous diseases of the skin or gastrointestinal tract. This case presents an unusual manifestation of feline pythiosis, representing the first involving the oral cavity in cats or dogs.

Nonsteroidal anti-inflammatory drugs (NSAIDs) constitute an important pharmacotherapeutic class that, over the past decade, have expanded in application to a panoply of medical conditions. They have been tested for neurodegenerative diseases such as Alzheimer’s to reduce inflammation and also in the attempt to abrogate amyloid deposition. However, the use of NSAIDs as aggregation inhibitors has not been extensively studied in pancreatic amyloid deposition. Pancreatic amyloidosis involves the misfolding of islet amyloid polypeptide (IAPP) and contributes to the progression of type-2 diabetes in humans and felines. To ascertain their antiamyloidogenic activity, several NSAIDs were tested using fluorometric thioflavin-T assays, circular dichroism, photo-induced cross-linking assays, and cell culture. Celecoxib, diclofenac, indomethacin, meloxicam, niflumic acid, nimesulide, phenylbutazone, piroxicam, sulindac, and tenoxicam reduced fibrillization at a molar ratio of 1:10. The circular dichroism spectra of diclofenac, piroxicam, and sulindac showed characteristic spectral signatures found in predominantly α-helical structures. The oligomerization of human IAPP was abrogated with diclofenac and sulindac at a molar ratio of 1:5. The cytotoxic effects of pre-incubated human IAPP on cultured INS-1 cells were noticeably reduced in the presence of diclofenac, meloxicam, phenylbutazone, sulindac, and tenoxicam at a molar ratio of 1:10. Our results demonstrate that NSAIDs can provide chemical scaffolds to generate new and promising antiamyloidogenic agents that can be used alone or as a coadjuvant therapy.

Background: The biologic attributes of the endocrine pancreas and the comparative endocrinology of islet amyloid polypeptide (IAPP) of fish are not well described in the literature. This study describes the endocrine pancreasof one teleostean fish. Ten captive Atlantic wolffish (Anarhichas lupus)from the Montreal Biodome were submitted for necropsy and their pancreata were collected. Results: Grossly, all the fish pancreata examined contained 1-3 nodules of variable diameter (1-8 mm). Microscopically, the nodules were uniform, highly cellular, and composed of polygonal to elongated cells. Immunofluorescence for pancreatic hormones was performed. The nodules were immunoreactive for insulin most prominent centrally, but with IAPP and glucagon only in the periphery of the nodules. Exocrine pancreas was positive for chromogranin A. Not previously recognized in fish, IAPP immunoreactivity occurred in α, glucagon-containing, cells and did not co-localize with insulin in β cells. The islet tissues were devoid of amyloid deposits. IAPP DNA sequencing was performed to compare the sequence among teleost fish and the potency to form amyloid fibrils. In silico analysis of the amino acid sequences 19-34 revealed that it was not amyloidogenic. Conclusions: Amyloidosis of pancreatic islets would not be expected as a spontaneous disease in the Atlantic wolffish. Our study underlines that this teleost fish is a potential candidate for pancreatic xenograft research.

To understand the mechanisms controlling platelet-derived growth factor receptor beta (PDGFR-beta) expression in malignancies, we have cloned and characterized the first functional promoter of the human PDGFR-beta gene, which has been confirmed by luciferase reporter gene assays. The transcription initiation sites were mapped by primer extension. Promoter deletion experiments demonstrate that the proximal, highly GC-rich region (positions -165 to -139) of the human PDGFR-beta promoter is crucial for basal promoter activity. This region is sensitive to S1 nuclease and likely to assume a non-B-form DNA secondary structure within the supercoiled plasmid. The G-rich strand in this region contains a series of runs of three or more guanines that can form multiple different G-quadruplex structures, which have been subsequently assessed by circular dichroism. A Taq polymerase stop assay has shown that three different G-quadruplex-interactive drugs can each selectively stabilize different G-quadruplex structures of the human PDGFR-beta promoter. However, in transfection experiments, only telomestatin significantly reduced the human PDGFR-beta basal promoter activity relative to the control. Furthermore, the PDGFR-beta mRNA level in Daoy cells was significantly decreased after treatment with 1 muM telomestatin for 24 h. Therefore, we propose that ligand-mediated stabilization of specific G-quadruplex structures in the human PDGFR-beta promoter can modulate its transcription.

PURPOSE. We investigated the involvement of MAPK signaling in the cell death mechanisms of classical microtubule interfering agents (MIA) and aryl-3-(2-chloroethyl)ureas (CEU) acting as antimitotics, along with CEU that don’t affect directly microtubules (non-MIA CEU). METHODS. To ascertain the activated signaling pathway profile of MIA and non-MIA CEU, Western blot, immunoprecipitation and transfection experiments were performed. RESULTS. Non-MIA CEU do not activate p38, as opposed to MIA, and the extent of ERK and JNK activation is lower than in response to MIA. The effect of MIA and non-MIA CEU on focal adhesion associated protein was also studied; MIA were shown to induce focal adhesion dismantlement associated with a sustained increase in paxillin phosphorylation and FAK cleavage, as opposed to non-MIA CEU. In addition, bcl-2 phosphorylation and AKT cleavage, induced by all MIA tested, was not observed in response to non-MIA CEU further emphasizing the differential cell death mechanisms induced by MIA and non-MIA CEU. Pharmacologic and genetic approaches emphasize that the ASK1-p38 pathway activation contributes to the cytotoxic mechanism of MIA, in contrast to non-MIA CEU. ASK1-p38 is important for increased paxillin phosphorylation and FAK cleavage, suggesting that ASK-1-p38 is an upstream event of FA structure dismantlement induced by MIA. Moreover, the endogen inhibitor of ASK-1, thioredoxin, is released from ASK-1 in response to MIA as opposed to non-MIA CEU. CONCLUSION. Our study supports that ASK1-p38 activation is an important signaling event, induced by MIA, which impairs focal adhesion structure and induces anchorage-dependent apoptosis or anoikis.

The anti-angiogenic approach is traditionally considered for solid malignancies, but increasing evidence has also highlighted its involvement in the progression of hematological oncologies. Angiogenesis, including expression of key proteins, is aberrant in blood cancers such as leukemias, lymphomas, and myelomas, in addition to many solid malignancies, including pancreatic cancer. The most critical mediator of oncogenic angiogenesis is vascular endothelial growth factor (VEGF), which is recognized by two high-affinity receptor tyrosine kinases, VEGFR1 and VEGFR2 (KDR gene). VEGFR2 is necessary for the survival, growth, and differentiation of endothelial cells as its downstream effects include activation of the MAPK and the PI3K pathways, and its upregulation is observed under conditions of pathological angiogenesis. While VEGF/VEGFR2 signaling is known to act in a paracrine manner on endothelial cells, autocrine signaling has been observed in both malignant cells and hematopoietic stem cells. For endothelial and hematological malignancies, both autocrine and paracrine pathways can be targeted with anti-VEGFR2 therapies, potentially doubling the clinical efficacy. Intriguingly, the promoter region of KDR contains a GC-rich region of DNA within its the core promoter located −120 to −31 basepairs (bp) upstream of the transcriptional start site (TSS), putatively able to form several G-quadruplexes. These are unique DNA secondary structures that often serve as transcriptional silencer elements. In the present study, G-quadruplex formation has been isolated −80 to −38 bp upstream of the TSS, which contains five strings of guanines that form a mixed parallel/anti-parallel G-quadruplex, as determined by circular dichroism. The cleavage pattern of DMS footprinting and the major stop product of the polymerase stop assay confirmed that the dominant isoform is a parallel G-quadruplex occurring within the four 3’ runs of guanines. Mutant studies were performed to further determine guanines integral to G-quadruplex formation. An electrophoretic mobility shift assay was used to confirm the formation of a biologically relevant intramolecular structure. High-throughput screening identified a novel KDR-interactive agent, NSC643735, which thermally stabilized the G-quadruplex. This compound is currently being tested in vitro, and has high potential to transcriptionally downregulate KDR mRNA and, subsequently, VEGFR2 protein. These data characterizing the formation of a unique secondary structure in an important oncogene are promising to develop novel and specific anti-KDR therapy for the treatment of hematological and solid malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1386.

Breast cancer is the most common form of cancer in women, whose lethality is only surpassed by lung cancer. Through the last quarter-century there have been significant advances in detection and treatment options, such as Herceptin for HER2/neu positive cancers. While the development of targeted biological agents has greatly improved patient outcome, up to 70% of patients remain de novo refractory, relapse on therapy, or develop resistance. There is a pressing and eminent need for novel therapies to synergize with, or resensitize to, standard treatments. Enter ADAM15, with known zymogen, secretase, and disintegrin activies. This catalytically active member of the ADAM family is normally expressed in early embryonic development and is aberrantly expressed in various cancers, including breast. ADAM15 promotes extracellular shedding of E-cadherin, a soluble ligand for the HER2/neu receptor, leading to activation, increased motility, and proliferation. Seven independent microarray studies have shown that ADAM15 and HER2/neu are simultaneously upregulated in several stages of breast cancer, where their overexpression correlates with more aggressive and invasive disease. We have examined a unique string of GC-rich DNA within the critical core promoter of ADAM15. This region of DNA consists of seven tandem runs of three or more consecutive guanines. Under superhelical stress produced by the transcriptional complex, this region can relax from duplex DNA to form an intrastrand secondary structure known as a G-quadruplex. These globular entities generally serve as silencing elements for gene transcription. Furthermore, the topology of each G-quadruplex is as unique as a fingerprint. This allows for specific therapeutic targeting, offering a unique opportunity for treatment and selectivity over normal cells thus providing a potentially wide therapeutic window. Circular dichroism (CD) studies have confirmed the formation of a mixed parallel/anti-parallel G-quadruplex. Further CD analysis of the minimal G-quadruplex forming fragments has shown the capability of forming four unique thermally stable species. Electrophoretic mobility shift assay indicates the strongest intramolecular G-quadruplex formation in the 5′- and 3′-end runs of guanine, with minor intermolecular formations in the 3′-mid sequence, and a relatively unstable 5′-mid formation. To specifically determine which guanines are involved in the formation of the G-quadruplex and predict biologically relevant three-dimensional structures, DMS footprinting was performed on the full-length sequence and each of the four dissected regions. The pursuant data was used to build a molecular model of the structure. Full characterization of the G-quadruplex species formed will allow for specific drug targeting and stabilization, and the further development of novel, targeted therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3681.